CN106011184A - Noncellular synthetic biology based preparation method of 2-phenylethanol and application - Google Patents

Abstract

The invention discloses a noncellular synthetic biology based preparation method of 2-phenylethanol and an application. According to the preparation method and the application, three recombinases including ARO8, ARO10 and ADH1 are utilized to constitute a co-reaction system, and a complicated metabolic process of an Ehrlich pathway requiring multi-step continuous reaction and multi-enzyme participation for synthesis of 2-phenylethanol in microorganisms is simplified to be performed in the same reaction system in vitro, so that the environmental pollution is reduced and the reaction process is shortened. Furthermore, the three recombinases are subjected to carrier-free co-immobilization with glutaraldehyde serving as a cross-linking agent, Combi-CLEAs (Combi-Cross-linked Enzyme Aggregates) are prepared, a target product is synthesized in vitro, accordingly, the enzymes are further recycled on the basis of the beneficial effects, and the production cost is reduced; the preparation method fills up the technical blank of multi-enzyme preparation of 2-phenylethanol in vitro, and important reference is provided for biological preparation of 2-phenylethanol.

Description

The acellular synthetic biology preparation method and application of 2 phenylethyl alcohol

Technical field

The present invention relates to biosynthesis technology field, more particularly, to the acellular synthesising biological of a kind of 2 phenylethyl alcohol Learn preparation method and application.

Background technology

2 phenylethyl alcohol (2-Phenylethanol, 2-PE) is also known as bata-phenethyl alcohol (β-Phenylethanol), chemical name 2- Phenylethanol (2-Phenylethyl alcohol, 2-PEA), molecular weight 122.16, structural formula is:

2 phenylethyl alcohol is a kind of aromatic alcohol with simple and elegant fine and smooth rose scent, is naturally occurring in the quintessence oil of many plants In, 2 phenylethyl alcohol fragrance the sweetest and, the most popular to people, be the second largest spice that at present whole world usage amount is only second to vanillin Composition, is widely used in food, cosmetics, Nicotiana tabacum L. and cosmetics of everyday use field, and it is not only the basic of all rose scent fragrance Component, and because it has synergism and potentiation, become the component needed for multiple odor type formula, it is the perfume (or spice) with very high value Material material；Pharmaceutically, 2 phenylethyl alcohol is traditional antibacterial.

At present, the annual production nearly ten thousand tons of whole world 2 phenylethyl alcohol, it is essentially all the cheap industrial chemicals benzene of employing or benzene second Alkene chemosynthesis, only has a seldom part and extracts from Oleum Rosae Rugosae.The big multipair health of raw material that chemosynthesis is used and Environment has a significant damage, and in synthesized product 2 phenylethyl alcohol often containing scent of is bad, be difficult to the impurity that removes, such as di- Benzene, β-chloro ethylbenzene, 2-chloroethyl alcohols etc., have a strong impact on product quality.Along with the change of consumption idea, people increasingly advocate " green Color ", the food of " natural " and additive.In American-European countries, flavoring agent and the aromatic that can be marked as " natural " must be to adopt Extract from natural material with physical method and enzyme catalysis or Production by Microorganism Fermentation.If extracting natural 2-benzene from Flos Rosae Rugosae Ethanol, every 5t fresh rose flower is only capable of extracting Flos Rosae Rugosae quintessence oil 1kg, and production cost is extremely expensive, it is impossible to large-scale production.International heaven So 2 phenylethyl alcohol is substantially and converts L-phenylalanine production with microorganism, is concentrated mainly on the flourishing state such as France, Germany, Japan Family, but volume of production is far from meeting consumption demand.

One important production ways of 2 phenylethyl alcohol is to use fermentable, this approach and means raw material and building-up process Environmental protection, reaction condition is gentle, product is safe, with short production cycle, can be mass-produced.2 phenylethyl alcohol in microbial bacteria body Biosynthesis has three approach: Article 1 approach is the shikimic acid pathway of de novo synthesis, is widely present in microbial body, but generation Thank to approach length, branch road is many, there is multiple suppression, and 2 phenylethyl alcohol yield is extremely low；Article 2 is that L-phenylalanine decarboxylation generates benzene second Amine, the reduction that deaminizes generate 2 phenylethyl alcohol, the less generation of this approach；Article 3 is Ehrlich approach, and L-phenylalanine is by turning Ammonia effect generates phenylpyruvic acid, again decarboxylation and forms hyacinthin, finally reduction generation 2 phenylethyl alcohol.Ehrlich approach is microorganism The first-selected approach of synthesis 2 phenylethyl alcohol, is respectively at aromatic series amino transaminases, phenylpyruvate decarboxylase and 3 kinds of enzymes of alcoholdehydrogenase Effect under carry out, it is conducted in-depth research on gene level by researchers, primary study 3 kinds of enzymes of coding Structural gene and transcribing and control methods.

Biosphere all substances metabolism has all been acted on jointly by multienzyme, and these different enzyme energy high-sequential ground are from group Dress forms multienzyme complex (multienzyme complexes, MECs) and participates in reaction, by improving intermediate product at different enzymes Between transhipment concentration, it is achieved efficient catalytic (Lopez-Gallego et al., 2010；Schoffelen et al.,2012； Zhang,2011).But there is the problem such as enzyme difficult separation and recycling, low, the less stable of recycling rate of waterused after multienzyme coreaction, The yield that there is also 2 phenylethyl alcohol is the most relatively low, and cost increases, and flavouring essence quality such as is affected at the more shortcoming.

The most not yet find outside multienzyme, to prepare the correlational study of 2 phenylethyl alcohol and document report.

Summary of the invention

The technical problem to be solved in the present invention is the acellular synthetic biology technology of preparing for existing 2 phenylethyl alcohol Deficiency, intends utilizing this advantage of multienzyme complex efficient catalytic, the most manually prepares multienzymatic reaction system, will efficient table The 3 kinds of recombinases of 3 kinds of enzymes reaching Ehrlich approach are placed in a suitable reaction system, build 3 kinds of external coreactions of recombinase Prepare the peak optimization reaction system of 2 phenylethyl alcohol, without metabolic system complicated in organism, the most external prepare target product 2 phenylethyl alcohol, sets up the acellular synthetic biology preparation method of a kind of 2 phenylethyl alcohol.

The purpose of the present invention is achieved by the following technical programs:

The acellular synthetic biology preparation method of a kind of 2 phenylethyl alcohol is provided, is by restructuring in same reaction system Transaminase I ARO8, phenylpyruvate decarboxylase ARO10 and the common catalyst system and catalyzing catalyzed conversion of alcoholdehydrogenase ADH1 composition, L-phenylpropyl alcohol Propylhomoserin, via phenylpyruvic acid, hyacinthin, is converted into 2 phenylethyl alcohol；Wherein, in described reaction system, it is L-phenylalanine and oxalyl Acetic acid is substrate, and recombinase ARO8, ARO10 and ADH1 press enzyme activity with 1:0.25:1,1:1:1,1:0.5:1 or 1:0.25:2 (U/U/U) proportioning determines, addition is 100 μ L, pH value be 5.5～7.5, Mg²⁺Concentration is in the reaction system of 1.0～5mM, Under the conditions of bath temperature is 30～40 DEG C, after reaction converts 4～7min, terminate reaction with isopyknic acetonitrile, i.e. prepare 2-benzene Ethanol.

The present invention creatively sums up and obtains by restructuring aromatic series transaminase I, phenylpyruvate decarboxylase and alcoholdehydrogenase group The free multi-enzyme system become, external can prepare 2 phenylethyl alcohol with L-phenylalanine and oxaloacetic acid as substrate, have catalytic efficiency High, simple to operate, be not required to purify the advantage such as intermediate product.

Under the conditions of described free multi-enzyme system, preferably L-phenylalanine and oxaloacetic acid ground mol ratio are 0.75:l.

Preferably ARO8, ARO10 and ADH1 are with 1:0.25:1 (U/U/U) proportioning.Preferably Mg²⁺Concentration is 1.0～2.5mM；Enter One step is preferably 1.5mM.Preferably, bath temperature is 33～40 DEG C；More preferably 37 DEG C, preferably at 37 DEG C of water-baths 5min.Preferably, pH value is 5.5～6.5；It is further preferred that described pH value is 6.Most preferably, in described free multienzyme Under the conditions of system, the mol ratio of described L-phenylalanine and oxaloacetic acid is 0.75:l, and recombinase ARO8, ARO10 and ADH1 press enzyme Vigor is with 1:0.25:1 proportioning, and addition is 100 μ L, pH value 6.0, Mg²⁺Concentration is 1.5mM, to wait body after 37 DEG C of water-bath 5min Long-pending acetonitrile terminates reacting and get final product.

It is inadequate that described multi-enzyme system also fails to be fully solved the productivity that intermediate character instability causes in actual applications After height, reaction there is certain difficulty in enzyme separation and recovery, the deficiencies such as recycling rate of waterused is low, less stable, for improving originally further Inventive technique scheme, described 3 kinds of recombinases are also secured in same carrier by the present invention further, improve the stability of enzyme And recycling rate of waterused, increase the local concentration of reaction to improve the yield of product.In practical study process of the test, only science Determine each factors such as the optimum temperature of enzyme effect, optimum pH, the co-immobilization ratio of enzyme, immobilization order action effect and Mechanism and affect rule and the relation of restriction each other, just can sum up the co-immobilization technical side of recombinase in described 3 Case.

Therefore the present invention provides a kind of preferably technical scheme to be first described restructuring transaminase I ARO8, phenylpyruvic acid to be taken off Carboxylic acid ARO10 and alcoholdehydrogenase ADH1 prepares catalyst system and catalyzing Combi-CLEAs altogether by common fixing means, then utilizes Combi- CLEAs carries out catalyzed conversion to L-phenylalanine, via phenylpyruvic acid, hyacinthin, is converted into 2 phenylethyl alcohol.

Under the conditions of common technique for fixing scheme, wherein, in described reaction system, it is to be with L-phenylalanine and oxaloacetic acid Substrate, preferable ph is 5.0, Mg²⁺Concentration is 1.0～5mM, and bath temperature is 40 DEG C, and reaction transformation time is 5min.

The preparation method of described altogether catalyst system and catalyzing Combi-CLEAs is: by ARO8, ARO10 and ADH1 according to 1:1:1,1: After 0.5:1 or 1:0.25:1 proportioning, after precipitant ammonium sulfate precipitation, form many enzyme aggregates, add glutaraldehyde and multienzyme is assembled Body cross-links, and prepares multienzyme fixed system altogether；Wherein, the saturation of ammonium sulfate is 60～90%, and pH value is 4～6, glutaraldehyde Addition according to its final concentration of 0.05～0.25% (V/V) determine.

Preferably, the saturation 70% of precipitant ammonium sulfate.Preferably, the pH value of precipitant ammonium sulfate is 5 preferably, The enzyme proportioning of ARO8, ARO10 and ADH1 is 1:0.5:1.Preferably, the addition of described glutaraldehyde is final concentration of according to it 0.1% (V/V) determines.Preferably, the temperature of described crosslinking is 20～35 DEG C, further preferred 30 DEG C.Preferably, described crosslinking Time be 30～150min, further preferred 120min.

Combi-CLEAs of the present invention has excellent repetitive operation, after reusing 4 times, remains to maintain 66% Initial enzyme live.

The method have the advantages that

The invention provides the acellular synthetic biology preparation method of a kind of new 2 phenylethyl alcohol, utilize 3 kinds of recombinases Constitute coreaction system, Ehrlich approach in microbial body needs multistep successive reaction, multiple enzyme participate in synthesizing 2-benzene The metabolic process that ethanol is so complicated, carries out in being reduced to same reaction system in vitro, thus decreases environmental pollution, contracting Short reaction process.Further, the present invention uses glutaraldehyde as cross-linking agent, 3 kinds of single enzymes of restructuring is carried out carrier-free solid altogether Fixedization, prepares co-crosslinking enzyme aggregate (Combi-CLEAs), external synthesis target product, thus at the base of above-mentioned beneficial effect Realize the recycling of enzyme on plinth further, reduce production cost.The present invention has filled up the skill preparing 2 phenylethyl alcohol outside multienzyme Art is blank, prepares, for biology, the reference that 2 phenylethyl alcohol provides important, opens one's minds for preparing natural 2-benzyl carbinol offer.

