Disaccharide exo-cleavage type beta-mannase hydrolase and application thereof

文档序号：1948423 发布日期：2021-12-10 浏览：17次中文

阅读说明：本技术 一种二糖外切型β-甘露聚糖水解酶及其应用 (Disaccharide exo-cleavage type beta-mannase hydrolase and application thereof ) 是由韩文君宓延红程媛媛古静燕胡玮张真庆于 2020-06-10 设计创作，主要内容包括：本发明涉及一种外切型β-甘露聚糖水解酶,该酶的氨基酸序列如SEQ ID NO:2所示。该酶既能降解葡甘聚糖,也能降解半乳甘露聚糖,其最终主产物是还原端以甘露糖单元为主的一系列寡糖。该酶降解纯甘露聚糖时,从非还原端开始,以甘露二糖(M2)为单元持续切割糖链,因此该酶适合专一性制备甘露二糖。此外,该酶的寡糖终产物甘露二糖或甘露糖的摩尔数与底物糖链的摩尔数一致,因此该酶具有分子计数器的功能。(The invention relates to an exo-type beta-mannanohydrolase, the amino acid sequence of which is shown in SEQ ID NO. 2. The enzyme can degrade glucomannan and galactomannan, and the final main product is a series of oligosaccharides with mannose unit as main reducing end. When the enzyme degrades pure mannan, the enzyme continuously cuts a sugar chain by taking the mannose (M2) as a unit from a non-reducing end, so the enzyme is suitable for specifically preparing the mannose. In addition, the number of moles of mannobiose or mannose, which is an oligosaccharide end product of the enzyme, coincides with the number of moles of sugar chains, which are substrates, and therefore the enzyme has a function of a molecular counter.)

1. An exo-type beta-mannase, the amino acid sequence of which is shown in SEQ ID NO 2.

2. The exo-type β -mannanase encoding nucleotide sequence of claim 1.

3. The nucleotide sequence of claim 2, wherein the nucleotide sequence is set forth in SEQ ID NO 1.

4. An expression vector comprising the nucleotide sequence of claim 2 or 3.

5. A host cell comprising the expression vector of claim 4 or having the nucleotide sequence of claim 2 or 3 integrated into its genome.

6. Use of the exo-beta-mannanase according to claim 1 for degrading mannan.

7. Use of the exo-type β -mannanase according to claim 1 for analysis, determination of the number of moles of sugar chains of its original polysaccharide substrate and judgment of the parity of the original substrate.

8. An exo-beta-mannanase which is the exo-beta-mannanase mutant of claim 1.

9. The exo-type β -mannanase according to claim 8 which has the amino acid sequence shown in SEQ ID NO 2, but has a mutation at position 188 and/or 296.

10. A mannan degradation product prepared by degrading mannan with the exo-type β -mannanase of claim 1.

Technical Field

The invention relates to the field of biotechnology; specifically, the invention relates to disaccharide exo-mannase, and a coding gene and application thereof.

Background

Hemicellulose is a structural polysaccharide that makes up the cell wall of plants, usually tightly bound to cellulose and lignin, forming lignocellulosic biomass. Among the many constituent types of hemicellulose, two polysaccharides, mannan (mannan) and xylan (xylolan), are closely related to human life, and they are mainly used in the industrial fields of food, medicine, textile, pulp and paper, biofuel, and the like. Mannan is hemicellulose with the second highest content in nature, and is a kind of polysaccharide with a complex structure, and can be divided into four types according to the difference of the composition structure of sugar units: pure mannan (pure mannan), glucomannan (glucomannan), galactomannan (galactomannan), and galactoglucomannan (galactoglucanan).

In addition to the common characteristic that mannose units are used as main components of the mannans, the polysaccharide skeleton structure of the mannans also has the complex characteristics of random heteropolymerization of various sugar units, local composition modularization and the like, and the side chain structure of the mannans also has similar characteristics. Thus, to completely degrade these glycans requires the synergistic action of multiple types of enzymes (enzyme systems).

