CN109055342A

CN109055342A - A kind of tendentious monosaccharide circumscribed-type algin catenase Aly-6 of M and its encoding gene and application

Info

Publication number: CN109055342A
Application number: CN201810887656.8A
Authority: CN
Inventors: 韩文君; 程媛媛; 李俊鸽; 王延辉; 古静燕; 李新; 卫洁
Original assignee: Ji'nan Enlightenment Biotechnology Co Ltd
Current assignee: Ji'nan Enlightenment Biotechnology Co Ltd
Priority date: 2018-08-06
Filing date: 2018-08-06
Publication date: 2018-12-21
Also published as: WO2020029379A1; AU2018435585B2; AU2018435585A1

Abstract

The present invention relates to a kind of tendentious monosaccharide circumscribed-type algin catenase Aly-6 of M and its encoding gene and applications.The amino acid sequence of the tendentious monosaccharide circumscribed-type algin catenase Aly-6 of M is as shown in SEQ ID NO.2；The nucleotide sequence of Aly-6 is encoded as shown in SEQ ID NO.1.Present invention firstly discloses a kind of tendentious monosaccharide circumscribed-type algin catenase Aly-6 of M is obtained in the genome by heat color Bacillus bacteria MY04, the enzyme is monosaccharide circumscribed-type algin catenase, and there is apparent M tendentiousness, enzyme Aly-6 can in such a way that monosaccharide is circumscribed guluronic acid segment of the sustaining degradation from algin, also can in such a way that monosaccharide is circumscribed sustaining degradation mannuronic acid segment, but be easier to degradation mannuronic acid segment；The enzyme sequence feature, degradation of substrates mode and oligosaccharides formation characteristic and existing known algin catenase have significant difference, and stable in physicochemical property, activity is high, have the potential quality of industrial application.

Description

A kind of tendentious monosaccharide circumscribed-type algin catenase Aly-6 of M and its encoding gene With application

Technical field

The present invention relates to a kind of tendentious monosaccharide circumscribed-type algin catenase Aly-6 of M and its encoding gene and application, Belong to gene engineering technology field.

Background technique

Algin is by α-L- guluronic acid (Guluronic acid, G) and beta-D-mannuronic acid (Mannuronic Acid, M) two kinds of sugar unit compositions linear polysaccharide, M sections of molecule inner injection, poly- G sections and MG or GM mixing section are alternately arranged^[1].It is brown Phycocolloid is usually processed by tangleweeds such as kelp, sargassum, bulk kelps and is made.It recent studies have shown that: with poly- M sections of algin oligosaccharide system Standby I class new drug candidates " GV971 " can inhibit the aggregation and cytotoxicity of beta amyloid cell, can be used for treating light moderate A Er Zi Haimo disease^[2], passed through III phase clinical research；It is saturated poly- G oligosaccharides and can be cooperateed with antibiotic and inhibit more drug resistance pathogenic bacteria^[3]。 This shows that the algin oligosaccharide of the size of specific aggregation degree and the ratio of M/G has significant application value and economic value, therefore Realize that the efficient preparation of this kind of oligosaccharides is of great significance.

Algin catenase is a kind of polysaceharide lyase (Polysaceharide Lyase, PL), passes through β-cancellation mechanism It is catalyzed the fracture of algin intramolecular glycosidic bond, and newly-generated non reducing end in product oligosaccharides forms C4=C5 insatiable hunger And double bond, the C=O (carboxylic carbonyl) with C5- form conjugated structure, produce to generate the oligosaccharides containing unsaturated ends (Δ) Object^[4].Restriction endonuclease generally produces the unsaturated product oligosaccharides of serial size after thoroughly degradation algin；Excision enzyme then usually produces Raw single oligosaccharides principal product, such as Δ unit.The algin catenase substrate different to M/G ratio has selectivity, desmoenzyme pair Enzyme synthesis can be divided at least six major class: restriction endonuclease, the excision enzyme of G- specificity by the inscribe of substrate/circumscribed degradation model difference, The restriction endonuclease of M- specificity, excision enzyme and difunctional restriction endonuclease, excision enzyme.With chemical method or physics biodegrading process phase Have many advantages, such as mild condition, easy to control than, the enzymic degradation of algin, and have certain substrate selective, thus has and push away The potential quality extensively applied^[5].It is current: (1) about the patent application of algin catenase^[6-8]Although more with scientific documents quantity, Focus primarily upon the primary researches such as the Resource exploitation of bacterium producing multi enzyme preparation and enzyme；(2) specific assays of enzyme are mostly with the limited poly- M of purity Section, it is poly- G section to test substrate, and the structure characteristic analysis of the product oligosaccharides about enzyme then lacks the mass spectrum about oligosaccharides final product With the spectral datas such as nuclear magnetic resonance, about oligosaccharides formation characteristic, that is, corresponding degradation of substrates mechanism (as minimum substrate, minimum product, Minimum product in substrate generation position and the substrate tendentiousness of enzyme etc.) explaination it is also relatively fewer；(3) molecular enzymology is ground Study carefully with gene cloning and heterogeneous expression, recombinase purifying and Function Identification based on, about active site mutation, functional module cut The research of short equal molecular modifications is less, about between molecular enzymology transformation and the algin degradation model or oligosaccharides formation characteristic of enzyme The cognition of regular sexual intercourse is less.Therefore, other with merchandized handling and widely applied β-agarase, cellulase etc. Polysaccharide degrading enzyme is compared, rare widely applied commercialization algin catenase.To sum up, the tradition research base of algin catenase Plinth weakness, the rare numbers of toolenzyme and mechanism are unintelligible, these deficiencies not only limit the accurate of existing algin catenase Using also seriously constraining the development that enzyme process prepares algin oligosaccharide technology.

Over nearly 5 years, patent inventor and seminar are directed to the important crucial requirement of above-mentioned industry, have been systematically carried out efficiently more Resource screening, gene order-checking and the Gene mining research of sugared degradation bacteria, emphasis is using algin catenase as object, network analysis The biochemical characters of multiple recombinases and its mutant, substrate selectivity, degradation of substrates mode and oligosaccharides formation characteristic, compare They are for the different values of oligosaccharides preparation and using characteristic.

Endo-type algin catenase such as inventor about the G- specificity in the source Flammeovirga sp.MY04 Aly5's research shows that^[9,10], the enzyme final principal product after algin of thoroughly degrading is degree of polymerization 2-7 containing unsaturated ends Series of oligosaccharides, in which: (1) the oligosaccharides final product of degree of polymerization 5-7 contains only Δ M non-reducing end, because be rich in M due to can not be by G- The restriction endonuclease Aly5 depth degradation of specificity can be considered unsaturated poly- M sections of series of oligosaccharides；(2) it is mono- to contain only Δ G for unsaturated disaccharides Member, the product oligosaccharides of degree of polymerization 3-4 contain Δ M or Δ G non-reducing end and rich in G, but are to be with disaccharides because being limited to Aly5 The restriction endonuclease and minimum substrate of minimum product are pentasaccharides, therefore are not digested thoroughly；(3) the non-catalytic area of Aly5 is amputated, though do not change Become the substrate selectivity and inscribe mode for truncating body protein, but the minimum substrate of truncate becomes larger, so that it is brown to degrade in truncate In final product after phycocolloid, the series of oligosaccharides content of degree of polymerization 5-7 is dramatically increased, and the series of oligosaccharides content phase of degree of polymerization 2-4 To reduction.This research confirms for the first time: using the algin restriction endonuclease of G- specificity, not only more can exclusively produce unsaturation The two class algin oligosaccharide products such as M sections poly-, unsaturated type is G section poly-, range of molecular weight distributions is dramatically different, and passes through molecule enzyme The characteristic that truncate generates larger oligose fragment can be enhanced by learning transformation.

Inventor also once had studied two M- specificity endo-type algins of pseudomonas aeruginosa, azotobacter vinelandii source Lyases AlgL^[11], discovery: in the oligosaccharides final product after they degrade algin, the oligosaccharides of the larger degree of polymerization (5-7) is contained only Δ G non-reducing end and it is rich in G, and can be considered unsaturated poly- G sections of series of oligosaccharides；And unsaturated disaccharides product mainly contains Δ M, Unsaturated trisaccharide, tetrose product mainly contain Δ M or the end Δ G.That is: the two M- specificity algin restriction endonuclease AlgL, drop Oligosaccharides final product after solving algin, the oligosaccharides final product with G- specificity restriction endonuclease Aly5, structure feature is on the contrary, but companion each other It is similar to each other with the Structure succession rule of degree of polymerization size.

