CN102154327A

CN102154327A - Thermomonospora curvata mycose synthetase gene and application thereof

Info

Publication number: CN102154327A
Application number: CN2010106148324A
Authority: CN
Inventors: 韦宇拓; 黄日波; 梁甲元; 杜丽琴
Original assignee: Guangxi University
Current assignee: Guangxi University
Priority date: 2010-12-30
Filing date: 2010-12-30
Publication date: 2011-08-17

Abstract

The invention discloses a thermomonospora curvata mycose synthetase gene and application thereof. The gene is cloned from thermomonospora curvata. Mycose synthetase expressed by the gene has two different transformation functions. The mycose synthetase can be used for transforming maltose into mycose and transforming cane sugar into kelp ketose under normal biochemical reaction conditions. The gene can be applied to the production of mycose by transforming maltose, and can be used for transforming cane sugar into kelp ketose. The mycose and the kelp ketose can be applied to the fields of food industry, beverages, functional foods, beautifying cosmetics, medical biological products and the like.

Description

A kind of crooked sporangium trehalose synthesize enzyme gene and application thereof

Technical field

The invention belongs to genetically engineered and modern technical field of enzyme engineering, be specifically related to a kind of crooked thermomonospora fusca trehalose synthesize enzyme gene and application thereof.

Background technology

Trehalose (Trehalose) is with α by two molecule glucoses, α-1, the non-reducing disaccharide that the 1-glycosidic link links to each other and forms, first it was extracted from the ergot of rye first by Wiggers in 1832, the trehalose of discovering subsequently is present in microorganism, shrimps, yeast saccharomyces cerevisiae, mushroom and various fungi, insect, plant etc. widely.Ectogenic trehalose has good non-specific provide protection to organism and biomacromolecule, microbial film equally; it can protect cell to avoid because environmental change; ooze the infringement that causes after the variation as dehydration, height; be embodied in effective protection, therefore be described as " sugar of life " microbial film, protein and DNA etc.The content of trehalose is very low in natural, and the past mainly is to extract from dry yeast, because content is low, leaching process is complicated, and cost is high.So expensive trehalose can only be confined to the application of the activity maintenance of some special dimension such as medical biotechnology goods, along with development of human society and progress, hope trehalose can be applied to daily life the delicate flavour of varieties of food items commonly used keep and improved texture, therefore better life and the health level that improves people must find cheapness and trehalose manufacture method efficiently as soon as possible.

Marine alga ketose (Trehalulose) and Palatinose (1somaltulose) all are the isomer of sucrose (sucrose), sucrose is with α, α-1, a kind of disaccharides of 2-glycosidic link be combined into, marine alga ketose be by a part glucose and a part fructose with α, α-1, a kind of disaccharides of 1-glycosidic link be combined into, Palatinose be by a part glucose and a part fructose with α, α-1, a kind of disaccharides of 6-glycosidic link be combined into.Marine alga ketose and Palatinose all are the isomerss of sucrose, but their chemical property and physical properties have very big difference, the Palatinose sugariness is 42% of a sucrose, suction hardly, it is generally acknowledged and do not have water-absorbent, even add 1.5～15% citric acid, its water absorbability can not increase yet, and therefore this characteristic that does not absorb water and be not clamminess is fit to food and the medicament in order to production needs kept dry very much.Different with Palatinose is that the marine alga ketose then has very high solubleness and water-absorbent, sugariness is 70% of a sucrose, its solubleness reaches more than 90% under the condition of room temperature, its solution almost can not be by crystallization, separates out as long as add the crystallization that a spot of marine alga ketose just can effectively reduce in the solution in separating out crystalline solution easily.The content of marine alga ketose in honey of occurring in nature is the highest, so pure honey density height also is not easy crystallization.This high-hydroscopicity and be difficult for the crystalline characteristic and be fit to very much in order to produce senior liquid drink, liquid pharmaceutical preparation and makeup etc.But the marine alga ketose of occurring in nature and trehalose equally all are the very low sugar of content, even at the highest honey content of content also below 1%, and because the high-hydroscopicity of marine alga ketose and be difficult for the crystalline characteristic, make its separation and purification very difficult, can produce the more syrupy preparation method of high purity marine alga ketose so need find.