Accompanying drawing explanation

The route schematic diagram of Fig. 1 multi-enzyme system catalysis L-phenylalanine synthesis 2 phenylethyl alcohol.After Fig. 2 adds three kinds of enzymes successively High performance liquid chromatography detection 2 phenylethyl alcohol result.The high performance liquid chromatography detection 2 phenylethyl alcohol result of Fig. 3 tri-enzyme coreaction.Figure The product 2 phenylethyl alcohol of 4 three enzyme coreactions is through GC-MS analysis result.Product 2-of Fig. 5 tri-enzyme coreaction Phenethanol is through ion mass spectrometry result.The response time of Fig. 6 tri-enzyme coreaction affects situation to many enzyme activities.Fig. 7 tri-enzyme The multienzyme proportioning of coreaction affects situation to reaction.The Mg of Fig. 8 tri-enzyme coreaction²⁺Concentration affects situation to many enzyme activities. The temperature of Fig. 9 tri-enzyme coreaction affects situation to many enzyme activities.The impact on many enzyme activities of the pH value of Figure 10 tri-enzyme coreaction Situation.The concentration of substrate of Figure 11 tri-enzyme coreaction affects situation to many enzyme activities.Figure 12 is high under the conditions of optimizing three enzyme coreactions Effect liquid phase chromatogram measures 2 phenylethyl alcohol result.During Figure 13 tri-enzyme is fixing altogether, precipitant affects situation to many enzyme aggregates vigor. During Figure 14 tri-enzyme is fixing altogether, ammonium sulfate saturation affects situation to many enzyme aggregates vigor.Figure 15 tri-enzyme fixing middle pH altogether is to many Enzyme aggregate vigor affect situation.During Figure 16 tri-enzyme is fixing altogether, multienzyme proportioning affects situation to Combi-CLEAs vigor.Figure During 17 3 enzymes are fixing altogether, glutaraldehyde concentration affects situation to Combi-CLEAs vigor.Figure 18 tri-enzyme fixing middle crosslinking temperature altogether Combi-CLEAs vigor affected situation.During Figure 19 tri-enzyme is fixing altogether, crosslinking time affects feelings to Combi-CLEAs vigor Condition.During Figure 20 tri-enzyme is fixing altogether, temperature affects situation to co-immobilization multienzyme and free many enzyme activities.Figure 21 co-immobilization is many Enzyme and the temperature stability of free multienzyme.Figure 22 pH is on co-immobilization multienzyme and the impact of free many enzyme activities.Figure 23 substrate is joined Comparison co-immobilization multienzyme and the impact of free many enzyme activities.Figure 24 co-immobilization multienzyme and the bin stability of free multienzyme. The operational stability of Figure 25 co-immobilization multienzyme.The clone identification result of Figure 26 Aro8 gene.The clone of Figure 27 Aro10 gene Qualification result.The clone identification result of Figure 28 ADH1 gene.Figure 29 expression vector p32-YT0801-Aro8 electrophoretic analysis figure.Figure 30 expression vector p32-YT0801-Aro10 electrophoretic analysis figures.Figure 31 expression vector p32-DV10-Adh1 electrophoretic analysis figure.

Detailed description of the invention

The present invention is further illustrated below in conjunction with specific embodiment.Following embodiment being merely cited for property explanation, it is impossible to reason Solve as limitation of the present invention.Unless stated otherwise, the raw material used in following embodiment obtains for conventional commercial or commercial sources The raw material obtained, unless stated otherwise, the method and apparatus used in following embodiment is method commonly used in the art and sets Standby.

Embodiment 1

Main agents: restructuring aromatic series amino transaminases I, phenylpyruvate decarboxylase and alcoholdehydrogenase, conventional commercial or ginseng Build according to prior art literature or build with reference to the embodiment of the present invention 5 method.Chemical standard product: L-phenylalanine, oxalyl second Acid, 2 phenylethyl alcohol, purchased from Sigma-Aldrich.Other chemical reagent are domestic analytical pure or chromatographically pure.Mainly Instrument: digital display thermostat water bath HH-2 (changzhou Guo Hua Electrical Appliances Co., Ltd), constant-temperature metal bath CHB-100 (Hangzhou, Jiangsu Bo Science and Technology Ltd.), accurate pH meter PHS-3C (Shanghai Precision Scientific Apparatus Co., Ltd), Mettler-Toledo electronics Balance AB204-N (Shanghai Mettler-Toledo Instrument Ltd.), Ultrasonic Cell Disruptor (Ningbo Science and Technology Ltd.), liquid phase Chromatograph (Shanghai Tian Mei company), gas chromatography mass spectrometer GC-MSQP2010Plus (Shimadzu Corporation of Japan).

Fig. 1 shows that L-phenylalanine is converted into the process of 2 phenylethyl alcohol.In same reaction system, pass sequentially through restructuring Transaminase I ARO8, phenylpyruvate decarboxylase ARO10 and the conversion of alcoholdehydrogenase ADH1, L-phenylalanine is via phenylpyruvic acid, benzene Acetaldehyde, is converted into 2 phenylethyl alcohol.All reaction systems are all 0.5mL, buffer comprises 0.01mM pyridoxal 5-phosphate, The diphosphothiamine of 0.2mM and the NADPH of 0.1mM.

1. the determination of activity of multienzyme coreaction: with L-phenylalanine and oxaloacetic acid as substrate, HPLC detection multienzyme shares Catalysis activity.Reaction system cumulative volume 500 μ L, contains: 10mM L-phenylalanine, 10mM oxaloacetic acid, 0.5mM Mg²⁺, 0.01mM phosphoric acid Vitamin B6,0.1mM diphosphothiamine, 0.1mM NADPH, tri-kinds of recombinases of ARO8, ARO10 and ADH1 each 10 μL.Add enzyme liquid mix homogeneously, start timing, 33 DEG C of reaction 3min, add 500 μ L acetonitriles and terminate reacting, filtering supernatant, HPLC measures the content of 2 phenylethyl alcohol.

Reaction point two groups: first group is by being sequentially added into three kinds of enzymes, i.e. ARO8 effect 10min the action time of chapter 3 list enzyme After, add ARO10 and fully react 30min, be eventually adding ADH1 effect 3min；Second group is simultaneously introduced three kinds of recombinases, mixing 3min is reacted after Jun Yun.Measure the content of 2 phenylethyl alcohol in two groups.

The enzyme activity definition of multienzyme coreaction: under optimal condition, every milliliter of be catalyzed generation of many enzyme mixations per minute 2 phenylethyl alcohol μm ol amount.Enzyme unit alive is μm ol/ (min mL).

2. 2 phenylethyl alcohol condition determination:

Instrument: sky U.S. chromatograph of liquid；Chromatographic column: Phenomenex luna C18 250mm × 4.6mm；Reference method, Flowing is acetonitrile-water (50/50, V/V) mutually, through 0.22 μm filtering with microporous membrane ultrasonic degassing before using；Detection wavelength is 216nm；Flow velocity 1.0mL/min；Column temperature is room temperature.20 μ l quantitative loop are quantitative.

3. the GC-MS testing conditions of 2 phenylethyl alcohol

Instrument uses GC-MSQP2010PlusD, pillar (0.25mm ID, 30m), split sampling.Heating schedule: sample introduction temperature Spend 250 DEG C, initial temperature 40 DEG C, retain 5min, 4.0 DEG C/min and rise to DEG C, then be heated to 250 DEG C of reservations with 30.0 DEG C/min 5min。

4. the reaction condition of multienzyme coreaction determines checking and test method

(1) time of multienzyme coreaction: the enzyme measuring multienzyme under the differential responses time respectively is lived, and records recombinase The enzyme of ARO8, ARO10, ADH1 live be respectively as follows: 0.87,0.22 and 1.75 μm ol/ (min mL) (assay method with reference to existing often Rule).Living as 100% with maximum enzyme, result is lived with relative enzyme and is represented.

(2) the enzyme proportioning of multienzyme coreaction: when designing multienzyme proportioning, fix the amount of one of them recombinase, the most solid Determine the amount of ARO8, according to enzyme activity, enzyme arranged in pairs or groups in varing proportions: 1:1:1,1:0.5:1,1:0.25:2,1:0.25:1,1: 0.25:0.5,1:0.15:0.5 and 1:0.15:0.25, the enzyme measured respectively under different ratio is lived, and lives as 100% with maximum enzyme, Result is lived with relative enzyme and is represented.

(3) Mg of multienzyme coreaction²⁺Concentration: set Mg different in reaction system²⁺Final concentration, measures under different condition Enzyme live.Set Mg the most respectively²⁺Final concentration of 0,0.3,0.5,0.6,1.0,1.5 and 2.0mM, different condition under enzyme activity determination Result is lived with relative enzyme and is represented, lives as 100% with the maximum enzyme measured.

(4) optimum temperature of multienzyme coreaction: measure multienzyme respectively at 30 DEG C, 33 DEG C, 35 DEG C, 37 DEG C and 40 DEG C of coreactions Time enzyme live, with maximum enzyme live for 100%, result with relative enzyme live represent.

(5) optimum pH of multienzyme coreaction: by three kinds of recombinases ARO8, ARO10, ADH1 according to enzyme activity with different ratio, Add in the 0.1M sodium phosphate buffer of different pH, mix homogeneously, place 30min for 4 DEG C, react under optimal condition, measure enzyme Live.PH system is respectively 5.0,5.5,6.0,6.5 and 7.0, and result is lived with relative enzyme and represented, with the maximum enzyme work measured is 100%.

(6) concentration of substrate impact on enzymatic activity: measure the different mol ratio at substrate L-phenylalanine Yu oxaloacetic acid Under the conditions of multienzyme enzyme live, result with relative enzyme live represent, with maximum enzyme live for 100%.For exempting to repeat, the present embodiment respectively with Substrate L-phenylalanine and oxaloacetic acid mol ratio be that 0.2:1,0.5:1,0.75:1,1:1,1.5:1 and 2:1 say Bright.All independent experiments repeat at least three times.

5. result of the test:

(1) activity identification of multienzyme coreaction: the present embodiment is with the substrate L-phenylalanine of ARO8 enzymatic reaction and oxalyl Acetic acid as starting material, using the catalysate 2 phenylethyl alcohol of ADH1 as end-product, reacts, detection in same system jointly The catalysis activity that three enzymes share, to verify the feasibility synthesizing 2 phenylethyl alcohol outside three enzyme sharing body.Reaction sets two groups: first simultaneously By being sequentially added into three kinds of enzymes the action time of single enzyme, (ARO8 response time when 30 DEG C is 10min to group, and ARO10 and ADH1 Action time be respectively 30min and 3min)；Second group is simultaneously introduced three kinds of recombinase reaction 3min.With reference to 2 phenylethyl alcohol mark Quasi-product, detect product with HPLC, and first group is only had substrate peak and the miscellaneous peak of other intermediate products, be not detected by product Existence, analyze be because formed intermediate product instability caused by；Second group with standard substance 2 phenylethyl alcohol appearance time phase Same position, i.e. occur in that a small peak at 4.650min, and substrate peak area reduces, and display system generates target product 2- Phenethanol, concentration is 11.51 μMs, as shown in Fig. 2 and Fig. 3.Show when ARO8, ARO10 and ADH1 are collectively reside in reaction system, There is catalysis activity.

The product of three enzyme coreaction systems is carried out GC-MS detection further, at 32.563min, is found to have target The existence of product 2 phenylethyl alcohol, as shown in Figure 4, carries out fragment ion analysis to this peak, by comparison, is further characterized by producing Thing is 2 phenylethyl alcohol, as shown in Figure 5.Result shows ARO8, ARO10 and ADH1 tri-enzyme coreaction, can success catalytic substrate L-benzene Alanine is converted into 2 phenylethyl alcohol.

(2) time of multienzyme coreaction accurately determines

The response time of single enzyme is different, and the ARO8 response time when 30 DEG C is 10min, and ARO10 and The action time of ADH1 is respectively 30min and 3min.The present invention researchs and analyses intermediate product character that multienzyme coreaction obtains not Stable, if enzyme action time is long, easily polymerization retrogradation is the most oxidized, it is therefore necessary to investigate the optimal the most anti-of multi-enzyme system Between Ying Shi.

With equimolar L-phenylalanine and oxaloacetic acid as substrate, Mg in reaction system²⁺Concentration is 0.5mM, adds enzyme Three kinds of recombinases that vigor is equal, react different time at 33 DEG C, measure containing of 2 phenylethyl alcohol in reactant liquor by HPLC method Amount, calculates relative enzyme and lives, show that the response time of three enzyme systems is 5min, as shown in Figure 6.