In addition, inventor is investigated the tendentious difunctional restriction endonuclease of tool G- in the source Flammeovirga sp.MY04 Aly1^[12,13]With Aly2^[14,115], it was demonstrated that they be more suitable for thoroughly degrading algin and efficiently produce unsaturated disaccharides (Δ G), The low molecular weights oligose fragment such as unsaturated trisaccharide.

To sum up, similar to domestic pharmaceutical industry research, the existing research emphasis of inventor around endo-type algin catenase into Analysis is gone.But the difference is that inventor and seminar also emphasis elaborate a series of substrate selectivity of restriction endonucleases, bottom Object degradation model and oligosaccharides generate feature, and by induction and conclusion, are the discovery that the substrate selectivity of enzyme and degradation of substrates mode are total With the product oligosaccharides formation characteristic for determining restriction endonuclease.Relevant molecular enzymology engineering research also explores transformation native enzyme, increases The effective way of its strong oligosaccharides formation characteristic.

Incessantly in this way, about the biochemical property of circumscribed-type algin catenase, catalyst mechanism, enzymatic property and dividing both at home and abroad The rare report of system research of son transformation.As in Chinese invention patent application about the application of circumscribed-type algin catenase only See Chinese patent literature CN107177612A (application number 201710367707X) in 2017, makes public for the first time thin from Yu Haiyang The basic biochemical property and enzymatic hydrolysis algin of the circumscribed-type algin catenase AlgL17 of bacterium microvesicle bacterium ALW1 can produce product: 4- Deoxidation-L- erythro -5- hexose uronic acid (DEH) does not elaborate the degradation of substrates characteristic of circumscribed-type algin catenase And the similar research of oligosaccharides formation characteristic.The country about difunctional algin catenase patent demand also rarely seen inventor about The published application of restriction endonuclease Aly-1, Aly-2^[12-15]。

But for the tendentious difunctional monosaccharide excision enzyme of M- and its related substrates degradation mechanism and oligosaccharides resulting from Product formation characteristic etc. does not have relevant report.

Summary of the invention

In view of the deficiencies of the prior art, the present invention provides a kind of tendentious monosaccharide circumscribed-type algin catenase Aly-6 of M And its encoding gene and application.

Technical scheme is as follows:

A kind of tendentious monosaccharide circumscribed-type algin catenase Aly-6 of M, amino acid sequence is as shown in SEQ ID NO.2.

The above-mentioned tendentious monosaccharide circumscribed-type algin catenase Aly-6 of M can degrade the gulose aldehyde from algin Mannuronic acid oligosaccharide segment can also drop in sour oligose fragment, therefore be facultative algin catenase.

The above-mentioned tendentious monosaccharide circumscribed-type algin catenase Aly-6 of M structural domain containing there are two, one of them is ranged AlgLyase superfamily, another ranges Heper_II_III superfamily.

For the enzyme during degrading brown alga glue polysaccharide, in the circumscribed mode of monosaccharide, gradually intermediates are (unsaturated Type sugar chain), therefore principal product is unsaturated monosaccharide unit (Δ) and remaining sugar chain.In the endless degradable algin of the enzyme, The generated unsaturated product oligosaccharides of series, common trait is that non-reducing end has contained only Δ G end structure.

The enzyme is after thoroughly degradation brown alga glue polysaccharide, in final remaining product oligosaccharides based on unsaturated three bglii fragments, Containing micro unsaturated tetrose, unsaturated disaccharides is had no.

The unsaturated oligosaccharide substrates of the minimum of the enzyme are disaccharides Δ M, and generate 2 molecule Δs after the Δ M that degrades.The enzyme micro can drop A molecule unsaturation disaccharides Δ G is solved, 2 molecule Δs are generated.The enzyme, which cannot degrade, is saturated disaccharides MM or GG.

To sum up, which includes unsaturated monosaccharide Δ, saturation monosaccharide M and G.

A kind of tendentious monosaccharide circumscribed-type algin catenase encoding gene aly-6 of M, nucleotide sequence such as SEQ Shown in IDNO.1.

Encoding gene aly-6, the total 2238bp of overall length of the above-mentioned tendentious monosaccharide circumscribed-type algin catenase Aly-6 of M, Coded protein contains 745 amino acid, and molecular weight is about 84.7kD.

A kind of recombinant expression carrier inserts the tendentious monosaccharide circumscribed-type algin cracking of above-mentioned M in expression vector The encoding gene aly-6 of enzyme Aly-6.

Preferred according to the present invention, the expression vector is selected from: coli expression carrier, Yeast expression carrier, withered grass Bacillus expression vector, lactic acid bacteria expression vectors, streptomyces expression vector, phage vector, filamentous fungi expression vector, plant table Up to carrier, insect expression vector or mammalian cell expression vector.

A kind of recombinant bacterium has been transferred to the tendentious monosaccharide circumscribed-type phycocolloid lyases Aly-6's of above-mentioned M in host cell Recombinant expression carrier, or the above-mentioned tendentious monosaccharide circumscribed-type algin catenase Aly-6 of M of expression.

Preferred according to the present invention, host cell is selected from: e. coli host cell, yeast host cells, withered grass bar Bacterium host cell, lactic acid bacteria host cell, actinomyces host cell, filamentous fungal host cell, insect cell or mammal Cell.

The encoding gene aly-6 of the above-mentioned tendentious monosaccharide circumscribed-type algin catenase Aly-6 of M, recombinant expression carrier, Recombinant bacterium is preparing the application in the tendentious monosaccharide circumscribed-type algin catenase rAly-6 of M.

The above-mentioned tendentious monosaccharide circumscribed-type algin catenase Aly-6 of M is in thoroughly degradation algin or degradation algin Oligosaccharides produces the application in unsaturated trisaccharide and unsaturated tetrose.

The above-mentioned tendentious monosaccharide circumscribed-type algin catenase Aly-6 of M is in endless degradable algin or degradation brown alga Answering in the unsaturated oligose fragment of biggish series and unsaturated monosaccharide Δ that glue oligosaccharides production non-reducing end contains the end Δ G With.

Beneficial effect

1, present invention firstly discloses the bases by heat color Bacillus bacteria (Flammeovirga yaeyamensis) MY04 Because obtaining a kind of tendentious monosaccharide circumscribed-type algin catenase Aly-6 of M in group, which is the cracking of monosaccharide circumscribed-type algin Enzyme, and have apparent M tendentiousness, enzyme Aly-6 can in such a way that monosaccharide is circumscribed gulose of the sustaining degradation from algin Aldehydic acid segment, also can in such a way that monosaccharide is circumscribed sustaining degradation mannuronic acid segment, but be easier to degradation mannuronic acid segment； The enzymatic property and existing known algin catenase have significant difference, and stable in physicochemical property, activity is high, have industrial application Potential quality.

2, the endless degradable algin of the tendentious monosaccharide circumscribed-type algin catenase Aly-6 of M of the present invention is more Mainly pass through circumscribed lasting generation unsaturated sugar unit Δ when sugared, and the remaining unsaturated product oligosaccharides piece of biggish series Section, structural features are that non-reducing end has contained only the end Δ G.Therefore, Aly-6 can be used for the endless high-fall of algin Solution, thus the unsaturated oligose fragment of serial size of the specificity preparation only containing the end Δ G；

3, the tendentious monosaccharide circumscribed-type algin catenase Aly-6 of M of the present invention is for algin of thoroughly degrading When polysaccharide, in final remaining product oligosaccharides, principal product be unsaturated trisaccharide, containing a small amount of unsaturated tetrose, have no unsaturated Disaccharides, and deep conversion is other products to generated primary product-unsaturation monosaccharide Δ-.Therefore, Aly-6 can be thorough Bottom digests algin, provides unsaturated monosaccharide carbon source for the growth of microorganism so that microorganism grows utilization；

4, the tendentious monosaccharide circumscribed-type algin catenase Aly-6 of M of the present invention is containing there are two structural domains (AlgLyase superfamily and Heper_II_III superfamily) has carried out truncating expression respectively, and as a result two are cut For short body protein matter without algin catenase activity, this shows that the functional module of two suppositions generates circumscribed-type by Molecular interaction Algin catenase activity, this is not completely identical as existing known algin catenase structure, for further research algin Lyases is laid a good foundation.