The TreP of number of different types and relevant gene thereof in multiple microorganism, have been separated at present, synthetic relevant enzyme of the trehalose of finding and gene show at present, the approach of trehalose synthesis can be divided into three kinds of approach in the microorganism, first kind of approach is the trehalose phosphorylase route of synthesis, mainly form by footpath 6-phosphotrehalose UDP-transglucosylase synthetic enzyme (Trehalose-6-phosphate synthase) and 6-phosphotrehalose UDP-transglucosylase esterase (Trehalose-6-phosphate phosphatase), at first by 6 phosphotrehalose UDP-transglucosylase synthetic enzyme catalysis UDP-glucose and the synthetic 6-phosphotrehalose UDP-transglucosylase of 6-glucose 1-phosphate1-, and then by obtaining trehalose behind the 6 phosphotrehalose UDP-transglucosylase esterase dephosphorylations; Second kind of approach is the malt oligosaccharide based mycose synthetase route of synthesis, mainly be by malt oligosaccharide based mycose synthetase (Maltooligosyltrehlosen, MTSase) and malt oligosaccharide based mycose lytic enzyme (Maltooligosyltrehalose trehalohydrolase, MTHase) form, be substrate by malt oligosaccharide based mycose synthetase with maltodextrin (Maltodextrin) earlier, by the intramolecularly transglycosylation the α between two glucose molecules of maltodextrin end, α-1,4 glycosidic links are transformed into α, α-1,1 glycosidic link forms malt oligosaccharide based mycose (Maltooligosyltrehalose).Then malt oligosaccharide based mycose under the catalysis of malt oligosaccharide based mycose lytic enzyme from the malt oligosaccharide based mycose molecule the next trehalose molecule of hydrolysis, maltodextrin originally then changes the new oligosaccharides that has reduced by two glucosyl groups into, and can be used as new substrate and carry out next round reaction, just maltodextrin can be changed into trehalose so repeatedly; The third approach is the TreP approach, not being both this approach with other trehalose route of synthesis, to have only a kind of enzyme be TreP (Trehalose synthase, Tre S) form, the substrate of its effect is maltose (Maltose), by changeing glycosyl in the catalysis maltose molecule, with α, the maltose that α-1,4 glycosidic link connects is converted into α, α-1,1 glycosidic link, maltose is transformed into trehalose.First kind of approach need use expensive high-energy phosphate compound UDP-glucose and the 6-glucose 1-phosphate1-is that substrate is produced trehalose, be difficult to the advantage of competing at production cost, second kind and the third approach are to be that substrate is produced trehalose with the hydrolysate maltodextrin of starch or maltose, has very strong competitive edge, the third approach is compared with second kind of approach, the third approach only needs a kind of enzyme, utilizing genetic engineering bacterium production recombinase, owing to need not produce two kinds of enzymes, do not need to consider whether two kinds reaction conditions is consistent, therefore the occupancy of equipment can be lower in process of production, production technique can be simpler, thereby have stronger competitive edge.

The purity of sucrose can reach more than 99%, is best suited for preparing the substrate of marine alga ketose, and product can directly apply to food and medicine.Document reports that openly the enzyme that can transform sucrose generation marine alga ketose mainly contains three types at present, a kind of is that sucrose isomerase also claims the alpha-glucosyl transferring enzyme, transformation efficiency to sucrose can reach 90%, but the product that it transforms sucrose is the mixing molasses of marine alga ketose and Palatinose equal proportion; Second kind is trehalose ketose synthetic enzyme, and it can transform sucrose and generate the marine alga ketose, and the sucrose inversion rate can reach more than 70%, but general speed of response is slower, generates trehalose but this fermentoid can not be substrate with maltose; The third can transform sucrose generation marine alga ketose is exactly TreP, discover that some TreP can be that substrate generates the marine alga ketose with sucrose, the transformation efficiency of sugarcane sugar was all lower when but at present report can transform TreP that sucrose generates marine alga ketone and is substrate with sucrose, generally all was lower than 5%.