(3) the enzyme proportioning of multienzyme coreaction accurately determines test

In multienzymatic reaction system, L-phenylalanine urging through transaminase I, phenylpyruvate decarboxylase and alcoholdehydrogenase successively Change, generate 2 phenylethyl alcohol, live because of the enzyme of three kinds of enzymes and enzymatic reaction speed is different, for making the conversion ratio of L-phenylalanine reach Maximum, the present embodiment further determines that the optimal proportion of three kinds of enzymes.Owing to transaminase I ARO8 is first enzyme of reaction, thus When discussing multienzyme proportioning problem, the enzyme amount of fixing ARO8, according to enzyme activity, three kinds of enzymes are arranged in pairs or groups in varing proportions, measure 7 altogether Plant the reaction of different proportion.All reaction systems are all 0.5mL, include the pyrophosphoric acid of the pyridoxal 5-phosphate of 0.01mM, 0.2mM Thiamine, the Mg of NADPH and 0.5mM of 0.1mM²⁺.After 33 DEG C of water-bath 5min, HPLC measures the content of 2 phenylethyl alcohol, calculates Enzyme relatively is lived.Fig. 7 show the experimental result display figure that the present invention is main.If ARO8 is not enough, then the phenylpyruvic acid concentration produced Low, the productivity of 2 phenylethyl alcohol is the lowest；If ARO8 is excessive and ARO10 and ADH1 is not enough, then phenylpyruvic acid concentration increases, and is easily caused product Thing suppresses, and the conversion ratio of L-phenylalanine is produced impact；Therefore, suitable multienzyme proportioning is conducive to the formation of product.By Fig. 7 Understanding, three enzymes are maximum with the vigor of 1:0.25:1 proportioning according to enzyme activity；And join with 1:1:1,1:0.5:1 and 1:0.25:2 respectively Although the enzyme of ratio is lived, relatively 1:0.25:1 takes second place, but is equally to ensure enough vigor, simply illustrate this ARO10 of three groups and ADH1 enzyme amount is the most saturated or excessive；And enzyme during 1:0.25:0.5,1:0.15:0.5 and 1:0.15:0.25 proportioning is lived relatively low, Illustrate that in these three groups, ADH1 and ARO10 enzyme amount is not enough.Therefore, during multienzyme coreaction, ARO8, ARO10 and ADH1 are with 1:1:1 (U/U/ U), 1:0.5:1 (U/U/U) and 1:0.25:2 (U/U/U) or 1:0.25:1 (U/U/U) is preferred, further, with 1:0.25:1 (U/U/U) it is optimum proportioning.

(4) Mg of multienzyme coreaction²⁺Concentration determines test

The present inventor researchs and analyses because of Mg²⁺It is the magnesium ion with two positive charges, may participate in the catalysis activity of enzyme so that Mg²⁺There is the catalytic action more higher than acid, the beneficially formation of enzyme active center and stablizing of space structure.Additionally, Mg²⁺Logical Cross electrostatic screen negative charge, the carrying out of reaction can be promoted.But meanwhile, the Mg of higher concentration²⁺Inhibited to enzyme, the present invention People researchs and analyses and is considered Mg²⁺It is centrally formed coordinate bond with electron rich, has blocked substrate and be combined with the active center of enzyme.Same with this Time, a large amount of previous experiments of the present inventor sums up Mg in ARO8 mono-enzyme catalysis system²⁺Concentration is the Mg of 0.6mM, ARO10 system²⁺For 0.5mM, and 5mM Mg²⁺Be conducive to the activity of ADH1, further demonstrate that the present inventor's researchs and analyses conclusion.Therefore, by one The multienzymatic reaction system that certainty ratio is mutually constituted must take into Mg²⁺Optimum concentration range.According to three kinds of single enzyme effects of restructuring Suitable Mg²⁺Concentration range, measures some groups of differences Mg²⁺The reaction of concentration.Fig. 8 shows wherein 6 groups of differences Mg²⁺Concentration Reaction result.ARO8/ARO10/ADH1 with 1:0.25:1 proportioning, 33 DEG C of water-bath 5min according to enzyme activity, measures each group of relative enzyme and lives. As shown in Figure 8, when in reaction system without Mg²⁺Time, recombinase ARO8 and ARO10 can not play its catalysis activity；Mg²⁺Dense Degree is when 1.0～2.5mM scope, and many enzymatic activitys maintain relatively high and stable level always, do not have change clearly；Mg²⁺Dense When degree continues to increase to 5.0mM, enzyme activity slightly declines, and shows that the activity of enzyme is by Mg²⁺Suppression；Mg²⁺When concentration is 1.5mM, The catalysis activity of multi-enzyme system is maximum.

(5) optimum temperature of multienzyme coreaction determines test

Test Summary, the optimum temperature of recombinase ARO8, ARO10 and ADH1 is respectively 30 DEG C, 37 DEG C and 35 DEG C, and Fig. 9 shows Show the enzyme activity situation of change reacted at different temperatures.In multienzymatic reaction system, ARO8/ARO10/ADH1 according to enzyme activity with 1:0.25:1 proportioning, Mg²⁺Concentration is 1.5mM, adds equal-volume acetonitrile and terminate reaction after water-bath 5min.

Result shows, within the temperature range of 30～40 DEG C, temperature is to the activity influence of three enzyme systems the most clearly.30℃ Being the optimum temperature of ARO8, but slightly affect ARO10 and ADH1 activity, the relative enzyme work of multi-enzyme system is 75%；40 DEG C right Three kinds of recombinases all have an impact, and the relative enzyme of multi-enzyme system is lived and reduced to 65%.The optimal reactive temperature that multienzyme shares is with ARO10's Optimum temperature is consistent, is all 37 DEG C, and analysis and summary thinks that ARO10 is the key enzyme of three enzyme systems, 30～40 DEG C of temperature ranges The effect that interior performance is higher.

(6) optimum pH of multienzyme coreaction determines test

The optimum pH of the single enzyme of Test Summary three restructuring is variant, and respectively 7.0,6.0,6.0, therefore the present inventor divides Analysis, the optimum pH of the multi-enzyme system that three is constituted, can be affected by three kinds of single enzyme pH differences, to this end, carried out difference Test, in the range of pH5.5～7.5 regulate pH, study the optimum pH of three enzyme systems.The result of the test of Figure 10 is with Mg²⁺Dense Degree for 1.5mM, ARO8/ARO10/ADH1 according to enzyme activity to illustrate under the conditions of 1:0.25:1 proportioning, 37 DEG C of water-bath 5min etc.. Test Summary combines Figure 10 and can be seen that, the relative enzyme when optimum pH scope of multi-enzyme system is 5.5～6.5, pH6.0 is lived maximum. In the range of pH5.5～7.5, the catalysis activity of multienzyme does not decline to a great extent, and minimum relative activity can keep about 66%.

(7) impact of enzymatic activity is tested by concentration of substrate

The concentration proportioning of research summary substrate L-phenylalanine and oxaloacetic acid can affect the ARO8 conversion ratio to substrate, continues And affect the conversion ratio of ARO10 and ADH1.Therefore, keep substrate oxaloacetic acid concentration be 10mM constant in the case of, the end of to Thing L-phenylalanine uses different mol ratios with oxaloacetic acid, after keeping 5min, surveys under the optimal condition of three enzyme coreactions Determining the content of 2 phenylethyl alcohol, calculate relative enzyme and live, result is as shown in figure 11.As seen from Figure 11, L-phenylalanine and oxalyl When acetic acid mol ratio is less than 0.75:l, the relative enzyme work of multienzyme coreaction increases with the increase of L-phenylalanine proportioning；When entering When one step increases the concentration of substrate L-phenylalanine, many enzyme activities the most significantly change, and maintain top level, explanation always L-phenylalanine concentration has reached saturation；Mol ratio is when 1.5:1 increases to 2:1, and relative enzyme is lived and slightly dropped to 96%, It is because caused by the inhibitory action of L-phenylalanine.As can be seen here, L-phenylalanine is 0.75:l with the mol ratio of oxaloacetic acid Time, relative enzyme is lived maximum.

Multienzyme coreaction synthesis 2 phenylethyl alcohol under embodiment 2 optimal condition

The present embodiment instrument, reagent and detection method etc. are with reference to embodiment 1.With L-phenylalanine and oxaloacetic acid as the end Thing, have studied restructuring aromatic series amino transaminases I ARO8, phenylpyruvate decarboxylase ARO10 and alcoholdehydrogenase ADH1, jointly acts on In the optimal condition of same system synthesis 2 phenylethyl alcohol, all reaction systems are all 0.5mL, and the phosphoric acid pyrrole comprising 0.01mM is trembled Aldehyde, the diphosphothiamine of 0.2mM and the NADPH of 0.1mM.

L-phenylalanine with mol ratio as 0.75:l and oxaloacetic acid, as substrate, synthesize 2-benzene according to aforementioned optimum condition Ethanol.Recombinase ARO8, ARO10 and ADH1 press enzyme activity with 1:0.25:1 proportioning, and addition is 100 μ L, at pH6.0, Mg²⁺Dense In the degree reaction system for 1.5mM, after 37 DEG C of water-bath 5min, terminate reaction with isopyknic acetonitrile, measure 2-benzene second with HPLC The content of alcohol, is 40.92 μMs, as shown in Figure 12.Under the conditions of the multienzyme coreaction that the present invention is optimal, 2 phenylethyl alcohol concentration reaches Higher value.The enzyme activity of restructuring multienzyme is 0.082 μm ol/ (min mL).

The coreaction test that fixed system participates in altogether of embodiment 3 three enzyme

Main agents: the conventional commercial or reference of restructuring aromatic series amino transaminases I, phenylpyruvate decarboxylase and alcoholdehydrogenase Field routine techniques builds, or builds with reference to the embodiment of the present invention 5 method.Chemical standard product: L-phenylalanine, oxalyl second Acid, 2 phenylethyl alcohol, purchased from Sigma-Aldrich.Other reagent routines are commercial, and above chemical reagent is domestic point Analyse pure or chromatographically pure.Key instrument: digital display thermostat water bath HH-2 (changzhou Guo Hua Electrical Appliances Co., Ltd), electric heating air blast is done Dry case 101A-3S (rich people observing and controlling Science and Technology Ltd. of Guangzhou Guangdong), (day science and technology is won in Hangzhou, Jiangsu to constant-temperature metal bath CHB-100 Company limited), Electrolux cold preservation freezing box BCD-263C (Yi Laite (Chinese) company limited), Eppendorf centrifuge 5810R (Eppendorf company of Germany), accurate pH meter PHS-3C (Shanghai Precision Scientific Apparatus Co., Ltd), Mettler- Toledo electronic balance AB204-N (Shanghai Mettler-Toledo Instrument Ltd.), (Ningbo science and technology is limited for Ultrasonic Cell Disruptor Company), chromatograph of liquid (Shanghai Tian Mei company).One, test method:

Enzyme activity determination method: with L-phenylalanine and oxaloacetic acid as substrate, HPLC detects co-immobilization multienzyme (Combi-CLEAs) catalysis activity.Reaction system cumulative volume 500 μ L, contains: 10mM L-phenylalanine, 10mM oxalyl second Acid, 1.5mM Mg²⁺, 0.01mM phosphoric acid Vitamin B6,0.1mM diphosphothiamine, 0.1mM NADPH, co-immobilization multienzyme 30 μ L. After adding enzyme liquid mix homogeneously, start timing, under optimum temperature, react 5min, be rapidly added 500 μ L acetonitriles and terminate reaction, supernatant Liquid is with after 22 μm membrane filtrations, and HPLC measures the light absorption value of 216nm wavelength, calculates the content of 2 phenylethyl alcohol.

The enzyme activity definition of three enzymes fixed system Combi-CLEAs altogether: under optimal condition, every milliliter of Combi-per minute The μm ol amount of the 2 phenylethyl alcohol of the be catalyzed generation of CLEAs mixed liquor.Enzyme unit alive is μm ol/ (min mL).

2 phenylethyl alcohol condition determination is with embodiment 1.

Enzymatic activity recovery calculates: it is initial free many that enzymatic activity recovery refers to that the vigor that Combi-CLEAs is showed accounts for The percent of enzyme liquid total activity.

1. prepared by co-immobilization multienzyme (Combi-CLEAs)

The preparation of many enzyme aggregates: restructuring transaminase I, phenylpyruvate decarboxylase and alcoholdehydrogenase press enzyme activity with 1: 0.25:1 ratio mixes, and takes 100 μ L mixed enzyme solutions and is loaded in test tube, is slowly added dropwise 900 μ L precipitant, simultaneously limit dropping wherein Limit gentle agitation, samples once every 30min, low-temperature centrifugation 5min under 8000rpm, takes supernatant mensuration enzyme and lives, until institute There is albumen precipitated.