Detailed description of the invention

The BLASTp that Fig. 1, recombination algin catenase rAly-6 functional module are constituted analyzes result photo；

The polyacrylamide gel electrophoresis figure (SDS-PAGE) of Fig. 2, expression and purification situation；

Wherein, the polyacrylamide gel electrophoresis figure (SDS- of A, recombination algin catenase rAly-6 expression and purification PAGE)；In figure: M, protein molecular weight standard, band from top to bottom size be 116kD, 66.2kD, 45kD, 35kD, 25kD, 18.4kD 14.4kD；Thallus before swimming lane 1, control strain broken wall, 10 μ L of applied sample amount, thallus before swimming lane 2, recombinant bacterium broken wall, loading Measure 10 μ L, supernatant after swimming lane 3, recombinant bacterium broken wall, 10 μ L of applied sample amount, swimming lane 4, the rAly-6 through ni-sepharose purification, 10 μ L of applied sample amount；

B, the polyacrylamide gel electrophoresis figure (SDS-PAGE) of truncate rAly-6Lm expression and purification；In figure: M, egg White matter molecular weight standard, size is 116kD, 66.2kD, 45kD, 35kD, 25kD, 18.4kD, 14.4kD to band from top to bottom；Swimming Thallus before road 1, control strain broken wall, 10 μ L of applied sample amount, thallus before swimming lane 2, recombinant bacterium broken wall, 10 μ L of applied sample amount, swimming lane 3, again Supernatant after group bacterium broken wall, 10 μ L of applied sample amount, swimming lane 4, the rAly-6 through ni-sepharose purification, 10 μ L of applied sample amount；

C, the polyacrylamide gel electrophoresis figure (SDS-PAGE) of rAly-6Hpm expression and purification；In figure: M, protein point Sub- amount standard, size is 116kD, 66.2kD, 45kD, 35kD, 25kD, 18.4kD, 14.4kD to band from top to bottom；It is swimming lane 1, right According to thallus before bacterial strain broken wall, 10 μ L of applied sample amount, thallus before swimming lane 2, recombinant bacterium broken wall, 10 μ L of applied sample amount, swimming lane 3, recombinant bacterium are broken Supernatant after wall, 10 μ L of applied sample amount, swimming lane 4, the rAly-6 through ni-sepharose purification, 10 μ L of applied sample amount；

Fig. 3, temperature are to the recombination active influence curve figure of algin catenase rAly-6；

Fig. 4, temperature are to the influence curve figure for recombinating algin catenase rAly-6 stability；

Fig. 5, pH value are to the influence curve figure for recombinating algin catenase rAly-6 activity and stability；

Fig. 6, metal ion and chemical reagent are on the recombination active influence column diagram of algin catenase rAly-6；

Molecular gel chromatography-the HPLC of product oligosaccharides during Fig. 7, recombination algin catenase rAly-6 degradation algin Analysis chart；

In figure: (1) UDP3, unsaturated trisaccharide；(2) UDP4, unsaturated tetrose；(3) UDP5, unsaturated pentasaccharides；(4) UDP6, six sugar of unsaturation

Unsaturation oligose fragment UDP3 prepared by Fig. 8, the recombination endless degradable algin of algin catenase rAly-6, The HPLC analysis chart of UDP4, UDP5, UDP6；

Fig. 9, with recombination the endless degradable algin of algin catenase rAly-6 prepared by unsaturation oligosaccharides piece UDP3, UDP4, UDP5, UDP6's¹H-NMR figure；

Figure 10, separating obtained UDP2, UDP3 in the product of the degradable algin of algin catenase rAly-1 are recombinated certainly HPLC analysis chart；

Figure 11, separate institute UDP2's from the product of the recombination degradable algin of algin catenase Pae-rEAlgL HPLC analysis chart；

Figure 12, with recombination algin catenase rAly-6 thoroughly degrade use rAlgL-6 prepared by oligosaccharides UDP3-UDP6 and The HPLC analysis chart of the UDP3 of rAly-1 preparation；

Figure 13, with excessive recombination algin catenase rAly-6 and four kinds of disaccharides formed containing heterogeneity (Δ M, Δ G, MM, GG) reaction product HPLC analysis chart；

Figure 14, divided with the HPLC of excessive recombination algin catenase rAly-6 and the reaction product for being saturated poly- M pentasaccharides (M5) Analysis figure (ultraviolet)；

Figure 15, divided with the HPLC of excessive recombination algin catenase rAly-6 and the reaction product for being saturated poly- G pentasaccharides (G5) Analysis figure (ultraviolet)；

Figure 16, the poly- M five of saturation marked with excessive recombination algin catenase rAly-6 degradative reduction end by 2-AB The HPLC analysis chart (fluorescence) of sugared (M5) process；

Figure 17, the poly- G five of saturation marked with excessive recombination algin catenase rAly-6 degradative reduction end by 2-AB The HPLC analysis chart (fluorescence) of sugared (G5) process；

Figure 18, with excessive recombination algin catenase rAly-6 degradative reduction end by 2-AB mark from The HPLC analysis chart (fluorescence) of unsaturated pentasaccharides (UDP5) process of rAly-6.

Specific embodiment

The elaboration of following embodiment is some common technologies how implemented for the comprehensive disclosure present invention, rather than is Limitation application range of the invention.Inventor tried one's best ensure in embodiment the accuracy of parameter (such as measure, Temperature, etc.), but some experimental errors and deviation should also pay attention to.Unless otherwise indicated, middle-molecular-weihydroxyethyl of the present invention is Refer to weight average molecular weight, temperature is degree Celsius.

Biological material source

Heat color bacillus (Flammeovirga yaeyamensis) MY04 bacterium source is in Chinese microorganism strain preservation pipe Reason committee common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences microbe research Institute, preservation date on November 27th, 2008, deposit number CGMCC NO.2777.

The extraction of embodiment 1, heat color bacillus (Flammeovirga yaeyamensis) MY04 strain gene group DNA

Heat color bacillus (Flammeovirga yaeyamensis) MY04 is seeded in fluid nutrient medium YT04,28 DEG C, under conditions of 200rpm, shaken cultivation to 600nm light absorption value (OD₆₀₀) it is 1.2；Take culture bacterium solution 10mL, 12,000 × g It is centrifuged 15min under the conditions of (g, Gravitational coefficient of the Earth), collects bacterial sediment；With bacteriolyze enzyme buffer liquid (the 10mM Tris- of 10mL HCl, pH 8.0) suspension thalline, it is centrifuged 15min under the conditions of 12,000rmp, collects bacterial sediment.

Aforesaid liquid culture medium YT04, every liter of component are as follows:

Tryptone 10g, yeast extract 5.0g, sodium chloride 30g dissolve with water and are settled to 1L, pH 7.2.

Into above-mentioned bacterial sediment, bacteriolyze enzyme buffer liquid (being purchased from Shanghai Sheng Gong bioengineering Co., Ltd) is added in every pipe 6.0mL obtains the bacterium solution of about 7.0mL, is separately added into each 280 μ L of lysozyme soln that concentration is 20mg/mL, keeps lysozyme dense eventually Degree is 800 μ g/mL；It is placed in 1.0h in ice-water bath, is then transferred in 37 DEG C of water-baths, warm bath 2h, until reaction system is sticky；It is added Concentration is the 30 μ L of Proteinase K Solution of sodium cetanesulfonate solution 0.41mL, 100mg/mL of 100mg/mL, in 52 DEG C of warm bath 1.0h；Equilibrated phenol/chloroform/isoamyl alcohol (volume ratio 25:24:1) the solution 7.5mL of Tris- is added, is gently mixed by inversion；? 10,000 × g, it is centrifuged 10min under the conditions of 4 DEG C, collects supernatant, and NaAc-HAc (pH5.2, the 3.0M) buffering of 1.0mL is added The dehydrated alcohol of liquid and 8.5mL, mixes well；Choose Filamentous DNA with pipette tips, is transferred in the centrifuge tube of 1.5mL, with 70% ethyl alcohol (stores in -20 DEG C), washs 2 times, discards supernatant after micro- centrifugation；10,2min 000 × g, is centrifuged under the conditions of 4 DEG C, it is thorough Bottom discards supernatant；It is dry that DNA is deposited in the drying of aseptic working platform apoplexy, then with aseptic deionized water in 4 DEG C of overnight dissolving DNAs Genomic DNA is made in sample.

The scanning and its sequence of embodiment 2, heat color bacillus (Flammeovirga yaeyamensis) MY04 strain gene group Column analysis.