At present existing many documents with patent report the trehalose synthesis related gene the clone with separate; and the relevant enzyme of these trehalose synthesize enzyme genes is used to prepare trehalose; but the route of synthesis difference of the synthetic relevant enzyme of these trehaloses; perhaps the bacterial classification source is different; caused dna sequence dna and aminoacid sequence to differ greatly; enzymatic property is different with enzyme activity, and major part does not also obtain the application of industrial scale.Also do not see relevant report at home about marine alga ketose synthase gene, the patent of producing about the application and the marine alga ketose of marine alga ketose synthetic enzyme does not yet have relevant report.

Summary of the invention

The object of the invention provides a kind of crooked thermomonospora fusca trehalose synthesize enzyme gene and application thereof.

The present invention is achieved through the following technical solutions above-mentioned purpose:

The inventor utilizes the bioinformation means that dna sequence dna huge among the GenBank and aminoacid sequence resource are carried out the homology search analysis, the dna sequence dnas of the thousands of kinds of microorganisms of finishing sequential analysis is carried out a large amount of nucleotide sequences to be analyzed and contrasts, found that note is the extremely useful gene of " agnoprotein " or " supposition is certain functional protein " in proteinic sequence library Genbank, infer the function of this sequence, find candidate gene sequence, carry out the Function Identification experiment then, final function and the character of determining gene, comprising people up to now and undiscovered trehalose synthesize enzyme gene (treS), and with this synthase gene through the clone with carry out recombinant expressedly, utilize recombinant expressed TreP to be used to transform maltose again and produce trehalose.

The inventor is in the process of research trehalose synthesize enzyme gene, to a kind of Chinese is crooked thermomonospora fusca, the genome sequence of Latin Thermomonospora curvata by name carries out the homology retrieval analysis, find that this section do not have the dna sequence dna of its function of bibliographical information as yet, this segment DNA sequence is noted as " trehalose synthesize enzyme gene of supposition " (putative trehalose synthase) in Genbank.Crooked thermomonospora fusca (Thermomonospora curvata) is a kind of thermophile bacteria, usually can in During High-Temperature Composting, can be separated to, has cellulolytic ability, growth temperature range is extensive, can under 28-65 ℃ temperature, grow, optimum growth temperature is 50 ℃, and what the extensive characteristic of this growth temperature range may be with trehalose synthesize enzyme gene is relevant.This segment DNA and the TreP that bibliographical information has been arranged by analysis, Glycosylase, the proteic gene of amylase family all has higher homology, homology reaches more than 70%, and the proteinic aminoacid sequence of the supposition of its coding and the TreP of having delivered, Glycosylase, the proteic amino acid of amylase family also has higher homology, amino acid identity reaches more than 80%, so be difficult to determine that it is which kind of enzyme by aminoacid sequence, must identify the function of this fragment gene through concrete experiment, confirm that what enzyme it is, at present the experimental identification relevant report of this gene function not yet.

Trehalose synthesize enzyme gene clone of the present invention is from crooked thermomonospora fusca (Thermomonospora curvata), and this bacterial classification can be bought from Chinese agriculture microbial strains preservation administrative center, also can obtain by field acquisition and other approach.

The TreP that crooked thermomonospora fusca (Thermomonospora curvata) clone obtains has following feature:

1, molecular weight is about: 69.35kD

2, iso-electric point is about: PI=4.7

3, the optimal reactive temperature of enzyme is 35～45 ℃

4, the pH of the reaction of enzyme is 6.0～7.5, and preferred pH is 6.5～7.0

5, work as Ni ²⁺, Fe ³⁺, Mn ²⁺, Mg ²⁺, Ca ²⁺When the 5m mol/L, the vigor of this enzyme is unaffected Deng the concentration of metal ion, but Fe ²⁺, Zn ²⁺, Cu ²⁺Metal ion can produce certain inhibition to the vigor of this enzyme when the concentration of 5m mol/L, the only surplus vigor that has about 85%.

6, after enzyme reached finite concentration, content of trehalose reached the highest in the reaction product, continue to add the content that excessive enzyme can reduce trehalose, and the content of by product glucose can raise.

7, this TreP can be that substrate generates the trehalose ketose with sucrose, and speed of response is that substrate is slow with maltose, but the reaction times surpass after 24 hours, the content of marine alga ketose reaches more than 83% in the reaction product.