The crosslinking of many enzyme aggregates: be slowly added to certain density friendship in the many enzyme aggregates suspension adding precipitant Connection agent glutaraldehyde, continues gentle agitation, cross-links certain time at a certain temperature, and 4000rpm is centrifuged 10min, abandons supernatant, with Being centrifuged after the disodium hydrogen phosphate of 1mL pH7.0/phosphate sodium dihydrogen buffer solution washing precipitation, the resuspended precipitation of 1mL buffer, in 4 DEG C Save backup.

2. multienzyme co-immobilization condition research

The determination of precipitant kind: be respectively adopted 90% ammonium sulfate, 90% isopropanol, 90% butanol, 60%PEG6000, 90% ethanol, 90% acetone, 90% tert-butyl alcohol and 90% acetonitrile, as precipitant, take 100 μ L with 1:0.25:1 (U:U:U) ratio The multienzyme liquid of mixing mixing, is separately added into the 900 above-mentioned precipitant of μ L, and 25 DEG C of precipitations 30min, 8000rpm are centrifuged 5min, washing, With the resuspended precipitation of disodium hydrogen phosphate/phosphate sodium dihydrogen buffer solution of 1mL pH7.0, measure each enzyme and live, calculate the enzyme response rate alive.

The determination of precipitant saturation: take 100 μ L mixed enzyme solutions, is slowly added to 900 μ L saturations wherein and is respectively 20%, the ammonium sulfate of the pH7.0 of 30%, 40%, 50%, 60%, 70%, 80% and 90%, 25 DEG C of precipitation 30min, survey Fixed each enzyme is lived, and calculates the enzyme response rate alive.

The determination of precipitation pH: take 100 μ L mixed enzyme solutions, be slowly added to 900 μ L, pH is respectively 1.0,2.0,3.0,4.0, 5.0, the saturated ammonium sulfate solution of 6.0,7.0 and 8.0,25 DEG C of precipitation 30min, measure each enzyme and live, calculate the enzyme response rate alive.

The determination of multienzyme proportioning: with reference to embodiment 1, fixes the enzyme amount of first enzyme ARO8, according to enzyme activity, ARO8, ARO10 and ADH1 respectively with 1:1:1,1:0.5:1,1:0.25:2,1:0.25:1,1:0.25:0.5,1:0.15:0.5 and 1: The many enzyme mixations of proportions of 0.15:0.25, respectively take 100 μ L mixed enzyme solutions, are slowly added to 900 μ L saturated ammonium sulfates wherein Solution, 25 DEG C of precipitation 30min, measure each enzyme and live, calculate the enzyme response rate alive.

The determination of crosslinker concentration: prepare many enzyme aggregates suspension according to aforementioned optimum condition, is added thereto to the denseest Degree is respectively the glutaraldehyde solution of 0.05%, 0.10%, 0.15%, 0.25%, 0.50% and 0.75% and stirs a little, 25 DEG C After crosslinking 120min, 4000rpm is centrifuged 10min, washs precipitation 3 times with the phosphate buffer of 10mLpH7.0, finally with 1mL The resuspended precipitation of this buffer, measures each enzyme and lives, and calculates the enzyme response rate alive.

The determination of cross-linking agent temperature: prepare many enzyme aggregates suspension, and add the glutaraldehyde solution of suitable concn, respectively 120min is cross-linked at 0 DEG C, 10 DEG C, 25 DEG C, 35 DEG C and 45 DEG C, centrifugal, washing, with the resuspended precipitation of 1mL buffer, measure each enzyme Live, calculate the enzyme response rate alive.

The determination of crosslinking time: prepare many enzyme aggregates suspension, adds glutaraldehyde solution, under optimum temperature, respectively Crosslinking 30,60,90,120 and 150min, centrifugal, washing, with the resuspended precipitation of 1mL buffer, measure each enzyme and live, calculate enzyme and live back Yield.

3. use Response surface methodology to verify multienzyme co-immobilization condition of the present invention

According to experiment of single factor result, use Box-Behnken EXPERIMENTAL DESIGN, aobvious on affect Combi-CLEAs activity Work factor carries out 3 factor 3 level design.Each factor coding and level are shown in Table 1.

After testing according to corresponding test table, data are carried out Quadratic Regression Fitting, obtain including first order, square Item and the quadratic equation of mutual item, analyze main effect and the interaction of each factor, finally ask in the range of certain level Good value.

Table 1Box-Behnken response surface design experiment factor level and coding

4. determine present invention process parameter from co-immobilization multienzyme (Combi-CLEAs) zymologic property research

(1) optimum temperature of Combi-CLEAs: respectively measure Combi-CLEAs 30 DEG C, 33 DEG C, 35 DEG C, 37 DEG C, 40 DEG C, enzyme at a temperature of 45 DEG C and 50 DEG C live, result is lived with relative enzyme and is represented, lives as 100% with the maximum enzyme measured.With free many The optimum proportioning 1:0.25:1 (U:U:U) of enzyme ARO8:ARO10:ADH1 is comparison.

(2) temperature stability of Combi-CLEAs: Combi-CLEAs is respectively placed in 10 DEG C, 30 DEG C, 50 DEG C, 70 DEG C and It is incubated 5h at 90 DEG C, measures residual enzyme and live.Measurement result is lived with relative enzyme and is represented, lives as 100% with uninsulated enzyme.With free The optimum proportioning 1:0.25:1 (U:U:U) of multienzyme ARO8:ARO10:ADH1 is comparison.

(3) optimum pH of Combi-CLEAs: Combi-CLEAs is added in the buffer solution of different pH, mix homogeneously, 4 DEG C place 30min, under the conditions of optimum temperature react, measure enzyme live.PH system is respectively 3.0,4.0,5.0,6.0,7.0,8.0 With 9.0, wherein pH3.0～5.0 is 0.1M sodium-acetate buffer, and pH6.0～7.0 is 0.1M sodium phosphate buffer, and pH8.0 is 0.1M Tris-HCl buffer；PH9.0 is 0.05M Gly-NaOH buffer.Result is lived with relative enzyme and is represented, to measure Big enzyme work is 100%.With the optimum proportioning 1:0.25:1 (U:U:U) of free multienzyme ARO8:ARO10:ADH1 for comparison.

(4) concentration of substrate impact on Combi-CLEAs activity: measure Combi-CLEAs at L-phenylalanine and oxalyl The mol ratio of acetic acid is respectively the enzyme under the conditions of 0.2:1,0.5:1,0.75:1,1:1,1.5:1 and 2:1 and lives, the relative enzyme of result Live and represent, live as 100% with the maximum enzyme measured.With the optimum proportioning 1:0.25:1 of free multienzyme ARO8:ARO10:ADH1 (U: U:U) for comparison.

(5) bin stability of Combi-CLEAs: take Combi-CLEAs and be respectively placed in 4 DEG C of Refrigerator store 40d, Mei Geyi Taking-up equivalent amounts of enzyme liquid of fixing time measures enzyme and lives, and lives as 100% with the maximum enzyme measured, and result is lived with relative enzyme and represented.With free The optimum proportioning 1:0.25:1 (U:U:U) of multienzyme ARO8:ARO10:ADH1 is comparison.

(6) operational stability of Combi-CLEAs: take equivalent and dissociate multienzyme and Combi-CLEAs acts on L-phenylpropyl alcohol respectively Propylhomoserin, leaches enzyme liquid and continues next batch reaction after terminating reaction, the enzyme measuring every batch reaction is lived, and with the maximum enzyme work measured is 100%, result is lived with relative enzyme and is represented.

Two, result of the test

1. multienzyme co-immobilization condition result of study

(1) selection of precipitant kind

Utilizing neutral salt, water-miscible organic solvent and nonionic high polymer precipitation resolvase is prepare CLEAs first Step, precipitant kind is different, and the enzyme conformation induced is the most different, the friendship that cross-linking reaction pausing mode subsequently is the most different, final The activity difference of connection enzyme aggregate is very big.Therefore enzyme is lived and is had a significant impact by the kind of precipitant.Screen through specific aim research And contrast test, the present embodiment is separately added into different 8 kinds of precipitant ((NH4) in the multienzyme liquid of equivalent₂SO₄, the tert-butyl alcohol, PEG6000, isopropanol, n-butyl alcohol, ethanol, acetone, acetonitrile), the enzyme recording various aggregation is lived as shown in figure 13, Tu13Zhong, CK: resolvase；A:90% (NH4)₂SO₄；The B:90% tert-butyl alcohol；C:60%PEG6000；D:90% isopropanol；The positive fourth of E:90% Alcohol；F:90% ethanol；G:90% acetone；H:90% acetonitrile).It can be observed from fig. 13 that various precipitant are to mixing multienzyme Sedimentation effect is each variant, higher as the activity of precipitant finally obtained enzyme aggressiveness using ammonium sulfate and the tert-butyl alcohol, and enzyme is lived The response rate reaches 68%.Ammonium sulfate is a kind of neutral salt, can destroy the hydrated sheath of protein in the solution and make protein coagulation Precipitation.The tert-butyl alcohol is a kind of water-miscible organic solvent albuminoid precipitant, and the principle of precipitation is mainly organic solvent can reduce water The dielectric constant of solution, reduces the polarity of solvent, weakens the interaction force between solvent molecule and protein molecule, increases charged Interaction between particle, increases attracting each other between protein molecule, causes easy coagulation between protein particulate；Secondly, This polar organic solvent can miscible with water, the hydrated sheath of deproteinising surrounding molecules, destroy protein molecule moisture film and It is that albumen precipitates.PEG 6000 is a kind of uncharged straight chain polymer, main by locus repulsive interaction with And the strongest hydrophilic, destroy the hydrated sheath on protein molecule surface, make enzyme assemble or be dehydrated and precipitate.Herein with 60% The aggregation of PEG 6000 preparation is lived relative to enzyme and is only had 43%, it may be possible to PEG concentration is the highest, the strongest to the steric exclusion effect of enzyme Caused, additionally, the viscosity of PEG solution is big, impact operation, test repeatability is bad.Sinking of other organic solvent such as acetone, acetonitrile Shallow lake effect is relatively good, but enzyme is lived the highest, this is because the impact that enzyme is lived in precipitation process by these organic solvents is very big, Enzyme part is caused to inactivate.Therefore, by analysis and verification experimental verification, the present invention chooses ammonium sulfate as preparing Combi-CLEAs's Precipitant.

(2) determination of precipitant saturation

The present embodiment have studied the precipitant concentration impact on many enzyme aggregates activity.Ammonium sulfate pair with different saturation Many enzyme mixations precipitation 30min, measures each aggregation activity after being centrifuged, with the free multienzyme enzyme of the equivalent response rate alive for 100%, Result is as shown in figure 14.Along with the rising of precipitant concentration, precipitated protein content also increases, thus the activity of many enzyme aggregates In rising trend；Precipitant concentration is the biggest, and the precipitation of pheron is the most complete, and the enzyme in aggregation is lived the highest.When ammonium sulfate is saturated Degree increases to 40% from 20%, and the settling velocity of multienzyme is the fastest, and aggregation relative activity is significantly increased to 58% from 18%；Along with The continuation of ammonium sulfate saturation increases, and relative enzyme is lived and gradually risen；When its saturation is 90%, overwhelming majority multienzyme is precipitated Getting off, relative enzyme is lived and is reached maximum, but 70%, 80% enzyme relative with 90% 3 species saturation is lived, difference is not notable, from preparation Cost consideration, the final saturation of precipitant is 70%.

(3) determination of pH is precipitated

Prepare many enzyme aggregates with the ammonium sulfate precipitation agent of different pH, measure enzyme and live, live back with the free multienzyme enzyme of equivalent Yield is 100%, and result is as shown in figure 15.As shown in Figure 15, along with the rising of pH value, enzyme work is in first rising the change declined again Trend；Relative enzyme in the range of pH4.0～6.0 is lived higher, is between 67%～77%；As pH5.0, aggregation vigor is Greatly, this is because the isoelectric point, IP of recombinase ARO8, ARO10 and ADH1 is respectively 5.81,4.91 and 5.06, thus attached at pH5.0 Nearly precipitation is the most complete.These enzyme aggregates got off by ammonium sulfate precipitation in aqueous, pass through non-covalent bond between enzyme molecule Combining, when enzyme aggregate is placed in aqueous phase reactions system, aggregation is decomposed because of unstable, and enzyme molecule is again Being dissolved in water, the space conformation of enzyme molecule is destroyed, thus affects enzyme activity, finds out from aforementioned result of the test, even if Completely under conditions of precipitation, the maximum enzyme relatively of aggregation is lived also less than 80%, therefore, and the most molten in order to avoid aggregation Solve, need to use certain density cross-linking agent to cross-link after multienzyme precipitates.