It is sequenced, genomic DNA made from embodiment 1 by upper using the scanning that pyrosequencing techniques carry out genome Hai Meiji biotech firm completes.With NCBI (National Center for Biotechnology Information, Http:// www.ncbi.nlm.nih.gov/) online software of website analyzes DNA sequencing result.Used NCBI The analysis software of website be Open Reading Frame Finder (ORF Finder, http: // ) and Basic Local Alignment Search Tool www.ncbi.nlm.nih.gov/gorf/gorf.html (BLAST,http://blast.ncbi.nlm.nih.gov/Blast.cgi)。

With the analysis of above-mentioned biological software the results show that heat color bacillus (Flammeovirga yaeyamensis) MY04 Encoding gene aly-6, the coding head of district 2238bp of gene aly-6 of an algin catenase are carried on strain gene group DNA, Nucleotide sequence is as shown in SEQ ID NO.1.The encoded recombination algin catenase rAly-6 of gene aly-6 contains 745 altogether A amino acid, amino acid sequence is as shown in SEQ ID NO.2.

With BLASTp software on-line analysis, the results show that with recombination algin catenase rAly-6's in ncbi database Amino acid sequence similarity is all protein of the function without identification greater than 30%.As shown in Figure 1, BLASTp analysis also pushes away The n-end of albumen for surveying Aly-6 contains the hypothesis catalyst structure domain AlgLyase guarded in algin catenase super families Superfamily, is named as Aly-6Lm, and C- contains at end the hypothesis catalytic structure guarded in Heparinase I I_III super families Domain Heper_II_III superfamily, is named as Aly-6Hpm.Divided with biological software BioEdit 7.0.5.3 The theoretical molecular weight of analysis, display a-protein ly-6 is about 84.7kD.With signal peptide on-line prediction software SignalP 4.1Server (http://www.cbs.dtu.dk/services/SignalP/) on-line analysis, the protein N-terminal 1-23 Amino acid is coding sequence of secretory signal peptide.

Embodiment 3, gene aly-6 and truncate aly-6Lm, aly-6Hpm are in e. coli bl21 (DE3) bacterial strain Recombinant expression

Using genomic DNA made from embodiment 1 as template, PCR amplification is carried out.Primer sequence is as follows:

Forward primer the Aly6-F:5 '-gg of aly-6 amplificationCATATGCAGAACGGAAATTTAATTACTTCCG-3’ (NdeI)；

Reverse primer the Aly6-R:5 '-gc of aly-6 amplificationTCTAGA AATTTCTTCCAATAGGTAAACCCC-3’(Xba I)；

Forward primer the Aly6Lm-F:5 '-gg of aly-6Lm amplificationCATATGCAGAACGGAAATTTAATTACTTCCGAG-3'(Nde I)；

Reverse primer the Aly6Lm-R:5 '-gc of aly-6Lm amplificationTCTAGA CAAACTTCTTTTTTGGTAAGTCGTTGC-3'(Xba I)；

Forward primer the Aly6Hpm-F:5 '-gg of aly-6Hpm amplificationCATATGTACTATTCAAGAGAATTGAAACTAGCC-3'(Nde I)；

Reverse primer the Aly6Hpm-R:5 '-gc of aly-6Hpm amplificationTCTAGAAATTTCTTCCAATAGGTAAACCCC- 3'(Xba I)；

Forward primer underscore mark is restriction enzyme Nde I site, and reverse primer underscore mark is limit Property restriction endonuclease Xba I site processed.High-fidelity DNA polymerase PrimeSTAR HS DNA Polymerase used is big purchased from China Lian Bao biotech firm, PCR reaction reagent used are operated according to the description of product that the said firm provides.

PCR reaction condition: 95 DEG C of initial denaturation 4min；94 DEG C of denaturation 40s, 60 DEG C of annealing 30s, 72 DEG C of extension 135s, 35 Circulation；72 DEG C of extension 10min；4 DEG C of stable 10min.

PCR product is subjected to double digestion with restriction enzyme Nde I and Xba I, is recycled by agarose gel electrophoresis PCR product after digestion.It will be purchased from the product pCold TF Plasmid DNA of DaLian, China treasured biotech firm, with Nde I and Xba I Double digestion carries out agarose gel electrophoresis and recycles the product segment after digestion.Restriction enzyme Nde I and Xba I are purchased In DaLian, China treasured biotech firm, the system of the reaction of enzyme-to-substrate used in digestion, temperature and time are mentioned according to the said firm The description of product of confession operates.

Will by the PCR product of Nde I and Xba I double digestion, and the pCold TF plasmid vector for also passing through double digestion, It is connect under the catalysis of DNA ligase；Connection product converts e.colistraindh5α, is coated on containing 100 μ g/mL ammonia benzyls On the Luria-Bertani culture medium solid plate of mycin, after 37 DEG C of culture 16h, picking monoclonal；Monoclonal access is contained It is cultivated in the liquid Luria-Bertani culture medium of 100 μ g/mL ammonia benzyl mycins, extracts plasmid；Plasmid is carried out with amplimer PCR verifying, as a result obtains the amplified production that size is 2.3kb, and the recombinant plasmid of preliminary proof building is correct；Then by the recombination Plasmid is sequenced, the results showed that, decibel inserts gene aly- between Nde I and Xba the I restriction enzyme site of pCold TF 6, aly-6Lm, aly-6Hpm, and direction of insertion is correct, so further proving that the recombinant plasmid of building is correct, by recombinant plasmid It is respectively designated as pCTF-Aly6, pCTF-Aly6Lm, pCTF-Aly6Hpm.

Recombinant plasmid pCTF-Aly6, pCTF-Aly6Lm, pCTF-Aly6Hpm are converted into coli strain BL21 (DE3) (being purchased from U.S. Invitrogen company), the operating procedure then provided according to the said firm use isopropylthiogalactoside (IPTG) inducing expression of recombination algin catenase rAly-6, rAly-6Lm, rAly-6Hpm is carried out.In 8,000 × g, 4 DEG C Under the conditions of be centrifuged 15min, collect thallus, and thallus, ultrasonication in ice water bath environment is resuspended with buffer solution A.15,000 × G, it is further centrifuged 30min under the conditions of 4 DEG C, collects water-soluble component, and with Ni- Ago-Gel to recombinating algin catenase RAly-6, rAly-6Lm, rAly-6Hpm are purified.With the buffer for containing imidazole concentration being 10,50,100,250,500mM A carries out gradient elution, and purification condition is operated according to the product manual of gel.With the solidifying denaturation gel electrophoresis detection recombination of polyacrylamide The purifying situation of algin catenase rAly-6, rAly-6Lm, rAly-6Hpm.As a result as shown in Figure 2 A: through IPTG inducing expression In BL21 (DE3) thallus afterwards, the yield of recombination algin catenase rAly-6 is not less than 200mg/L thalline culture；Purifying Recombination algin catenase rAly-6 afterwards is in single band on running gel, and position matches with the molecular weight of prediction, It is in single band on running gel, and position matches with the molecular weight of prediction；As a result as shown in Fig. 2 B, 2C, recombination algin is split Water soluble expression is also presented in solution enzyme rAly-6Lm, rAly-6Hpm, and the recombination of recombination algin catenase after purification after purification is brown Phycocolloid lyases rAly-6 is in single band on running gel, and position matches with the molecular weight of prediction.By weight after purification Group algin catenase rAly-6, rAly-6Lm, rAly-6Hpm sample is packed into the bag filter that smallest molecule interception is 10kD, It dialyses in 4 DEG C of environment to buffer solution A.The ingredient of described buffer solution A is 50mM Tris, 150mM NaCl, pH 7.9, The recombination pure enzyme solution of algin catenase rAly-6, rAly-6Lm, rAly-6Hpm is made.

The measurement of embodiment 4, recombinase rAly-6, rAly-6Lm, rAly-6Hpm optimum temperature

The algin that mass-volume concentration is 0.1-1.2% is prepared with deionized water, after heating for dissolving, is placed in 0 DEG C, 10 DEG C, 20 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, cool down in 50 DEG C of water baths 1h.It is added respectively into every 900 μ L substrate solution The 100 μ L of dilution of the recombination algin catenase rAly-6 obtained of embodiment 3, recombinates the dilution of algin catenase rAly-6 Liquid concentration be 10 μ g/mL, the reaction was continued after mixing, every when sample.3 parallel samples, are gone out under the conditions of each temperature with boiling water bath Recombination enzyme preparation living is control group.RAly-6Lm and rAly-6Hpm measures its optimum temperature also referring to the above method.