The experimental technique that the inventor adopted comprises well-known polymerase chain reaction (PCR) technology; Dna molecular operative techniquies such as extraction, the enzyme of DNA cut, connection, the dna sequence dna that this segment mark is designated as the trehalose synthesize enzyme gene of supposition is connected on the expression vector, on expression plasmids such as pSE380, importing intestinal bacteria then efficiently expresses, through cytoclasis, after for example utilizing smudge cellses such as N,O-Diacetylmuramidase, ultrasonic wave or mechanical disruption method, the target protein that obtains this genetic expression is studied, determine its function and enzymatic property, what confirmed this dna fragmentation coding is the diverse TreP that a kind of property feature and forefathers find.These different property features have comprised the different of aminoacid sequence and dna sequence dna, the molecular size difference of enzyme, the optimal reactive temperature of enzyme is different with optimal pH, the reaction conditions difference, enzyme gets specificity and maltose is converted into the characteristics such as difference of trehalose ratio substrate, so judges it is a kind of new discovery.

The gene clone of TreP of the present invention is from crooked thermomonospora fusca (Thermomonospora curvata), and this enzyme is a kind of intracellular enzyme in natural crooked thermomonospora fusca, need just can be checked through with cytoclastic method; And the content of this enzyme is also very low in natural bacterium, nutrient solution need be concentrated 1000 times and concentrate the above vigor that could check TreP, can't satisfy industrial production requirement.The using gene engineering bacterium is expressed trehalose synthesize enzyme gene of the present invention, and the vigor that can make enzyme can be used for the production of trehalose and marine alga ketose than high thousands of times of natural bacterial strain.

The gene (treS) of TreP of the present invention of encoding is one section dna fragmentation of not determining function, and length is 1803 base pairs, 601 amino acid of encoding, and GC content is 68%.

The present invention compared with prior art has substantive distinguishing features and significant advantage:

1, have equally and transform the function that maltose generates trehalose, but belong to the less class TreP of molecular weight, the TreP from Pimerlobacter sp.R48 of molecular weight and the former institute of Japanese woods patent report, with the Chinese patent patent No. be that the Thermobifida fusca TreP of the report of ZL200410013007.3 is more or less the same, but the trehalose synthase from aquatic thermophile bacteria Thermus aquaticus than the former institute of Japanese woods report lack 363 amino acid, is that the TreP of the Tengchong thermophilc anaerobe reported of ZL200410073883.5 has lacked 179 amino acid than China Patent No..The proteolytic enzyme of small molecular weight is more prone to carry out industrialized production by gene recombination and expression technology, thereby more the market competitiveness is arranged.

2, the optimal reactive temperature of enzyme is 35～45 ℃, it is moderate optimal reactive temperature, with the Chinese patent patent No. be that a kind of TreP of report of ZL00710100350.5 and the optimal reactive temperature of application thereof are 20 ℃, with the Chinese patent patent No. be that the optimal reactive temperature of Thermobifida fusca TreP of the report of ZL200410013007.3 is 25-30 ℃, being a kind of thermally-stabilised TreP of ZL200410073883.5 report and encoding gene thereof with China Patent No. compares with the most suitable temperature of reaction 60-70 ℃ of application.Under substrate conversion efficiency and the essentially identical condition of speed of response, under 35～45 ℃ of relatively mild reaction conditionss, not needing on producing excessively heats up or lower the temperature keeps the temperature of production unit, has superiority on save energy.

3, have two kinds of substrates conversion capability efficiently, can not only transform the trehalose of maltose generation more than 70%, can also be that substrate generates 80% above marine alga ketose with sucrose, can be reaction substrate with maltose and sucrose simultaneously so just, will obtain in the reaction product with trehalose and trehalose ketose be main component and also contain maltose simultaneously, sucrose, the mixing molasses of glucose and fructose, this syrup can further utilize transglycosylase to react again, just can produce and obtain containing the maltose trisaccharide, Isomaltotriose, panose, trehalose glucose trisaccharide, trehalose fructose trisaccharide, multiple three sugar mixture syrup such as marine alga ketose glucose trisaccharide and marine alga ketose fructose trisaccharide.Mix oligosaccharides and have stronger health-care effect than single oligosaccharides, the mixing molasses of this multiple trisaccharide is also referred to as the honey syrup, will consider two kinds of reaction conditionss that enzyme is different if prepare mixing molasses with two kinds of enzymes.