(4) determination of multienzyme proportioning

When determining multienzyme proportioning, fix the enzyme amount of first enzyme ARO8, according to enzyme activity to three kinds of enzymes in varing proportions Collocation, and at 25 DEG C with pH5.0, the ammonium sulfate precipitation 30min of 70% saturation, after being centrifuged, in each aggregation, add appropriate penta Dialdehyde cross-links, and prepares Combi-CLEAs, measures its enzyme and lives, with the free multienzyme enzyme of the equivalent response rate alive for 100%, and knot Fruit is as shown in figure 16.Multienzyme proportioning is different, and the vigor of enzyme is different；When ARO8:ARO10:ADH1 is 1:0.5:1 (U/U/U), relatively Enzyme is lived maximum, and this has certain deviation with optimum proportioning during aforementioned free three enzymes combination, it may be possible to three enzymes are with 1:0.5:1 proportioning Time, the cross-linked aggregates granular size of three kinds of recombinases formation that molecular diameter, space conformation are different is suitable, and substrate molecule is easy Diffuse into inside aggregation, and the phenylpyruvic acid that enzyme intermolecular distance specific ionization enzyme produces closer to, ARO8 is easily by ARO10 in situ Decarboxylation, hyacinthin easily by ADH1 dehydrogenation in situ, therefore, obtain with Combi-CLEAs prepared by the enzyme liquid of 1:0.5:1 proportioning High vigor.

(5) determination of crosslinker concentration

Glutaraldehyde is suitable for immobilization system of the present invention, and the crosslinking of enzyme is mainly by aldehyde radical and enzyme molecular surface by it Amido react, obtain insoluble enzyme cross-linked aggregates.But the enzyme cross-linked aggregates of preparation is had by the consumption of glutaraldehyde Different impacts.ARO8, ARO10 and ADH1 according to enzyme activity with 1:0.5:1 proportioning after, through 70% saturation ammonium sulfate precipitation After, many enzyme aggregates are cross-linked by the glutaraldehyde adding different volumes, make the final concentration of glutaraldehyde in solution be respectively as follows: 0.05%, 0.10%, 0.15%, 0.25%, 0.50% and 0.75% (V/V), prepares various Combi-CLEAs, measures its enzyme Living, result is as shown in figure 17.As shown in Figure 17, along with the increase of glutaraldehyde concentration, under enzyme activity is in the most slightly rising the most persistently The variation tendency of fall, when glutaraldehyde concentration is 0.10%, relative enzyme is lived and is reached the highest；Glutaraldehyde concentration is increased to by 0.50% When 0.75%, relative enzyme is lived and drastically be have dropped 30%.Showing within the specific limits, glutaraldehyde consumption increases, the Combi-obtained The amount of CLEAs also increases, and enzyme activity increases, but glutaraldehyde consumption is excessive, can cause the excessive crosslinking to enzyme, cause enzymatic activity Heavy losses.This is because glutaraldehyde mainly reduces the number of dropouts of enzyme by crosslinked action, when low concentration, only have a small amount of Enzyme molecule can crosslink, the insoluble cross-linked enzyme aggregate amount obtained is little, has substantial amounts of enzyme not to be crosslinked, Washing process leaks, runs off, thus enzyme activity is low；But when the concentration is too high, glutaraldehyde and the aminoacid of enzyme molecule self Occur many sites to combine, make the higher structure of protein that a certain degree of destruction to occur, not only cause the loss of enzyme activity, and And form hard-packed aggregation substrate molecule is difficult to close to the active center of enzyme, thus affect enzyme molecular activity center Effectiveness, therefore embody relatively low enzyme activity.

(6) determination of crosslinking temperature

Temperature is also the key factor affecting multienzyme cross-linked aggregates vigor.Final concentration is added in many enzyme aggregates Be 0.10% glutaraldehyde cross-link, be respectively placed in cross-linking reaction 120min at 0,10,20,25,30,35,45 DEG C, measure each The vigor of Combi-CLEAs, calculates relative enzyme and lives.As can be seen from Figure 18, temperature in time being increased to 30 DEG C for 0 DEG C, enzyme activity Constantly increase；Enzyme in the range of 20～30 DEG C is lived and is increased significantly；When 30 DEG C, relative enzyme is lived maximum, reaches 74%.Along with temperature Continuation raise, enzyme activity loss increase, when 45 DEG C, the relative activity of Combi-CLEAs reduces to 43%.Therefore prepare multienzyme to hand over The optimum temperature of connection aggregation elects 30 DEG C as.

(7) determination of crosslinking time

Crosslinking time affects the activity of many enzyme aggregates.The glutaraldehyde of final concentration of 0.10% cross-links multienzyme at 30 DEG C and gathers The time of collective is respectively 30,60,90,120 and 150min, measures the vigor of each Combi-CLEAs, calculates relative enzyme and lives.Knot Fruit as shown in figure 19, along with the increase of crosslinking time, live and the most steadily increase by the relative enzyme of Combi-CLEAs, this is because crosslinking Time is the longest, cross-links the most thorough, and the amount of multienzyme cross-linked aggregates is the most, and the vigor of enzyme is the biggest；When more than 120min, enzyme is lived and is opened Beginning to decline, during crosslinking 150min, relative enzyme is lived and is dropped to 41%, this is because crosslinking time is long, too much glutaraldehyde covers The amount of activated position of enzyme, affects enzyme activity；Additionally, crosslinking time is the most long, easily there is the reactions such as polymerization and oxidation in glutaraldehyde, from And affect enzyme and live and cross-linking effect.Therefore, crosslinking time is extremely important to stability and the activity of Combi-CLEAs, therefore determines 120min is suitable crosslinking time.

Three, the response surface research checking test of multienzyme co-immobilization condition

1.Box-Benhnken test results and analysis

The present invention uses Box-Behnken EXPERIMENTAL DESIGN, and with (NH4) 2SO4 as precipitant, choosing multienzyme proportioning is 1: 0.5:1, precipitation pH are 5.0, crosslinking time is 120min, and the notable factor affecting multienzyme CLEAs activity is carried out 3 factor 3 water Flat design, with 3 times test gained enzymatic activity recoveries meansigma methods as response value (Y), concrete EXPERIMENTAL DESIGN and result such as table 2 Shown in.

Table 2Box-Behnken EXPERIMENTAL DESIGN and result thereof

(2) Quadratic Regression Fitting and variance analysis

It is many that applied statistics soft SA S 8.0 (Statistics Analysis System) carries out secondary to the data of table 2 Item regression fit, significance statistically is determined by T inspection, and the coefficient of determination and the coefficient of variation are determined by F test.By Box- The quadratic regression equation that Behnken test data fitting meets with a response between value (Y) Yu each factor (A, B, C) is:

Y=-1024.29+17.18A+519.85B+31.61C+8.63AB-0.079AC+11.78BC-0.11A²- 7488.50B²-0.47C²

Above-mentioned binary regression equation is carried out significance analysis and variance analysis, the results are shown in Table 3.

The variance analysis of table 3Box-Behnken EXPERIMENTAL DESIGN regression equation

Between each factor and response value, the significance of linear relationship is judged by F value inspection, probability P (F ＞ F_α) value more Little, then the significance of explanatory variable is the highest.Lose plan item (Lack of fit) and be used to an important number of Estimate equation reliability According to, if significantly, show that equation simulation is bad；If not notable, show that equation simulation is relatively good, after can analyzing well Data, i.e. actual experimental point can be replaced experimental result to be analyzed and predicts with this regression equation.As shown in Table 3, mould The significant level of type is 0.0001 (< 0.01), shows that model is extremely notable on the impact of response value (enzymatic activity recovery), this model The most meaningful.Crosslinking temperature impact is extremely notable, and 3 factors are crosslinking temperature on the significance that affects of enzymatic activity recovery > ammonium sulfate saturation > glutaraldehyde concentration；Mutual item A (ammonium sulfate concentrations) and B (glutaraldehyde concentration), A (ammonium sulfate concentrations) and C (crosslinking temperature) significant interaction, illustrates that these 3 factors are all the crucial governing factors in Combi-CLEAs preparation process； Quadratic term A²、B²、C²Affect the most notable.Lose and intend item P=0.6311 (> 0.05), there is no significant, illustrate that data do not have Abnormity point, it is not necessary to introduce the item of more high reps, model is suitable.

The Analysis on confidence of table 4 model

Std.Dev.	2.58	R-Squared	0.9864
				Mean	46.63	Adj R-Squared	0.9688
C.V.%	5.54	Pred R-Squared	0.9153
				PRESS	290.63	Adeq Precision	19.485

As can be seen from Table 4, coefficient R²=0.9864, illustrate the change of response value have 98.64% derive from selected Three variablees.Correction coefficient of determination R²(Adj)=0.9688 (> 0.80), illustrate that the variability of the experimental data of 96.88% can be used This model explanation；And relatively low coefficient of variation C.V.% (5.54%) shows, only having 5.54% variation in model can not be by this mould Type is explained, i.e. has the variation of 94.45% can be explained by by this model, shows that this model is highly significant.Signal to noise ratio More than 4 the most rationally, this model signal to noise ratio is 19.485 to Adeq Precision, shows that model can reflect real experiment very well Value.To sum up analyzing, this regression equation can preferably describe the true relation between each factor and response value, it is possible to use it is true Determine the optimum preparating condition of Combi-CLEAs.

3. checking test

Learning according to the Box-Behnken analysis that designs a model, multienzyme co-immobilization condition optimizing technological parameter is: (NH₄)₂SO₄Saturation is 71.57%, and glutaraldehyde concentration is 0.10%, and crosslinking temperature is 28.55 DEG C.With this understanding, the enzyme response rate alive Predictive value is 67.29%.In order to determine whether the model of foundation is consistent with result of the test, according to practical operation, by fixing chemical industry Skill parameters revision is (NH₄)₂SO₄Saturation is 72%, and crosslinking temperature is 29 DEG C, and glutaraldehyde concentration is still 0.10%；In this parameter Under, carrying out 3 groups of experiments, the enzyme the obtained response rate alive is 65.76 ± 3.27%, close with model predication value.Institute's fitting data Model has the preferable goodness of fit with practical solution of the present invention well.

4. co-immobilization multienzyme zymologic property research test

(1) impact of temperature enzymatic activity many on co-immobilization

Under high temperature, the activity conformation of enzyme dissociates, and is that enzymatic activity reduces or the basic reason of forfeiture, and immobilization technology By the connection of the various covalent bonds between enzyme molecule or between enzyme and carrier, it is effectively reduced dissociating of this conformation.Therefore Most of enzymes are after immobilization, and optimum temperature relatively resolvase can increase.This experiment determines under different temperatures respectively Combi-CLEAs and the activity of resolvase, result is as shown in figure 20.As shown in Figure 20, the optimum temperature of resolvase is 37 DEG C, The optimum temperature of Combi-CLEAs has slightly rising, is 40 DEG C；And the appropriate effect temperature range of Combi-CLEAs is more free Enzyme width, in the range of 37～50 DEG C, its enzyme activity is held at more than 86% that the highest enzyme is lived.Show to cross-link immobilization skill Art improves the catalytic temperature of enzyme.Its reason is that the cross-linked enzyme aggregate structure comparison that cross-linking agent glutaraldehyde is formed is fine and close, to enzyme Active group have certain protective effect.From figure it can also be seen that, with spending the further rising of temperature, immobilized enzyme and trip Activity from enzyme is all gradually lowered, therefore, except determining its optimum temperature, it is also contemplated that the heat stability of immobilized enzyme.