Concentration (the OD of newly-generated reduced sugar in each reaction system is measured with DNS- reducing sugar method₅₄₀), and average value is calculated, Carry out variance analysis.The corresponding reaction temperature of obtained the maximum absorption is the optimum temperature of recombinase, opposite enzyme activity (RA) is defined as: each The percentage of absorption value and obtained the maximum absorption.As a result as shown in Figure 3: when measuring enzyme activity as substrate using algin as described above, weight Group algin catenase rAly-6 reaches maximum vigor in 40 DEG C of reactions, this shows to recombinate algin catenase rAly-6's Optimal reactive temperature is 40 DEG C.

But the activity of rAly-6Lm and rAly-6Hpm is measured under above-mentioned different temperatures, discovery is without activity.This Illustrate Aly6 two hypothesis catalyst structure domain single expressions be it is inactive, Aly6 activity need two structural domains it is common Cooperation can just show.Further illustrate that truncating expression easily inactivates circumscribed-type algin catenase Aly-6.

Embodiment 5, the measurement for recombinating algin catenase rAly-6 optimal pH

Respectively with concentration be 50mM NaAc-HAC buffer, 50mM NaH₂PO₄-Na₂HPO₄Buffer, 50mM Tris-HCl buffer prepares the algin substrate that mass-volume concentration (g/mL) is 0.1~1.2%, institute with algin respectively Corresponding pH value is respectively 5,6,6,7,8,7,8,9,10 3 sections, and each pH value is set up under optimum temperature.Substrate is molten Xie Hou is placed in optimum temperature and is incubated for 1h, and recombination algin cracking made from embodiment 3 is then added into every 900 μ L substrate The 100 μ L of dilution of enzyme rAly-6, starts to react after mixing, every when sample.3 parallel samples under the conditions of each pH, with boiling water The recombination enzyme preparation of bath inactivation is control group.Concentration of reduced sugar newly-generated in each reaction system is measured with DNS- reducing sugar method (OD₅₄₀), and calculate average value and deviation.Opposite enzyme (RA) is living is defined as: the percentage of each group mean absorbance and obtained the maximum absorption Than.The corresponding pH of obtained the maximum absorption is the optimal pH of recombinase.As a result as shown in Figure 5: recombination algin catenase rAly-6's Optimal reaction pH is 6.0.

Embodiment 6, the temperature stability analysis for recombinating algin catenase rAly-6

By weight made from the embodiment 3 after heat treatment different time lower at 0 DEG C, 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C Group algin catenase rAly-6 enzyme solution is respectively 0.1~1.2% with the mass-volume concentration (g/mL) configured with distilled water Algin is mixed in the ratio of 1:9 (volume ratio), then remnant enzyme activity is measured under optimum temperature, without Overheating Treatment Enzyme solution enzyme activity is defined as 100% relative activity.As a result as shown in Figure 4: temperature of the recombination algin catenase rAly-6 at 40 DEG C After lower pretreatment 1h, still there is > 50% residual activity, show that the enzyme has certain thermal stability.

Embodiment 7, the pH stability analysis for recombinating algin catenase rAly-6

The enzyme solution of algin catenase rAly-6 will be recombinated made from embodiment 3, in optimum temperature (40 DEG C), different pH After distinguishing preincubate 2h in (pH5~10) environment, the algin substrate for being 0.1~1.2% with mass-volume concentration (g/mL) is molten Liquid is mixed in the ratio of 1:9 (volume ratio), then remnant enzyme activity is measured under optimum temperature, without pretreated enzyme solution enzyme Work is defined as 100% relative activity.As a result as shown in figure 4, pre-processing 2h in the range of pH6~7, rAly-6 enzyme activity is still kept 90% or more.

Embodiment 8, metal ion and chemical reagent are on the recombination active influence of algin catenase rAly-6

It is that 0.1~1.2% algin substrate, recombination made from embodiment 3 are brown by the mass concentration configured with deionized water After phycocolloid lyases rAly-6 enzyme solution and water are mixed in the ratio of 5:1:4 (volume ratio), then added not into reaction system Same metal ion, the ion of addition final concentration of 1mM or 10mM, then in 40 DEG C of reaction 4h, by DNS- reduced sugar above-mentioned The vigor of method survey enzyme.The activity (being set as 100%) that control group is rAly-6 when any metal ion is not added.As a result such as Fig. 6 institute Show, under 1mM or 10mM concentration: (1) K⁺、Li⁺、Na⁺Three kinds of monovalent metal reagents are to the activities present of rAly-6 in 10mM Facilitation, but Ag⁺With significantly inhibiting effect；(2)Co²⁺、Mg²⁺、Mn²⁺Equal divalent metals reagent has enzyme activity in 10mM There is facilitation, and remaining divalent, trivalent metal ion are inhibited to enzyme activity；(3) glycerol, imidazoles and DTT in 1mM and There is promotion activity to the activity of rAly-6 when 10mM, inhibiting effect is presented to enzyme activity in remaining reagent.

The enzyme activity of embodiment 9, DNS- reducing sugar method measurement recombination algin catenase rAly-6

By the mass concentration configured with deionized water be 0.1-1.2% algin, the recombination brown alga that concentration is 10 μ g/mL Glue lyases rAly-6 enzyme solution, HAc-NaAc (pH 6.0) buffer of 150mmol/L and water are by 2:1:3:4 (volume ratio) After ratio mixing, 4h is reacted at 40 DEG C.Reaction product is heated into 10min in boiling water bath, is transferred to 5min in ice-water bath, 12, 000 × g, it is centrifuged 15min under the conditions of 4 DEG C, collects supernatant；By the supernatant of certain volume, (3,5- is to nitro two with isometric DNS Toluene) mixing of-reaction solution, 10min is heated in boiling water bath, is down to room temperature, measures absorption value in 540nm.With the pure grape of analysis Uronic acid sodium lactone makees standard items, and the molar concentration and OD of glucurone are drawn in same method operation₅₄₀Between amount Imitate relation curve.Algin cracking is recombinated with the quantification of protein kit measurement for being purchased from Shanghai Sheng Gong bioengineering Co., Ltd Protein content in enzyme rAly-6 enzyme solution.The unit of activity for calculating enzyme is defined according to international standard, i.e., at the standard conditions, often Enzyme amount needed for generating 1 μm of ol product in minute is 1 IU.The result shows that: recombination rAly-6 can be digested using algin as substrate And reduced sugar product is generated, enzyme activity is 726 ± 2.2U/mg.

Embodiment 10, the efficient liquid phase (HPLC) for the product for recombinating algin catenase rAly-6 degradation algin are analyzed

The algin substrate that mass-volume concentration (g/mL) is 0.1~1.2% is prepared with deionized water, after heating for dissolving, The 1h that cools down is placed in 40 DEG C of water baths.The dilution of recombinase rAly-6 made from embodiment 3 is added into every 100 μ L substrate 10-100 μ L is supplied when less than 200 μ L volume with aseptic deionized water；The reaction was continued after mixing, every when sample.Reaction is produced Object heats 10min in boiling water bath, is transferred to 5min in ice-water bath.12,000 × g, it is centrifuged 15min under the conditions of 4 DEG C, in collection Clearly.

The NH for being 0.20mol/L with concentration₄HCO₃Solution, balance Superdex Peptide 10/300GL (GE company) point Sub- gel chromatographic columns, flow velocity 0.40mL/min, at least 2 column beds.By the sample of the different enzymolysis times of above-mentioned algin, with automatic Sample injector loads 100 μ g/ samples, and other conditions are constant, 235nm detection.With HPLC operating software, the product of each oligosaccharide compositions is analyzed Facet product, calculates relative molar concentration.

As shown in fig. 7, under the above conditions, after algin substrate is degraded, with the increase of enzyme digestion reaction time, having The increase at any time of the product oligosaccharides product oligosaccharides degree of polymerization of 235nm characteristic absorption and reduce, being finally converted into appearance time is The unsaturated trisaccharide of 40.5'.This tentatively shows that recombinase rAly-6 is algin catenase.

Embodiment 11, recombinate the endless degradable algin of algin catenase rAly-6 oligosaccharides principal product molecular weight mirror It is fixed

According to described in embodiment 10, total 200mg algin is not exclusively digested with recombination algin catenase rAly-6, sample Product in batches loading, cross Superdex Peptide 10/300GL molecular gel chromatographic column (GE company), and according to appearance time point Not Shou Ji appearance time be 34.8 ', 36.0 ', 38.2 ' and 40.5 ' four oligosaccharide samples.By be repeatedly collected into four oligosaccharides After sample is concentrated respectively, it is freeze-dried repeatedly with desalination.Gained oligosaccharide sample is dissolved with aseptic deionized water, carries out first mass spectrometric (MS) it analyzes, determines the relative molecular weight of each oligosaccharides.Heavy water (D is used again₂O resulting oligosaccharide sample) is dissolved, is freeze-dried repeatedly, To complete hydrogen deuterium exchange, and carry out¹H-NMR analysis, finally determines the chemical structure and feature of each oligosaccharides.