Description of drawings

Fig. 1 is the reaction product analysis of substrate with maltose for TreP.

Fig. 2 is the reaction product analysis of substrate with sucrose for TreP.

Fig. 3 is that the optimal reaction pH of TreP is worth analyzing.

Fig. 4 is the mensuration of the optimal reactive temperature value of TreP.

Fig. 5 is the mensuration that metal ion is lived and influenced the TreP enzyme.

Fig. 6 is various sugared content analyses in the TreP reaction product of conversion maltose differential responses time.

Fig. 7 is for adding different enzyme amounts to each sugared content analysis in the reaction product of TreP conversion maltose.

Fig. 8 is each sugared content analysis in the product of TreP conversion sucrose reaction different time.

Embodiment

By the following examples technical scheme of the present invention is described further, the described content of following experiment and example belongs to main contents of the present invention, but be not restricted to these contents, though some conspicuous expansion content concerning the professional of this area does not occur in specification sheets of the present invention, should belong to patent request scope of the present invention yet.

1, the cultivation of crooked thermomonospora fusca

Crooked thermomonospora fusca (Thermomonospora curvata, strain number is 41067) purchase of Chinese agriculture microbial strains preservation administrative center, be inoculated in following substratum (grams per liter): glucose 4.0 grams, yeast extract 10 grams, malt extract 10 gram L, transfer pH to 7.2 with KOH, on 50～55 ℃ of constant temperature shaking tables, vibrated 48 hours then, be used for the extraction of total DNA.

2, the preparation of the clone of crooked thermomonospora fusca trehalose synthesize enzyme gene, expression and thick enzyme

According to note in the Thermomonospora curvata gene order of announcing among the Genbank for may be the sequence of trehalose synthesize enzyme gene, called after Tct, design this gene of corresponding primer amplification and be connected to and import intestinal bacteria on the expression vector then and express, the design primer is as follows:

Upstream primer (Sense primer): 5 '-CTGCC ATGGAGATGACCGGGGACCCC '

Downstream primer (Antisense Primer): 5 '-TCA AAGCTT TCACTTCGCCGTCTGCCG-3 '

5 of upstream and downstream primer ' end is designed Nco 1 and Hind III restriction enzyme site respectively, be used for the PCR product is connected to expression vector pSE380, may be the dna sequence dna Tct of the sequence of trehalose synthesize enzyme gene with the primer amplification that designs, amplified production reclaims the purpose amplified fragments with the test kit purifying, utilize Nco 1 and Hind III to carry out double digestion then, be connected with the expression vector pSE380 that utilizes Nco 1 and Hind III to carry out double digestion equally, obtain Tct expression of gene recombinant plasmid pSE380-Tct through screening verification then, be used for expressing the preparation TreP then.

The expression plasmid pSE380-Tct of reorganization is transformed in the competent cell of e. coli strain bl21, picking list colony transformation is in the LB liquid nutrient medium, in 37 ℃ of shaking tables, cultivate, when OD600 reaches 0.8 left and right sides, add IPTG and make its final concentration reach 1mmol/L, continue to cultivate 20h and carry out abduction delivering.The centrifugal collection thalline of the cultured fermented liquid of abduction delivering, utilize broken born of the same parents' damping fluid washing thalline once, under condition of ice bath, utilize ultrasonic wave to break born of the same parents after utilizing the broken resuspended thalline of born of the same parents' damping fluid again, broken born of the same parents clarify to bacterium liquid, centrifugal 15 minutes centrifugal collection supernatants of 12000rpm/min, collected supernatant liquor is crude enzyme liquid, can be used for transforming the test of maltose.

3, crooked thermomonospora fusca TreP transforms the analysis of maltose product

Utilize high performance liquid chromatograph (HPLC) to carry out the analysis that TreP transforms maltose and sucrose after product, analysis condition, chromatographic column: Alltima Amino 100A 5u post (4.6mm * 250mm); Detector: Alltech 2000ES type light scattering detector; Moving phase: 70% acetonitrile/30%H ₂O; Flow velocity: 1mL/min, column temperature: 35 ℃.