(2) temperature stability of co-immobilization multienzyme

Heat stability is the important indicator investigating immobilized enzyme performance.To immobilized enzyme and resolvase in different temperatures Lower process 5h, measures its residual enzyme activity, and result is as shown in figure 21.In figure it can be seen that along with temperature improve, Combi- The activity of CLEAs and free multienzyme is the most on a declining curve, but the activity decrease amplitude of resolvase is bigger；After 50 DEG C of insulation 5h, The relative enzyme of Combi-CLEAs is lived and is remained at 80%, and resolvase only 50%；After 90 DEG C process 5h, the former is at residual activity 25%, the latter is less than 10%.As can be seen here, this process for fixation of co-crosslinking enzyme aggressiveness can be effectively improved the heat stability of enzyme, Substantially reduce heat inactivation degree.Its reason is that after resolvase forms CLEAs, space structure changes, amount of activated group quilt Being embedded in inside aggregation, thus be protected, therefore thermostability has strengthened；Additionally, higher temperature is conducive to substrate Molecule diffuses to aggregation inside and reacts with enzyme.

(3) impact of pH enzymatic activity many on co-immobilization

The vigor of enzyme is easily affected by environment pH, and only under certain pH, enzyme could show maximum vigor.Due to carrier pair The impact of microenvironment ionic strength, the optimum pH of immobilized enzyme is often drifted about.The present embodiment determines pH 3.0 ～9.0 buffer in Combi-CLEAs and the activity of free multienzyme, result is shown in Figure 22.As seen from the figure, Combi-CLEAs Optimum pH is 5.0, and the optimum pH of free multienzyme is 6.0, and Combi-CLEAs oxytropism offset by a unit, its appropriate effect PH scope also offsets to acid.Being possibly due in cross-linking process, the aldehyde radical in glutaraldehyde molecules occurs with the amino in enzyme molecule Necleophilic reaction so that the negative charge of protein molecule side-chain radical increases, pH in external solution must oxytropism skew These negative charges can be neutralized.

(4) impact of concentration of substrate enzymatic activity many on co-immobilization

When the concentration proportioning of research substrate affects co-immobilization multienzyme (Combi-CLEAs) activity, keep substrate oxalyl second Acid concentration be 10mM constant in the case of, substrate L-phenylalanine and oxaloacetic acid are used different mol ratios, at Combi- After keeping 5min under the optimal condition of CLEAs, measuring enzyme and live, result is as shown in figure 23.As seen from the figure, substrate proportioning is to fixing altogether Change multienzyme consistent with the impact of free multienzyme, when L-phenylalanine is less than 0.75:l with oxaloacetic acid mol ratio, the phase of the two Enzyme is lived and all increases with the increase of L-phenylalanine concentration, but the relative enzyme of immobilized multienzyme is lived slightly higher；When substrate mol ratio During more than 1.5:1, because of the suppression of L-phenylalanine, the two vigor is slightly decreased, and immobilized multienzyme decrease speed is the fastest.Thus may be used Seeing, when the mol ratio of L-phenylalanine and oxaloacetic acid is 0.75:l, immobilized multienzyme enzyme relative with free multienzyme is lived maximum.

(5) bin stability of co-immobilization multienzyme

The bin stability of enzyme is extremely important to its commercial applications.Free multienzyme and co-immobilization multienzyme CLEAs are put In 4 DEG C of Refrigerator store 40d, measuring enzyme at regular intervals and live, result is as shown in figure 24.In the front 10d of storage, both work Power does not all have significant change；After 20d, immobilized enzyme maintains the activity of 98%, and free enzyme activity is 95%；After 40d, enzyme activity is all Having declined, but the downward trend specific ionization enzyme of Combi-CLEAs is slow, both activities maintains 87% He that initial enzyme is lived respectively 70%.Showing that the bin stability of immobilized enzyme is preferable, this is primarily due to the precipitation in Combi-CLEAs preparation process and friendship Connection step has a purification effect to enzyme, it is to avoid foreign protein causes the hydrolysis of enzyme.Additionally, enzyme molecule is with multiple spot covalent cross-linking form jail Jail is locked in a region, it is to avoid enzyme leaks in solution with free state form.

(6) operational stability of co-immobilization multienzyme

Repeatable usability is also the important indicator weighing immobilized enzyme performance.After the reaction of each batch terminates, Combi-CLEAs leaches continuation next batch reaction, and result is shown in Figure 25.Immobilized multienzyme after reusing 4 times, substrate Conversion ratio is 23%, and the initial enzyme maintaining 66% is lived, and resolvase is because being difficult to reclaim, and after using 1 time, enzyme is lived and only remained 4%, illustrate that Combi-CLEAs has preferable repetitive operation；After using 8 times continuously, immobilized multienzyme vigor fall Greatly, only remaining the vigor of 19%, main cause is to be repeated several times to affect its stability, next to that Combi-CLEAs granule is tiny, Impact is reclaimed.

Embodiment 4 multienzyme fixed system altogether is applied to coreaction and prepares 2 phenylethyl alcohol

The optimal procedure parameters drawn according to response surface analysis, carries out multienzyme and fixes altogether, synthesizes 2-benzene second according to optimum condition Alcohol.Combi-CLEAs addition is 100 μ L, after pH5.0,40 DEG C of water-bath 5min, terminates reaction with isopyknic acetonitrile, uses It is 28.61 μMs that HPLC records the content of 2 phenylethyl alcohol.The enzyme activity of co-immobilization multienzyme is 0.057 μm ol/ (min mL).

The construction method of three kinds of recombinases that embodiment 5 optimizes

Be not particularly illustrated in following construction method is with reference to this area routine techniques, does not has the biomaterial of explanation It is biomaterial commercial or commonly used in the art.

One, from the different strains of saccharomyces cerevisiae, clone has obtained with 3 keys of Ehrlich approach synthesis 2 phenylethyl alcohol 11 genes of enzyme.

(1) 3 aromatic series amino transaminases I gene.The a length of 1503bp of its DNA sequence, relative molecular mass size is 56.2kD, theoretical isoelectric point, IP is 5.81.The Genbank accession number of 3 genes be respectively KC422721.1, KC422722.1 and KC422723.1。

(2) 3 phenylpyruvate decarboxylase genes.The a length of 1908bp of its DNA sequence, relative molecular mass size is 71.4kD, theoretical isoelectric point, IP is 6.10.Wherein 2 genes are registered in Genbank, accession number be respectively KC422719.1 and KC422720.1。

(3) 5 alcohol dehydrogenase genes.Be respectively as follows: 2 alcoholdehydrogenase Adh1 genes, 1 alcoholdehydrogenase Adh2 gene, 1 Alcoholdehydrogenase Adh3 gene and 1 alcoholdehydrogenase Sfa1 gene.Wherein, the DNA sequence length of Adh1 and Adh2 gene is all 1047bp, it is 6.07, EC1118-Adh1 that relative molecular mass size is about the theoretical isoelectric point, IP of 36.8kD, DV10-Adh1 gene It is all 6.21 with DV10-Adh2 theory isoelectric point, IP.The Genbank accession number of EC1118-Adh1 gene and DV10-Adh2 is respectively KC491732.1 and KC491733.1.The a length of 1128bp of DNA sequence of YT0801-Adh3 gene, relative molecular mass size For 40.4kD, theoretical isoelectric point, IP is 8.65, and its Genbank accession number is KC422718.1.The DNA sequence of YT0801-Sfa1 gene Arranging a length of 1161bp, relative molecular mass size is 41kD, and theoretical isoelectric point, IP is 6.51, and its Genbank accession number is KC491734.1。

Material: saccharomyces cerevisiae (Saccharomyces cerevisiae) bacterial strain and escherichia coli (Escherichia Coli) bacterial strain DH5, conventional bacterial strain, it is possible to use the bacterial strain that other laboratory normal experiments use, the bacterium that the present embodiment uses Strain is the experiment bacterial strain of this laboratory routine preservation.Cloning vehicle pGEM-T Easy Vector, Promega Products.

TakaRaEx Taq enzyme is purchased from Dalian treasured biological engineering company limited；Plain agar gel DNA reclaim test kit and The little extraction reagent kit of plasmid is purchased from Tian Gen biochemical technology company limited；Conventional reagent is domestic analytical pure.

According to the genes of interest sequence information reported, primer 5 and oligo 6.0 is utilized to design specific primer, by Shanghai Jierui Biology Engineering Co., Ltd synthesizes, and primer sequence information is shown in Table 5.

Table 5 primer sequence information table

The cultivation of saccharomyces cerevisiae and the extraction of STb gene:

Extraction genomic DNA from different Wine brewing yeast strains respectively, reference Snailase overnight facture (Wang Ping etc., 2008；Zhao Hongyu etc., 2011).With saccharomyces cerevisiae STb gene as template, above-mentioned specific primer is utilized to carry out PCR reaction amplification mesh The DNA full length fragment of gene, pcr amplification reaction system is:

PCR response parameter: 94 DEG C of denaturations 5min, 94 DEG C of degeneration 45s, 46 DEG C of annealing 45s, 72 DEG C extend 90s；Totally 33 Circulation；72 DEG C extend 10min, preserve at PCR primer 4 DEG C.

The recovery of purpose fragment pcr amplification product: amplification PCR primer, after 1.0% agarose gel electrophoresis, cuts purpose Band, reclaims test kit with TIANGEN plain agar sugar gel DNA and reclaims.

The connection of PCR primer: the PCR primer of recovery is connected with carrier T-easy Vector, recombinant plasmid transformed large intestine bar Bacterium E.coli DH5 α.Coupled reaction system is as follows:

Connect mixture and connect overnight at 4 DEG C, convert E. coli competent DH5 α (CaCl₂Method).

Connect the conversion of product and the preparation of competent escherichia coli cell with reference to existing conventional techniques.

The conversion connecting product comprises the following steps:

Step one: the preparation of competent escherichia coli cell

(1) one single bacterium colony of picking from 37 DEG C of fresh plate cultivating 16h, forwards to equipped with 100mL LB culture medium In 250mL triangular flask, it is aggressively shaken 3h (220rpm) in 37 DEG C.

(2) aseptically escherichia coli are transferred to the 50mL centrifuge tube of a pre-cooling aseptic, disposable In, place 10min on ice.

(3) in 4 DEG C with GS3 rotary head with 4000rpm, centrifugal 10min collects cell.

(4) pour out culture fluid, pipe is inverted 1min, make the trace culture fluid of final residual flow to end.

(5) with 10mL ice-cold 0.1M CaCl₂Resuspended every part of precipitation, after ice bath 30min, then is centrifuged with 4000rpm 10min collects cell.

(6) most supernatant is abandoned, and with the 0.1M CaCl of 2mL ice pre-cooling₂Resuspended precipitation, 4 DEG C of placements, use within 72h；Or At 0.1M CaCl₂On the basis of add glycerol to final concentration 20%, in-70 DEG C of preservations after subpackage, the most standby.

Step 2: the qualification of competent escherichia coli cell

(1) draw the suspension of 100 μ L competent cells, add the 2 μ L plasmid (anti-Amp) without genes of interest, mixing, Ice bath 30min.Make blank one simultaneously, the most additionally draw the suspension of 100 μ L competent cells, be not added with plasmid.

90s is placed in (2) 42 DEG C of water-baths, is quickly transferred on ice, makes cell cool down rapidly 1～2min.

(3) adding the LB liquid of 400 μ L, 37 DEG C of water-baths recover 5min, proceed to 37 DEG C of shaking table 200rpm shaken cultivation 45min。

(4) in 100mL is cooled to the LB solid medium of 46 DEG C, adding the Amp (50 μ g/ μ L) of 200 μ L, mixing is all Even, inject plate.

(5) 100 μ L (3), even spread are added.

(6) after drying, it is inverted flat board, 37 DEG C of overnight incubation, observed result.

(7) comparison being not added with plasmid produces without bacterium colony；The bacterium colony uniformly gathered that has adding plasmid produces.Illustrate that competence is thin Born of the same parents have preferable conversion capability, and the miscellaneous bacteria of nonreactive Amp.

Step 3: connect the conversion of product

(1) from-70 DEG C of refrigerators, take 200 μ L competent cell suspensions, make it thaw under room temperature, after defrosting, be immediately placed on ice On；

(2) add connection mixed liquor 10 μ L, shake up gently, place 30min on ice；

Thermal shock 90s in (3) 42 DEG C of water-baths, is immediately placed in cooled on ice 3～5min after thermal shock；

(4) add the 1mL LB fluid medium without Amp, mix rear 37 DEG C of shaken cultivation 1h, make antibacterial recover normal raw Long status, and the antibiotics resistance gene (Amp of expression plasmid coding⁺)；

(5) take 100 μ L after above-mentioned bacterium solution being shaken up to be spread evenly across containing Amp⁺Screening flat board on, face up placement 0.5h is so that liquid is absorbed；

(6) after bacterium solution is cultured base absorption completely, it is inverted flat board, cultivates 16～24h in 37 DEG C；

(7) after bacterium colony fully develops the color, clone's (white macula) of picking band recombiant plasmid cultivates, solid at LB with sterile toothpick Body culture medium is chosen the single bacterium colony of the white after conversion, is inoculated in the LB fluid medium that 2mL contains antibiotics ampicillin, 37 DEG C of shaken cultivation 8～12h.