As shown in figure 8, few with unsaturation prepared after the recombination endless degradable algin of algin catenase rAly-6 Bglii fragment UDP6, UDP5, UDP4, UDP3 carry out HPLC analysis, and product has characteristic absorption in 235nm as the result is shown, when appearance Between be respectively 34.8 ', 36.0 ', 38.2 ' and 40.5 ', purity is all larger than 99%, meets further experiment requirement.

Further pass through¹H-NMR data analyze the structure feature of above two oligosaccharides: as shown in figure 9, at 5.65ppm Characteristic chemical shifts value shows to recombinate the non-of four kinds of products after the endless degradable algin of algin catenase rAly-6 and goes back Originality end is entirely Δ G unit.

Embodiment 12, recombination algin catenase rAly-1 thoroughly degrade algin product UDP2, UDP3 preparation

According to patent application (application number 201610838337.9, a kind of facultative endo-type recombination algin catenase rAly- 1 and its encoding gene and application) in described in embodiment 11, total 200mg algin is thoroughly digested with recombinase rAly-1, Product in batches loading, cross molecule gel column Superdex Peptide 10/300GL (GE company), and distinguish according to appearance time Collect two oligosaccharide samples that appearance time is 40.5' and 43.2'.After the two oligosaccharide samples are repeatedly collected, are concentrated respectively, It is freeze-dried repeatedly with desalination.

As shown in Figure 10, oligose fragment UDP2, UDP3 of preparation is tested and analyzed through HPLC, and product is in 235nm as the result is shown With characteristic absorption, purity is all larger than 99%, meets further experiment requirement.Above-mentioned patent application (application number 201610838337.9, a kind of facultative endo-type recombination algin catenase rAly-1 and its encoding gene and application) in implement Example 10 proves that the UDP2 almost all by rAly-1 degradation algin preparation is Δ G component；The non reducing end of UDP3 has two Seed type: Δ G and Δ M, molar ratio are about 5:4.

Embodiment 13, recombination algin catenase Pae-rEAlgL thoroughly degrade algin product UDP2 preparation

According to patent application, (application number 2017106518145, algin catenase are preparing answering in series of oligosaccharides product With) in described in embodiment 11, total 200mg algin is thoroughly digested with recombinase Pae-rEAlgL, product in batches loading, Molecule gel column Superdex Peptide 10/300GL (GE company) is crossed, and collects single oligosaccharides respectively according to appearance time Segment.After these oligosaccharide samples are repeatedly collected, are concentrated respectively, it is freeze-dried repeatedly with desalination.

As shown in figure 11, the oligose fragment UDP2 of preparation is tested and analyzed through HPLC, and product has spy in 235nm as the result is shown Sign absorbs, and purity is all larger than 99%, meets further experiment requirement.Above-mentioned patent application (application number 2017106518145, it is brown Phycocolloid lyases is preparing the application in series of oligosaccharides product) in embodiment 12 prove, by Pae-rEAlgL degradation preparation UDP2 almost all is Δ M component.

The product analysis of embodiment 14, recombination algin catenase rAly-6 degradation oligosaccharides

The oligose fragment prepared by deionized water and embodiment 11, embodiment 12 and embodiment 13, comprising: unsaturation five Sugared (UDP5), unsaturated tetrose (UDP4), unsaturated trisaccharide (UDP3: containing only the end Δ G substrate), unsaturated trisaccharide (UDP3: Δ M and the end Δ G mixed substrates), unsaturated disaccharides (UDP2: Δ G), the saturation of unsaturated disaccharides (UDP2: Δ M) and purchase it is few Bglii fragment (guluronic acid disaccharides (GG), mannuronic acid disaccharides (MM)).Substrate solution is respectively configured in these oligosaccharide samples, HAc-NaAc (pH6.0) buffer of the recombination algin catenase rAly-6 enzyme solution for being 10 μ g/mL with concentration, 150mmol/L And after water is mixed in the ratio of 2:1:3:4 (volume ratio), 40 DEG C of reactions are for 24 hours.According to operation condition of chromatogram described in embodiment 10, Carry out HPLC analysis.

As a result as shown in Figure 12,13, herein described recombination algin catenase rAly-6:

(1) unsaturated pentasaccharides (UDP5) of degrading generates the unsaturated trisaccharide and a small amount of unsaturated disaccharides of equivalent later；

(2) unsaturated tetrose (UDP4) of degrading generates the unsaturated trisaccharide and a small amount of unsaturated disaccharides of equivalent later；

(3) the unsaturated disaccharides of equivalent is generated after micro unsaturated trisaccharide (UDP3: containing only the end Δ G substrate) of degrading, Degradation rate about 5%；

(4) unsaturated trisaccharide (UDP3: Δ M and the end Δ G mixed substrates) of degrading generates the unsaturated disaccharides of equivalent later, Degradation rate about 60%；

(5) the unsaturated disaccharides (UDP2: Δ G) of micro degradation generates unsaturated monosaccharide Δ, degradation rate about 5%；

(6) unsaturated disaccharides (UDP2: Δ M) of degrading generates unsaturated monosaccharide Δ, degradation rate about 90%；

(7) front and back is reacted with saturation disaccharides (MM and GG) without significant changes.

These the result shows that: rAly-6 can effectively degrade the oligose fragment of trisaccharide and the above size；The enzyme cannot degrade full With disaccharides GG or MM；Unsaturated two bglii fragments (Δ M or Δ G) are the minimum oligosaccharide substrates for recombinating algin catenase rAly-6, Wherein Δ M is easier to be degraded than Δ G, but the two cannot be completely degraded；Saturation monosaccharide M, G or unsaturated monosaccharide Δ etc. are Recombinate the minimum product oligosaccharides type of algin catenase rAly-6.In conclusion recombination algin catenase rAly-6 is one The kind tendentious algin catenase of M, the above size of trisaccharide that is easy to degrade or containing the end Δ M sugar chain substrate, but be not easy In degradation trisaccharide, the especially sugar chain substrate containing the end Δ G, GG or MM of disaccharides size.

Embodiment 15, high performance liquid chromatography (HPLC) analysis for recombinating algin catenase rAly-6 enzyme cleavage patterns

Take the solution that poly- M pentasaccharides (M5), poly- G pentasaccharides (G5) are saturated containing 30 μ g, the HAc-NaAc (pH 6.0) of 150mmol/L The dilution and water of buffer, embodiment 3 recombination algin catenase rAly-6 obtained, mix according to volume ratio 2:1:3:4, It is placed in 40 DEG C of water-baths and reacts 12h.Reaction system is set into 10min in boiling water bath, goes to ice-water bath 5min, 12,000 × g, 4 At least 15min is centrifuged under the conditions of DEG C.Supernatant is collected, the oligosaccharides catabolite as recombination algin catenase rAly-6.With pre- The recombination algin catenase rAly-6 enzyme solution first inactivated in boiling water bath, does negative control reaction.

According to chromatographic condition described in embodiment 10, recombination algin catenase rAly-6 enzymatic hydrolysis is saturated poly- M pentasaccharides (M5), the sample of poly- G pentasaccharides (G5) loads 20 μ g/ samples with autosampler, and other conditions are constant, 235nm detection.With HPLC operating software analyzes the integral area of each oligosaccharide compositions, calculates relative molar concentration.Referring to molecular weight standard object, determine The relative molecular weight of each oligosaccharides.

Such as Figure 14, recombinates after algin catenase rAly-6 degradation is saturated poly- M pentasaccharides (M5) and generate unsaturated trisaccharide UM3 With unsaturated disaccharides UM2.As shown in figure 15, recombination algin catenase rAly-6 degradation generates after being saturated poly- G pentasaccharides (G5) Unsaturated trisaccharide UG3 and unsaturated disaccharides UG2.The remnants of this disaccharides cannot thoroughly degrade disaccharides with the enzyme shown in Figure 13 The result of Δ M or Δ G are consistent.However, series of oligosaccharides product caused by rAly-6 degradation M5, the absorption value at 235nm Intensity (area integral) is 8-10 times of degradation G5 product.This sufficiently shows: recombinase rAly-6 can degrade the oligosaccharides rich in M Segment, can also degrade the oligose fragment rich in G, but have M- tendentiousness on the whole.