(1) maltose is the reaction product analysis of substrate

Get 10% maltose substrate, the 96 μ L+4 μ L enzyme liquid of pH6.5,37 ℃ are reacted, carry out the analysis of reaction product then with HPLC, the results are shown in Figure 1, compare with the trehalose standard specimen, purpose product peak appears in maltose reaction master drawing 1D, shows with maltose to be that substrate reactions can generate trehalose, and reaction product has glucose to generate and unconverted maltose.

(2) sucrose is the reaction product analysis of substrate

Get 10% sucrose substrate, the 96 μ L+4 μ L enzyme liquid of pH6.5,37 ℃ are reacted, carry out the analysis of reaction product then with HPLC, the results are shown in Figure 2, compare with the standard specimen of marine alga ketose, purpose product peak appears in sucrose reaction master drawing 2D, shows with sucrose to be can generate the marine alga ketose in the substrate reactions product, also have the generation of glucose and fructose in the reaction product, and unconverted sucrose.

(3) analysis of optimal reaction pH value

Get the enzyme liquid (protein content reach 4 μ gs) of 96 μ L respectively with the maltose substrate+4 μ L of the phosphate buffered saline buffer preparation 10% of different pH, at 37 ℃ of reaction 1h, use the product component of HPLC analytical reaction then, represent enzyme activity with the content of trehalose in the reaction product.Different pH influence the result as shown in Figure 3 to enzyme activity, and the optimal reaction pH that shows enzyme is 6.0～7.5.

(4) optimal reactive temperature is measured:

Get 10% maltose substrate, the 96 μ L+4 μ L enzyme liquid (protein content reaches 4 μ g) of pH6.5, insulation reaction 1h under different reaction temperature uses the product component of HPLC analytical reaction then, represents enzyme activity with the trehalose percentage composition in the reaction product.Differing temps influences the result as shown in Figure 4 to enzyme activity, and the optimal reactive temperature that shows enzyme is 35～45 ℃.

(5) metal ion influence that enzyme is lived:

Get 10% maltose substrate, 96 μ L+4 μ L enzyme liquid (protein content reaches 4 μ g)+10 μ L metal ions (making the metal ion final concentration is 5mmol/L) of pH6.5, react 1h down at 37 ℃, reflect the variation of enzyme activity by the consumption of substrate, with the enzyme activity that adds phosphate buffered saline buffer is 100%, different metal ions enzyme vigor influence the result as shown in Figure 5, show under the condition of the concentration of 5m mol/L, Ni2+, Fe3+, Mn2+, Mg2+, divalent-metal ions such as Ca2+ do not have restraining effect to the vigor of this enzyme, Fe ²⁺, Zn ²⁺, Cu ²⁺Vigor to this enzyme has partial inhibition, the enzyme activity of loss about 15%.

(6) the differential responses time, various sugared content in the product:

Get 10% maltose substrate (pH6.5)+4 μ L enzyme liquid (protein content reaches 4 μ g) of 96 μ L, react the different time down at 37 ℃, the content of various sugar in the analytical reaction product is then just represented the most suitable reaction times of enzyme with the content of trehalose in the reaction product.Different time influences the result as shown in Figure 6 to various sugared content in the product, show with 10% maltose be substrate reactions about 100 minutes content of trehalose just can reach the highest, content can reach more than 70%.

(7) different enzyme amounts are reacted each sugared content in 1 hour after product

Phosphate buffered saline buffer (pH7.0) with 100mM is prepared 1% maltose substrate, reaction volume is that 100ul (80ul substrate+different enzyme amount+damping fluids) reacts 1h down at 37 ℃, boils 5min, and is centrifugal, with each sugared content behind the HPLC assaying reaction, result such as Fig. 7.After showing that adding enzyme reaches finite concentration, content of trehalose reaches the highest in the reaction product, continues to add the content that excessive enzyme can reduce trehalose, and the glucose by product can increase.

(8) with sucrose be each sugared content in the substrate reactions different time product

With sucrose is substrate pH6.5, and 35 ℃ of times that reaction is different, each sugared content results is seen Fig. 8 in the assay products, and the result shows the prolongation along with the reaction times, and the content of marine alga ketose can improve in the product, can reach more than 80%.