The extraction of recombiant plasmid: carry out, to converting bacterial strain, matter of recombinating with reference to the little extraction reagent kit of description TIANGEN plasmid Grain extracts.

The enzyme action of recombiant plasmid is identified: endonuclease reaction system is:

37 DEG C of insulation 3h, carry out 1.0% agarose gel electrophoresis.

PCR identifies and enzyme action identifies that errorless clone carries bacterial strain, by Beijing six directions Hua Da limited public affairs of Gene science share Department's Guangzhou Branch order-checking.

Nucleic acid sequence alignment is carried out on NCBI BLAST (http://blast.ncbi.nlm.nih.gov), aminoacid Sequence alignment is carried out on EBI-BLAST (http://www.ebi.ac.uk), and nucleic acid sequence analysis is with DNassist 2.0 He NCBI Spidey completes, and nucleic acid completes with aminoacid sequence drafting DNAMAN, and the prediction of protein structure and function exists There is provided on Swissprot (http://www.expasy.ch/sprot/) website and carry out on the platform of link.

The present embodiment extracts the STb gene of 17 different Wine brewing yeast strains respectively, carries out with ARO8F and ARO8R for primer PCR expands, and the PCR primer size that result obtains from 3 yeast strains may each be about 1500bp, is consistent with expection.Convert E.coli DH5 α, extracts positive recombiant plasmid, identifies through EcoR I enzyme action, and Insert Fragment size about 1500bp, with target Gene is close, as shown in Figure 26.Wherein in Figure 26, left (a) is the electrophoretic analysis result of Aro8 gene PCR product；Right figure (b) is The enzyme action qualification result of recombiant plasmid.M:MakerⅢ；1～the PCR of 3:YT0801-Aro8, EC1118-Aro8 and DV10-Aro8 Product；1₀～3₀: recombiant plasmid；1₁～3₃:1₀～3₀The digestion products of number plasmid.

The present embodiment extracts the STb gene of 17 strain difference Wine brewing yeast strains, carries out PCR with ARO10F and ARO10R for primer Amplification.The PCR primer size that result obtains from 3 saccharomycete strains may each be about 1900bp, is consistent with expection.Reclaim PCR primer, Convert DH5 α competent cell, containing Amp⁺LB flat board on carry out the screening of blue white macula.Extract positive recombiant plasmid, EcoR I enzyme Cut qualification.Enzyme action result display electrophoretic band is clear, and Insert Fragment size about 1900bp is close with target gene, sees figure Shown in 27, in figure, left figure (a) is the electrophoretic analysis of Aro10 gene PCR product；Right figure (b): the enzyme action of recombiant plasmid is identified.M: MakerⅢ；1～the PCR primer of 3:YT0801-Aro10, EC1118-Aro10 and DV10-Aro10；1₀～3₀: recombiant plasmid；1₁ ～3₃:1₀～3₀The digestion products of number plasmid.

The present embodiment extracts the STb gene of 17 different Wine brewing yeast strains, carries out PCR amplification.Obtain from 2 strain yeast with The PCR primer (about 1050bp) that Adh1 gene size is consistent, is obtained the PCR primer being consistent with Adh2 gene size by 1 strain yeast (about 1050bp), is obtained the PCR primer (about 1150bp) being consistent with Adh3 gene size by 1 strain yeast, by 1 strain yeast obtain with The PCR primer (about 1150bp) that Sfa1 gene size is consistent.Reclaim PCR primer, Transformed E .coli DH5 α, carry out blue white macula sieve Choosing, extracts positive recombiant plasmid, identifies through EcoR I enzyme action, and Insert Fragment size is all close with target gene, sees Figure 28 institute Show.In Figure 28, left figure (a) is the electrophoretic analysis of PCR primer；Right figure (b) is that the enzyme action of recombiant plasmid is identified, M:Maker III；1 ～the PCR primer of 5:DV10-Adh1, EC1118-Adh1, DV10-Adh2, YT0801-Adh3 and YT0801-Sfa1；1₀～5₀: Recombiant plasmid；1₁～5₅:1₀～5₀The digestion products of number recombiant plasmid.

Two, e. coli bl21 (DE3) is utilized respectively the albumen coded by 3 key genes to be carried out prokaryotic expression, Detecting through SDS-PAGE, the molecular size range of 3 kinds of albumen is basically identical with the size using bioinformatics method prediction.

(1) YT0801-Aro8 gene is cloned into expression vector pET-32a (+) on, construct aromatic series amino transaminases I engineering strain.At 30 DEG C, inducing 8h through IPTG, engineered strain can express recombinase ARO8.Purified, fusion protein The concentration of ARO8 is 462.3mg/L.Detecting through SDS-PAGE, its molecular size is about 70kDa.

(2) YT0801-Aro10 gene is cloned into expression vector pET-32a (+) on, construct phenylpyruvate decarboxylase Engineering strain.At 28 DEG C, inducing 3h through IPTG, engineered strain can express recombinase ARO10.Purified, fusion protein The concentration of ARO10 is 202.2mg/L.Detecting through SDS-PAGE, its molecular size is about 90kDa.

(3) DV10-Adh1 gene is cloned into expression vector pET-32a (+) on, construct alcohol dehydrogenase gene engineering bacteria Strain.At 28 DEG C, inducing 3h through IPTG, engineered strain can express recombinase ADH1.Purified, fusion protein ADH1 concentration is 252.6mg/L.Detecting through SDS-PAGE, its molecular size is 61kDa.

Material: plastid transformation recipient bacterium E.coli DH5 α, E.coli BL21 (DE3) are by Zhongshan University school of life and health sciences Liu Beautiful shining professor's present, it would however also be possible to employ this kind of recipient bacterium commonly used in the art.PMD18-T carrier is purchased from Promega company, PET-32a (+) carrier presented by Zhongshan University school of life and health sciences professor Liu Yuhuan, it would however also be possible to employ commonly used in the art should Carrier.

Ampicillin, isopropyl-β-D-thiogalactoside (IPTG), the bromo-4-of 5-chloro-3-indole-β-D-galactose Glycosides (X-gal), trishydroxymethylaminomethane (Tris), acrylamide (Acrylamide), methylene diacrylamide (BIS), β- Mercaptoethanol, bromophenol blue (BPB), agarose, agar powder are import subpackage, purchased from Beijing limited public affairs of Pu Boxin biotechnology Department.Agarose gel DNA reclaims test kit and the little extraction reagent kit of plasmid and is purchased from Tian Gen biochemical technology company limited；Protein purification tree Fat His bind resin and purification column are purchased from Merck company (German)；Various restricted enzyme, T4DNA ligase, low Molecular weight protein standard, purchased from precious biological (Dalian) Engineering Co., Ltd.DNAMarkerⅢ、1kb plus DNA Ladder (MD113)

, protein Marker, purchased from TIANGEN Biotech (Beijing) Co., Ltd..L-phenylalanine, oxaloacetic acid, Radix Asparagi The chemical standard product such as propylhomoserin, phenylpyruvic acid, hyacinthin, 2 phenylethyl alcohol are purchased from Sigma-Aldrich.Other chemistry examinations Agent is domestic analytical pure or chromatographically pure.

Culture medium: LB (Luria-Bertani) culture medium (1000ml): 10g tryptone, 5g yeast extract, 10gNaCl, pH are natural (solid medium need to separately add 20g agar powder), sterilizing 15min at 121 DEG C.It is cooled to room temperature and adds ammonia Benzylpcnicillin in culture medium, final concentration of 1g/L.LB culture medium-X-gal-IPTG (1000ml): 10g tryptone, 5g Yeast extract, 10gNaCl, pH are natural, 20g agar powder, sterilizing 15min at 121 DEG C.It is cooled to the ammonia that room temperature adds 100 μ L Benzylpcnicillin (100mg/mL), adds the IPTG (24mg/mL) of 10 μ L in the medium, the X-gal (20mg/mL) of 200 μ L.

(1) design of primers

Known array information according to aim sequence two ends, designs pcr amplification primer thing, as shown in table 6.

Table 6 primer sequence information table

Primer is synthesized by Shanghai Jierui Biology Engineering Co., Ltd.DNA sequencing is completed by Hua Da gene company limited.

(2) the PCR amplification of clone gene

With extract cloned plasmids as template, carry out pcr amplification reaction, reaction system is as follows:

YT0801-Aro8 gene PCR response parameter: 94 DEG C of denaturations 5min, 94 DEG C of degeneration 45s, 59 DEG C of annealing 45s, 72 DEG C extend 90s；Totally 33 circulations；72 DEG C extend 10min, PCR primer 4 DEG C preservation.

YT0801-Aro10 gene PCR response parameter: 94 DEG C of denaturations 5min, 94 DEG C of degeneration 45s, 60 DEG C of annealing 45s, 72 DEG C extend 120s；Totally 35 circulations；72 DEG C extend 10min, PCR primer 4 DEG C preservation.

DV10-Adh1 gene PCR response parameter: 94 DEG C of denaturations 5min, 94 DEG C of degeneration 45s, 58 DEG C of annealing 45s, 72 DEG C Extend 60s；Totally 33 circulations；72 DEG C extend 10min, PCR primer 4 DEG C preservation.

(3) pcr amplification product recovery, connect with convert

After pcr amplification product reclaims, at 4 DEG C, connect pMD18-T carrier overnight, Transformed E .coli DH5 α.Coupled reaction System is as follows:

(4) double digestion of recombiant plasmid is identified and sequencing

Extracting recombiant plasmid, carry out double digestion qualification, enzyme action system is as follows:

The recombiant plasmid containing Aro8 gene involved by the present embodiment EcoR I and Xho I enzyme action are identified, containing Aro10 and The recombiant plasmid of Adh1 gene BamH I and Xho I enzyme action are identified, product should sized by two fragments, enzyme action is identified errorless Recombiant plasmid order-checking.

(5) carrier pET-32a (+) and the connection of recombiant plasmid and conversion

Double digestion carrier pET-32a (+) and recombiant plasmid, 37 DEG C of water-bath 3h, electrophoresis.Enzyme action system is as follows:

It is separately recovered DNA fragmentation little, big with agarose gel, sets up linked system, the amount of Insert Fragment and the amount of carrier Depending on the size and DNA concentration thereof of fragment, typically take ratio 3:1.Add T4 ligase (350U/ μ L, 1.0 μ L) and connect Enzyme buffer liquid (1.0 μ L), supplements system to 10 μ L with deionized water, flicks mixing, and 16 DEG C connect overnight, Transformed E .coli DH5 α, extracts recombiant plasmid, identifies and sequencing through double digestion.According to the source of gene, expression vector plasmid is respectively designated as P32-YT0801-Aro8, p32-YT0801-Aro10 and p32-DV10-Adh1.Electrophoretic analysis such as Figure 29, Figure 30 and Figure 31 institute Show.And this recombiant plasmid is carried out order-checking qualification, sequencing result is completely the same with genes of interest sequence.Show expression vector p32- YT0801-Aro8, p32-YT0801-Aro10 and p32-DV10-Adh1 successfully construct.In Figure 29, M:Maker III；1: matter Grain；2: digestion products.In Figure 30, M:MD113；1～6: plasmid；1₀,5₀: the digestion products of No. 1 and No. 5 plasmid.In Figure 31,1～ 5: plasmid；1₀₂,2₀,4₀: 1, the digestion products of 2 and No. 4 plasmids.

(6) conversion of expression vector: expression vector Plastid transformation expression strain E. coli BL21 (DE3).Carry Take recombiant plasmid, identify through double digestion.

The abduction delivering of recombiant protein and purification:

(1) abduction delivering of recombiant protein, mainly comprises the steps that (1) goes bail for recombinant clone deposited in containing There are the LB plate streaking of 100 μ g/mLAmp, 37 DEG C of overnight incubation.(2) picking list bacterium colony, is inoculated into containing 100 μ g/mLAmp's In 15mL LB liquid medium, 37 DEG C, 200r/m overnight incubation.(3) take 500 μ L bacterium solution and be transferred to the 50mLLB training containing Amp Supporting in base (1:100), 37 DEG C, 200rpm amplification culture to OD is 0.8 (about 2.5h), adds the IPTG of 100mM to its final concentration For 1mM.Abduction delivering certain time at a certain temperature.(4) 8000rpm is centrifuged 15min, collects thalline and supernatant respectively ,- 20 DEG C save backup.(5) ultrasonication carry out protein electrophoresis, observes protein expression situation.