Embodiment 16, fluorescence-high performance liquid chromatography (HPLC) analysis for recombinating algin catenase rAly-6 enzyme cleavage patterns

The unsaturated pentasaccharides for taking the poly- M pentasaccharides (M5) of the saturation containing 10 μ g, the solution of poly- G pentasaccharides (G5), embodiment 10 to prepare (UDP5), rotation is evaporated.Dimethyl sulfoxide (DMSO) solution containing excessive anthranilamide (2-AB), boron nitrilation sodium is added, It mixes in 60 DEG C of water-baths of postposition and incubates 2h.Rotation is evaporated, and 500 μ L deionized water dissolving samples is added, by sample and 200 μ L chloroforms Resonance is swung, and supernatant is collected in centrifugation.Continue to be extracted repeatedly with chloroform, no less than 7 times, obtains reducing end under neutral and be fluorescently labeled The poly- M pentasaccharides (2AB-M5) of saturation, the poly- G pentasaccharides (2AB-G5) of saturation of fluorescent marker, fluorescent marker unsaturated pentasaccharides (2AB- UDP5)。

Take above-mentioned 2AB-M5,2AB-G5,2AB-UDP5 sample, embodiment 3 are obtained to recombinate algin catenase rAly-6 Dilution, 150mmol/L HAc-NaAc (pH 6.0) buffer and water, according to volume ratio 2:1:3:4 mix, be placed in 40 DEG C 12h is reacted in water-bath.Reaction system is set into 10min in boiling water bath, goes to ice-water bath 5min, 12,000 × g, under the conditions of 4 DEG C It is centrifuged at least 15min.Supernatant is collected, the oligosaccharides catabolite as recombination algin catenase rAly-6.In advance in boiling water The recombination algin catenase rAly-6 enzyme solution that 10min is heated in bath, does negative control reaction.

According to chromatographic condition described in embodiment 10, by 2AB-M5,2AB-G5,2AB-UDP5 and its label of enzymolysis product Sample loads 50-200ng/ sample with autosampler, and other conditions are constant, 330nm excitation, 420nm detection.It is grasped with HPLC Make software, analyze the integral area of each oligosaccharide compositions, calculates relative molar concentration.Referring to molecular weight standard object, each oligosaccharides is determined Relative molecular weight.

As shown in figure 16, under the above conditions, it after 2AB-M5 is degraded, with the increase of enzyme digestion reaction time, gradually gives birth to At 2AB-UM4,2AB-UM3,2AB-UM2, and ultimately forming oligosaccharides final product is 2AB-UM2, and relative amount finally tends to be steady It is fixed.This show recombinate algin catenase rAly-6 be to cut 2AB-M5 in a manner of monosaccharide excision enzyme, from non reducing end by Step one molecule of cutting is saturated monosaccharide M, two molecule unsaturation monosaccharide Δs, until one molecule 2AB-UM2 of final residue.

It is also shown in FIG. 17, after being reacted with excessive recombination algin catenase rAly-6 with 2AB-G5, about 45% bottom Object is degraded, and generates the 2AB-UG2 of equimolar amounts.After 2AB-G5 is degraded, with the increase of enzyme digestion reaction time, gradually give birth to At 2AB-UG4,2AB-UG3,2AB-UG2, and ultimately forming oligosaccharides final product is 2AB-UG2, and relative amount finally tends to be steady It is fixed.This show recombinate algin catenase rAly-6 be to cut 2AB-G5 in a manner of monosaccharide excision enzyme, from non reducing end by Step one molecule of cutting is saturated monosaccharide G, two molecule unsaturation monosaccharide Δs, until one molecule 2AB-UG2 of final residue.

As shown in figure 18, after being reacted with excessive recombination algin catenase rAly-6 with 2AB-UDP5, as enzymatic hydrolysis is anti- Increase between seasonable, increasingly generates 2AB-UDP4,2AB-UDP3,2AB-UDP2, and ultimately forming oligosaccharides final product is 2AB- UDP2, relative amount finally tend towards stability.This shows that recombinating algin catenase rAly-6 is cut in a manner of monosaccharide excision enzyme A molecule 2AB-UDP5 is cut, three molecule unsaturation monosaccharide Δs are gradually cut from non reducing end, until a final remaining molecule 2AB-UDP2。

Further, comparison diagram 14 with Figure 16's as a result, and Figure 15 and Figure 17's as a result, explanation: (1) 2-AB is to saturation Pentasaccharides end mark, it is suppressed that enzyme rAly-6 to the degrading activity of oligosaccharides, especially make enzyme can not degrade after 2-AB label two Sugared Δ M；(2) rAly-6 is monosaccharide circumscribed-type algin catenase, is greater than polyG to the Preference of polyM.

Bibliography involved in specification:

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Sequence table

<110>Jinan Wu Tong biotechnology Co., Ltd

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cagtgtttaa acgatgccaa ttggttggtt tatacaagtc aggcgtacag ttcgatttac 480

gaatggttgg atgctggtac tagagataaa ttaaacaagg aattatttag accttatgct 540

gatttcttat ctgtacaaac gcctcaattc tttaaccgtc tgcataacca tagtacttgg 600

gcaaatgcag cagtaggtat gattggtttg gtcatacacg atgaagattt aattcagaaa 660

tctttatacg gtcttccttt tgaaaaccta tacgcaaaag acaacgatgg aggagatatt 720

attagagctg atcagaaaaa agcgggcttc cttgccaaca tcgatcatgc tttttctcct 780

gatggatatt atttcgaagg tccctactac caaagatatg cgatgtatcc tttcttgatg 840

tatgcggtag cattggatag aaataaaaag gaacttgata ttttcaacta cgacaataag 900

atccttctaa aaggtatcga aacgcttgtt cagttatcga atgagaaagg ggaattcttc 960

ccattaaacg atgcacaaaa aggaatgtct tactattcaa gagaattgaa actagccatt 1020

tctatcgctt acgaacacgg tgatgtacat agaaatggtc ttttatcgat tgtaaaagaa 1080

ttgaacgcag ttcctctaaa ctatggtggt ttcttagaat caaatgctat tgcacagggt 1140

aaagcaacga cttaccaaaa aagaagtttg ttggtcactg atggtaataa aggtgataaa 1200

ggagcgatag gcattttaag aggaagccgt caggagtaca tcatgaagtt tacccaacac 1260

ggtatgggac atggtcattt cgaccgcctt tcttatttga tgtacaataa aggagatgag 1320

gtgttacaag attatggtgc tgctcgttat gtcaacatca accaaaaaga tggtggcgga 1380

tacttaaaag aaaataaaac atgggcgaaa caaactgtag ctcataacac cttagtgatt 1440

ggcgatcgta cgcaatgcaa tgccaaggtt gaaaaagcgg aatcggctac tcctgaattg 1500

tattacttca atgctgagga tgaaaatgtc cagatcatga gtgctaaaga cagtattgct 1560

tatgatggcg ttcatcaaca aagaacttta gtattaatta aagacaatgc atttgaaaat 1620

cctttattac ttgatttatt ccgagcagac cttccttctg atcagaaatt aagaatgcct 1680

tataattata aaggtcagat gatgtattct actttcgata atcaaccatt aaattcttta 1740

ccggtgatgg gtaaaaaaga tggtttccaa tatttatgga aagtaggtga agcgccatcc 1800

aaaggggatt ttaatcaggt caactggttt aacaataaag tgttctatac actcaatatg 1860

gccacttctg cagaggacag catcaatttt gttcttcttg gcgccaacga tcctaaaatg 1920

aaccttagaa acgacaaggc tttccactgg ttaaaaacag gtaaaaaagg aagtaattta 1980

tatgcatccg tattagaaac tcacggaggt tattcttaca tcacggagtt ggctcctcac 2040

acgtacagta gtattaaaaa tgtaaatgta gtgtacaata ccaatgcgta ttctgctgtg 2100

actttcgaaa ataaagaagg caagaaatgg agatttataa tgtgcaataa tgatagctcc 2160

tcttcatcaa aacataaatt gaaaataggt gagactacct taaaatggaa aggggtttac 2220

ctattggaag aaatttaa 2238

<210> 2

<211> 745

<212> PRT

<213>heat color bacillus (Flammeovirga yaeyamensis)