4, prepare trehalose from malt syrup

Get and commercially availablely prepare maltose content with starch and be about 70% malt syrup, use 5mM, pH is that the damping fluid that 6.5 Sodium phosphate dibasics and SODIUM PHOSPHATE, MONOBASIC are made into is mixed with the reaction substrate (pH is about 6.5) that contains 30% maltose, getting 100 liters of reaction substrates that prepare is positioned in the stainless steel reaction jar, get 10 liters of the TreP crude enzyme liquids that above-mentioned the 2nd one step process makes, join in 100 liters of reaction substrates that contain 30% maltose, under 37 ℃ of conditions, reacted 24 hours then, with HPLC reaction product is analyzed result such as table 1 then:

Each component concentration (%) in 24 hours afterreaction liquid of table 1:30% maltose substrate reactions

Trehalose	Maltose	Glucose
			17.1％	5.8％	4.4％

The result shows, the enzyme liquid of 100 liter 30% maltose+10 liter, and the concentration of substrate of real reaction approximates 27.3%; The content of trehalose is about 17.1% in the reaction product, so total transformation efficiency is 17.1%/27.3% ≈ 62.6%), that is to say that the maltose with 27.3% is reaction substrate, had an appointment in the reaction product in 24 hours 62.6% maltose of reaction is converted into trehalose under 25 ℃ of conditions, has promptly formed about 18.8 kilograms of trehaloses in 110 liters total reaction volume altogether.

5, prepare the marine alga ketose from sucrose

Use 5mM, pH is that damping fluid that 6.5 Sodium phosphate dibasics and SODIUM PHOSPHATE, MONOBASIC are made into is mixed with 30% sucrose solution reaction substrate, getting 100 liters of reaction substrates that prepare is positioned in the stainless steel reaction jar, get 10 liters of the TreP crude enzyme liquids that above-mentioned the 2nd one step process makes, join in 100 liter 30% the sucrose reaction substrate, under 35 ℃ of conditions, reacted 48 hours then, with HPLC reaction product analyzed result such as table 1 then:

Each component concentration (%) in 24 hours afterreaction liquid of table 1:30% sucrose substrate reactions

The marine alga ketose	Sucrose	Glucose	Fructose
				22.1％	1.1％	2.1％	2.1％

The result shows, the enzyme liquid of 100 liter 30% sucrose+10 liter, and the concentration of substrate of real reaction approximates 27.3%; The content of trehalose is about 22.1% in the reaction product, so total transformation efficiency is 22.1%/27.3% ≈ 80.1%), that is to say that the sucrose solution with 27.3% is a reaction substrate, had an appointment in the reaction product in 48 hours 80.1% sucrose inversion of reaction becomes the marine alga ketose under 35 ℃ of conditions, has promptly formed about 24 kilograms of marine alga ketoses in 110 liters total reaction volume altogether.

The above embodiment is for better explanation the present invention, and should not be construed as limiting the invention, and described content belongs to main contents of the present invention, but is not restricted to these contents.Though some content that can expand does not apparently occur, also should belong to patent request scope of the present invention in specification sheets.

Claims

1. a crooked thermomonospora fusca trehalose synthesize enzyme gene is characterized in that, it is characterized in that, its nucleotide sequence is shown in SEQ ID NO.1.

2. according to the described crooked thermomonospora fusca trehalose synthesize enzyme gene of claim 1, it is characterized in that its encoded protein matter has the aminoacid sequence shown in the SEQ ID NO.2 in the sequence table.

3. according to the described crooked thermomonospora fusca trehalose synthesize enzyme gene of claim 1, it is characterized in that its molecular weight is 69.35kD, iso-electric point is PI=4.7, and the suitable temperature of reaction of enzyme is 35～45 ℃, and the suitable reaction pH of enzyme is 6.0～7.5.

4. according to the described crooked thermomonospora fusca TreP of claim 1, it has the ability that maltose is changed into trehalose or sucrose inversion generated the marine alga ketose.

5. the application of the described TreP of claim 1 in transforming maltose production trehalose or marine alga ketose is characterized in that, may further comprise the steps:

1) trehalose synthesize enzyme gene is made up genetic engineering bacterium and carry out efficiently expressing of algae sugar synthetic enzyme;

2) collect the TreP that preparation is recombinated;

3) malt syrup with maltose or starch preparation is that raw material is made trehalose with TreP, perhaps is that raw material is made trehalose with TreP with sucrose.