(2) extraction of recombiant protein: wash thalline 2～3 times with PBS, is added in the ratio of original bacteria liquid volume 1/5～1/10 Lysate, carries out thalline resuspended.Using ice-bath ultrasonic to crush thalline, ultrasound condition is power 400W, ultrasonic 3s, is spaced 2s, broken Broken 60 times.12000rpm is centrifuged 5min, is collected by supernatant centrifuge tube, carries out SDS-PAGE analysis.

(3) purification of recombiant protein, mainly comprises the steps that 1mL50%Ni-NTAHis bind resin is hanged by (1) Liquid joins 4mL1 × Ni-NTA and combines in buffer, softly mixes.(2) after resin natural subsidence, draw on 4mL with rifle head Clearly, adding the supernatant that 4mL prepares, softly mix, 4 DEG C combine 60min.

(3) lysate Ni-NTAHis Bind resin compound is added in the empty chromatographic column of lower end closed, remove lower end Closure cap, collects effluent (through peak), preserves, for SDS-PAGE electrophoretic analysis.(4) slow with 4mL1 × Ni-NTA rinsing Rush liquid to rinse twice, collect rinse component, for SDS-PAGE electrophoretic analysis.(5) with 0.5mL1 × Ni-NTA lavation buffer solution Eluting destination protein 4 times, is divided into 4 portion collection by elution fraction, and carries out SDS-PAGE electrophoretic analysis and respectively preserve component, finally Determine destination protein.(4) the SDS-PAGE electrophoretic analysis of recombiant protein: the electrophoretic analysis of recombiant protein in the present invention, according to it Molecular weight select 8% SDS-PAGE (> 10kDa) electrophoresis protocols.The dye of the formula of running gel, electrophoretic parameters and gel Color, discoloration method equal reference standard experimental technique is carried out.

(5) concentration of method (1998) the mensuration recombiant protein of reference Ao Sibai:

The mensuration of recombinant protein A RO8 concentration: making Ox blood serum standard curve, equation is Y=0.0009X-0.0086, side Journey correction coefficient is 0.9967, and linear fit is preferable.The concentration of mensuration supernatant total protein and after purification destination protein, is respectively 1149.2mg/L and 462.3mg/L, destination protein accounts for the 40.2% of total protein.

The mensuration of recombinant protein A RO10 concentration: the absorbance of mensuration supernatant total protein and after purification ARO10 albumen, According to BSA standard curve, calculate the concentration learning total protein and ARO10 after purification, respectively 789.9mg/L and 202.2mg/L, Destination protein accounts for the 25.6% of total protein.

The mensuration of recombinant protein A DH1 concentration: the absorbance of mensuration supernatant total protein and after purification ADH1 albumen, root According to BSA standard curve, calculate the concentration learning total protein and ADH1 after purification, respectively 880.1mg/L and 252.6mg/L, mesh Albumen account for the 28.7% of total protein.

Three, identify the biological activity of 3 kinds of recombinases, and study the zymologic property of recombinase respectively.

(1) detecting through HPLC and LC-MS, recombinase ARO8 has the normal biological activity of aromatic series transaminase I, it is possible to urge Change L-phenylalanine and generate phenylpyruvic acid.The optimum temperature of ARO8 and optimum pH are respectively 30 DEG C and 7.0, better heat stability. Its catalysis activity needs Mg²⁺Participation, Fe³⁺、Fe²⁺And Cu²⁺Suppress its activity.The organic solvent pair such as low concentration methanol and DMSO Its enzyme is lived and is had facilitation.30 DEG C, under the conditions of pH7.0, recombinase ARO8 acts on Michaelis constant K of L-phe_mFor 6.577mM, maximum reaction rate V_maxIt is 10.352 μMs/min；Act on Michaelis constant K of oxaloacetic acid_mFor 15.779mM, Big reaction rate V_maxIt is 16.863 μMs/min.Enzyme activity under optimal condition is 0.87 μm ol/ (min mL).

(2) detecting through GC-FID and GC-MS, recombinase ARO10 has the normal biological activity of phenylpyruvate decarboxylase, energy Enough catalysis phenylpyruvic acids generate hyacinthin.The optimum temperature of ARO10 and optimum pH are respectively 37 DEG C and 6.0, and heat stability is good Good.ARO10 need to be with ThPP and Mg²⁺As cofactor, Cu²⁺And Mn²⁺Enzyme is lived and has facilitation, Fe³⁺Suppress Deng metal ion Enzyme is lived.The organic solvents such as methanol all suppress its enzyme to live.37 DEG C, under the conditions of pH6.0, restructuring ARO10 acts on phenylpyruvic acid K_mFor 8.239mM, maximum reaction rate V_maxIt is 2.994 μMs/min.Enzyme activity under optimal condition is 0.22 μm ol/ (min mL)。

(3) detecting through HPLC and LC-MS, restructuring alcoholdehydrogenase ADH1 has the normal biological activity of this enzyme, it is possible to by benzene Acetaldehyde reduction is 2 phenylethyl alcohol.The optimum temperature of ADH1 and optimum pH are respectively 35 DEG C and 6.0, to thermo-responsive.Zn²⁺And Mg²⁺Promote Enter it active, and Fe³⁺Its activity is suppressed Deng metal ion.The ethanol of organic solvent methanol and low concentration also can promote that enzyme is lived.? Under the conditions of pH6.0 and 35 DEG C, ADH1 acts on Michaelis constant K of hyacinthin_mFor 3.612mM, maximum reaction rate V_maxFor 18.587μM/min.Enzyme activity under optimal condition is 1.75 μm ol/ (min mL).

SEQUENCE LISTING

<110>Agricultural University Of South China

<120>the acellular synthetic biology preparation method and application of 2 phenylethyl alcohol

<130>

<160> 18

<170> PatentIn version 3.3

<210> 1

<211> 25

<212> DNA

<213>primer ARO8F

<400> 1

atgactttac ctgaatcaaa agact 25

<210> 2

<211> 25

<212> DNA

<213>primer ARO8R

<400> 2

ctatttggaa ataccaaatt cttcg 25

<210> 3

<211> 23

<212> DNA

<213>primer ARO10F

<400> 3

ttaagcatgg cacctgttac aat 23

<210> 4

<211> 25

<212> DNA

<213>primer ARO10R

<400> 4

gcgcccacaa gtttctattt tttat 25

<210> 5

<211> 25

<212> DNA

<213>primer ADH1F

<400> 5

gcttatttag aagtgtcaac aacgt 25

<210> 6

<211> 25

<212> DNA

<213>primer ADH1R

<400> 6

atgtctatcc cagaaactca aaaag 25

<210> 7

<211> 25

<212> DNA

<213>primer ADH2F

<400> 7

atgtctattc cagaaactca aaaag 25

<210> 8

<211> 26

<212> DNA

<213>primer ADH2R

<400> 8

ttatttagaa gtgtcaacaa cgtatc 26

<210> 9

<211> 21

<212> DNA

<213>primer ADH3F

<400> 9

atgttgagaa cgtcaacatt g 21

<210> 10

<211> 24

<212> DNA

<213>primer ADH3R

<400> 10

ttatttacta gtatcgacga cgta 24

<210> 11

<211> 25

<212> DNA

<213>primer SFA1F

<400> 11

atgtccgccg ctactgttgg taaac 25

<210> 12

<211> 32

<212> DNA

<213>primer SFA1R

<400> 12

ctattttatt tcatcagact tcaagacggt tc 32

<210> 13

<211> 30

<212> DNA

<213>primer p32-ARO8F

<400> 13

cggcggaatt catgacttta cctgaatcaa 30

<210> 14

<211> 28

<212> DNA

<213>primer p32-ARO8R

<400> 14

ggcctcgagc tatttggaaa taccaaat 28

<210> 15

<211> 31

<212> DNA

<213>primer p32-ARO10F

<400> 15

tggatccatg gcacctgtta caattgaaaa g 31

<210> 16

<211> 34

<212> DNA

<213>primer p32-ARO10R

<400> 16

gcgctcgagc tattttttat ttcttttaag tgcc 34

<210> 17

<211> 32

<212> DNA

<213>primer p32-ADH1F

<400> 17

cggatccatg tctatcccag aaactcaaaa ag 32

<210> 18

<211> 31

<212> DNA

<213>primer p32-ADH1R

<400> 18

cgctcgagtt atttagaagt gtcaacaacg t 31

Claims (10)

1. the acellular synthetic biology preparation method of a 2 phenylethyl alcohol, it is characterised in that be logical in same reaction system Cross the common catalyst system and catalyzing catalyzed conversion of restructuring transaminase I ARO8, phenylpyruvate decarboxylase ARO10 and alcoholdehydrogenase ADH1 composition L-phenylalanine, L-phenylalanine is converted into 2 phenylethyl alcohol via phenylpyruvic acid, hyacinthin；Wherein, in described reaction system, it is With L-phenylalanine and oxaloacetic acid as substrate, recombinase ARO8, ARO10 and ADH1 press enzyme activity with 1:0.25:1,1:1:1, 1:0.5:1 or 1:0.25:2 (U/U/U) proportioning determines, pH value be 5.5～7.5, Mg²⁺Concentration is the reaction system of 1.0～5mM In, under the conditions of bath temperature is 30～40 DEG C, after reaction converts 4～7min, terminate reaction with isopyknic acetonitrile, i.e. prepare 2- Phenethanol.

The acellular synthetic biology preparation method of 2 phenylethyl alcohol the most according to claim 1, it is characterised in that described L-benzene The mol ratio of alanine and oxaloacetic acid is 0.75:l.

The acellular synthetic biology preparation method of 2 phenylethyl alcohol the most according to claim 1, it is characterised in that described The proportioning of ARO8, ARO10 and ADH1 is 1:0.25:1.

The acellular synthetic biology preparation method of 2 phenylethyl alcohol the most according to claim 1, it is characterised in that described Mg²⁺ Concentration is 1.0～2.5mM；It is preferably 1.5mM.

The acellular synthetic biology preparation method of 2 phenylethyl alcohol the most according to claim 1, it is characterised in that described water-bath Temperature is 33～40 DEG C；Response time is 5min.

The acellular synthetic biology preparation method of 2 phenylethyl alcohol the most according to claim 1, it is characterised in that described pH value It is 5.5～6.5；The most described pH value is 6.

The acellular synthetic biology preparation method of 2 phenylethyl alcohol the most according to claim 1, it is characterised in that be first by institute State restructuring transaminase I ARO8, phenylpyruvate decarboxylase ARO10 and alcoholdehydrogenase ADH1 and prepare catalysis altogether by common fixing means System Combi-CLEAs, then utilizes Combi-CLEAs that L-phenylalanine is carried out catalyzed conversion, via phenylpyruvic acid, benzene second Aldehyde is converted into 2 phenylethyl alcohol.

The acellular synthetic biology preparation method of 2 phenylethyl alcohol the most according to claim 7, it is characterised in that described reaction In system, pH value is 5.0, Mg²⁺Concentration is 1.0～5mM, and bath temperature is 40 DEG C, and reaction transformation time is 5min.

The acellular synthetic biology preparation method of 2 phenylethyl alcohol the most according to claim 7, it is characterised in that described urge altogether The preparation method of change system Combi-CLEAs is: ARO8, ARO10 and ADH1 are joined according to 1:1:1,1:0.5:1 or 1:0.25:1 After Bi, after precipitant ammonium sulfate precipitation, form many enzyme aggregates, add glutaraldehyde and many enzyme aggregates are cross-linked, prepare many Enzyme fixed system altogether；Wherein, the saturation of ammonium sulfate is 60～90%, and pH value is 4～6, and the addition of glutaraldehyde is according to it eventually Concentration is 0.05～0.25% to determine.

The acellular synthetic biology preparation method of 2 phenylethyl alcohol the most according to claim 9, it is characterised in that described heavy The saturation 70% of shallow lake agent ammonium sulfate；The pH value of precipitant ammonium sulfate is 5；The enzyme proportioning of ARO8, ARO10 and ADH1 is 1:0.5: 1；The addition of described glutaraldehyde final concentration of 0.1% determines according to it；The temperature of described crosslinking is 20～35 DEG C；Described crosslinking Time be 30～150min.