<400> 2

Met Lys Met Lys Asn Asn Leu Tyr Thr Ala Val Leu Leu Ile Leu Leu

1 5 10 15

Ser Val Ser Asn Val Phe Ser Gln Asn Gly Asn Leu Ile Thr Ser Glu

20 25 30

Ala Gln Leu Gln Glu Met Lys Leu Ala Leu Lys Ser Asp Asn Leu Phe

35 40 45

His Ser Ala Val Asn Asp Ala Ile Glu Glu Val Ala Pro Tyr Leu Lys

50 55 60

Ser Gly Leu Asp Val Pro Val Pro Lys Asp Leu Ala Gly Gly Tyr Thr

65 70 75 80

His Ser Gln His Lys Leu Asn Tyr Thr Ile Met Gln Lys Ala Gly Met

85 90 95

Leu Tyr Gln Leu Thr Gly Asn Glu Gln Tyr Ala Glu Leu Ile Lys Asn

100 105 110

Thr Leu Leu Lys Tyr Ala Glu Leu Phe Pro Thr Phe Asp Arg His Pro

115 120 125

Ala Thr Arg Ser Tyr Ser Pro Gly Lys Phe Phe Trp Gln Cys Leu Asn

130 135 140

Asp Ala Asn Trp Leu Val Tyr Thr Ser Gln Ala Tyr Ser Ser Ile Tyr

145 150 155 160

Glu Trp Leu Asp Ala Gly Thr Arg Asp Lys Leu Asn Lys Glu Leu Phe

165 170 175

Arg Pro Tyr Ala Asp Phe Leu Ser Val Gln Thr Pro Gln Phe Phe Asn

180 185 190

Arg Leu His Asn His Ser Thr Trp Ala Asn Ala Ala Val Gly Met Ile

195 200 205

Gly Leu Val Ile His Asp Glu Asp Leu Ile Gln Lys Ser Leu Tyr Gly

210 215 220

Leu Pro Phe Glu Asn Leu Tyr Ala Lys Asp Asn Asp Gly Gly Asp Ile

225 230 235 240

Ile Arg Ala Asp Gln Lys Lys Ala Gly Phe Leu Ala Asn Ile Asp His

245 250 255

Ala Phe Ser Pro Asp Gly Tyr Tyr Phe Glu Gly Pro Tyr Tyr Gln Arg

260 265 270

Tyr Ala Met Tyr Pro Phe Leu Met Tyr Ala Val Ala Leu Asp Arg Asn

275 280 285

Lys Lys Glu Leu Asp Ile Phe Asn Tyr Asp Asn Lys Ile Leu Leu Lys

290 295 300

Gly Ile Glu Thr Leu Val Gln Leu Ser Asn Glu Lys Gly Glu Phe Phe

305 310 315 320

Pro Leu Asn Asp Ala Gln Lys Gly Met Ser Tyr Tyr Ser Arg Glu Leu

325 330 335

Lys Leu Ala Ile Ser Ile Ala Tyr Glu His Gly Asp Val His Arg Asn

340 345 350

Gly Leu Leu Ser Ile Val Lys Glu Leu Asn Ala Val Pro Leu Asn Tyr

355 360 365

Gly Gly Phe Leu Glu Ser Asn Ala Ile Ala Gln Gly Lys Ala Thr Thr

370 375 380

Tyr Gln Lys Arg Ser Leu Leu Val Thr Asp Gly Asn Lys Gly Asp Lys

385 390 395 400

Gly Ala Ile Gly Ile Leu Arg Gly Ser Arg Gln Glu Tyr Ile Met Lys

405 410 415

Phe Thr Gln His Gly Met Gly His Gly His Phe Asp Arg Leu Ser Tyr

420 425 430

Leu Met Tyr Asn Lys Gly Asp Glu Val Leu Gln Asp Tyr Gly Ala Ala

435 440 445

Arg Tyr Val Asn Ile Asn Gln Lys Asp Gly Gly Gly Tyr Leu Lys Glu

450 455 460

Asn Lys Thr Trp Ala Lys Gln Thr Val Ala His Asn Thr Leu Val Ile

465 470 475 480

Gly Asp Arg Thr Gln Cys Asn Ala Lys Val Glu Lys Ala Glu Ser Ala

485 490 495

Thr Pro Glu Leu Tyr Tyr Phe Asn Ala Glu Asp Glu Asn Val Gln Ile

500 505 510

Met Ser Ala Lys Asp Ser Ile Ala Tyr Asp Gly Val His Gln Gln Arg

515 520 525

Thr Leu Val Leu Ile Lys Asp Asn Ala Phe Glu Asn Pro Leu Leu Leu

530 535 540

Asp Leu Phe Arg Ala Asp Leu Pro Ser Asp Gln Lys Leu Arg Met Pro

545 550 555 560

Tyr Asn Tyr Lys Gly Gln Met Met Tyr Ser Thr Phe Asp Asn Gln Pro

565 570 575

Leu Asn Ser Leu Pro Val Met Gly Lys Lys Asp Gly Phe Gln Tyr Leu

580 585 590

Trp Lys Val Gly Glu Ala Pro Ser Lys Gly Asp Phe Asn Gln Val Asn

595 600 605

Trp Phe Asn Asn Lys Val Phe Tyr Thr Leu Asn Met Ala Thr Ser Ala

610 615 620

Glu Asp Ser Ile Asn Phe Val Leu Leu Gly Ala Asn Asp Pro Lys Met

625 630 635 640

Asn Leu Arg Asn Asp Lys Ala Phe His Trp Leu Lys Thr Gly Lys Lys

645 650 655

Gly Ser Asn Leu Tyr Ala Ser Val Leu Glu Thr His Gly Gly Tyr Ser

660 665 670

Tyr Ile Thr Glu Leu Ala Pro His Thr Tyr Ser Ser Ile Lys Asn Val

675 680 685

Asn Val Val Tyr Asn Thr Asn Ala Tyr Ser Ala Val Thr Phe Glu Asn

690 695 700

Lys Glu Gly Lys Lys Trp Arg Phe Ile Met Cys Asn Asn Asp Ser Ser

705 710 715 720

Ser Ser Ser Lys His Lys Leu Lys Ile Gly Glu Thr Thr Leu Lys Trp

725 730 735

Lys Gly Val Tyr Leu Leu Glu Glu Ile

740 745

Claims

1. a kind of tendentious monosaccharide circumscribed-type algin catenase Aly-6 of M, amino acid sequence is as shown in SEQ ID NO.2.

2. a kind of tendentious monosaccharide circumscribed-type algin catenase encoding gene aly-6 of M, nucleotide sequence such as SEQ IDNO.1 It is shown.

3. it is brown to insert the tendentious monosaccharide circumscribed-type of M as claimed in claim 2 in expression vector for a kind of recombinant expression carrier The encoding gene aly-6 of phycocolloid lyases Aly-6.

4. recombinant expression carrier as claimed in claim 3, which is characterized in that the expression vector is selected from: Bacillus coli expression Carrier, Yeast expression carrier, hay bacillus expression vector, lactic acid bacteria expression vectors, streptomyces expression vector, phage vector, Filamentous fungi expression vector, plant expression vector, insect expression vector or mammalian cell expression vector.

5. a kind of recombinant bacterium has been transferred to the weight of the tendentious monosaccharide circumscribed-type phycocolloid lyases Aly-6 of above-mentioned M in host cell Group expression vector, or the above-mentioned tendentious monosaccharide circumscribed-type algin catenase Aly-6 of M of expression.

6. recombinant bacterium as claimed in claim 5, which is characterized in that host cell is selected from: e. coli host cell, saccharomycete Host cell, hay bacillus host cell, lactic acid bacteria host cell, actinomyces host cell, filamentous fungal host cell, insect Cell or mammalian cell.

7. the encoding gene aly-6 of the tendentious monosaccharide circumscribed-type algin catenase Aly-6 of M described in claim 2, right are wanted Ask recombinant bacterium described in 3 recombinant expression carriers, claim 4 in the preparation tendentious monosaccharide circumscribed-type algin catenase of M Application in rAly-6.

8. the tendentious monosaccharide circumscribed-type algin catenase Aly-6 of M described in claim 1 is in thoroughly degradation algin or degradation Algin oligosaccharide produces the application in unsaturated trisaccharide and unsaturated tetrose.

9. the tendentious monosaccharide circumscribed-type algin catenase Aly-6 of M described in claim 1 is in endless degradable algin or drop Solution algin oligosaccharide production non-reducing end contains in the unsaturated oligose fragment of biggish series and unsaturated monosaccharide Δ of the end Δ G Application.