Compared with a chemical or physical method, the strategy for preparing the oligosaccharide by degrading the mannan polysaccharide by the enzyme method has the advantages of mild reaction conditions, strong controllability, clear product due to clear substrate selectivity and the like. Therefore, the mannanase as a tool enzyme for preparing the oligosaccharide has important research and development values and economic values. At present, the beta-mannase is widely applied to various fields of food, animal breeding, paper making, biofuel, oil drilling and the like. In the food industry, the beta-mannase is used as a feed additive, can act on mannan in food and assist in producing mannan oligosaccharide prebiotics. In the detergent industry, many types of household products and food products, such as hair sprays, shampoos, conditioners, toothpastes, ice creams, and barbequing pastes, contain certain amounts of mannan components as thickeners or stabilizers, which are often difficult to remove once they form stains. If the treatment is performed by hydrolyzing mannan with beta-mannanase, the oligosaccharide fragments become easily water-soluble from the poorly soluble polysaccharides, thereby making it easier to remove stains. In the paper, pulp and paper industries, the use of beta-mannanase can facilitate the removal of lignin from pulp, produce results comparable to alkaline pretreatment, and can reduce environmental pollution to a large extent.

At present, although there are a lot of patent applications and scientific research documents related to beta-mannanase, there are relatively few basic researches on a substrate selection mechanism, a degradation mode of a polysaccharide/oligosaccharide substrate, a generation characteristic of an oligosaccharide product, and an intrinsic connection between the oligosaccharide product and a catalytic mechanism of the beta-mannanase. This deficiency creates two industry difficulties: (1) at present, although a certain amount of mannanase products are already industrialized and commercialized, the number and types of tool enzymes which can be accurately applied are extremely rare; (2) the deep development and the directed modification of enzyme resources lack guiding theories and technical references, which limits the development level of tool enzymes to a certain extent.

In order to meet the important application requirements of the tool-type mannan exonuclease, the research and exploration of the related aspects and levels such as increasing the development of new enzyme resources, enhancing the core application value and application characteristic analysis of the enzyme, discussing new guidance theory and technical approach required by molecular enzymology modification and the like are needed to be comprehensively developed.

Disclosure of Invention

Aiming at the difference of the prior art, the invention provides a disaccharide excision-type beta-mannanohydrolase Man02066, and a coding gene and application thereof.

In a first aspect, the invention provides an exo-type beta-mannanase, the amino acid sequence of which is shown in SEQ ID NO 2.

In a preferred embodiment, the exo-type β -mannanase degrades mannan having sugar chain branches or linear mannan without sugar chain branches; preferably, the exo-type β -mannanase degrades linear mannans without sugar chain branching.

In a preferred embodiment, the mannan is glucomannan, galactomannan, pure mannan; pure mannans are preferred.

In a preferred embodiment, the exo-type β -mannanase is capable of degrading konjac-derived glucomannan (KGM), locust bean gum-derived galactomannan (LBG); preferably, the polysaccharide substrate of the exo-type β -mannanase is konjac-derived glucomannan.

In a preferred embodiment, the exo-type β -mannanase consists of only one Glycoside carbohydrate family 26domain module.

In a preferred embodiment, the reaction temperature for degrading mannan by the exo-type beta-mannase is 0-40 ℃ and the pH is 6.0-8.0.

In a preferred embodiment, the optimum temperature for the endo-beta-mannanase to degrade Konjac Glucomannan (KGM) and Locust Bean Gum (LBG) is 40 ℃ and the optimum pH is 6.0.

In a preferred embodiment, the exo-type β -mannanase may also degrade mannooligosaccharides, the smallest substrate capable of degradation being mannotriose (M3) and the smallest product being mannomonosaccharide (M).

In a second aspect, the invention provides a nucleotide sequence encoding the exo-type β -mannanase of the first aspect.

In a specific embodiment, the nucleotide sequence is set forth in SEQ ID NO 1.

In a third aspect, the present invention provides an expression vector comprising the nucleotide sequence of the second aspect.

In a fourth aspect, the present invention provides a host cell comprising an expression vector according to the third aspect or a genome thereof into which has been integrated a nucleotide sequence according to the second aspect.

In a preferred embodiment, the host cell is a host cell for the production of the exo-type β -mannanase or for the degradation of β -mannans.

In a fifth aspect, the invention provides the use of the exo-type β -mannanase of the first aspect for degrading mannan.

In a preferred embodiment, the mannans comprise sugar chain branched mannans or linear mannans without sugar chain branches; preferably, the mannan is a linear mannan without sugar chain branches.

In a preferred embodiment, the mannan is glucomannan, galactomannan, pure mannan; pure mannans are preferred.

In a sixth aspect, the present invention provides the use of the exo-type β -mannanase of the first aspect for the analysis, determination of the number of moles of sugar chains of its original polysaccharide substrate and determination of the parity of the original substrate.

In a seventh aspect, the invention provides an exo-type β -mannanase which is an exo-type β -mannanase mutant according to the first aspect.

In a specific embodiment, the amino acid sequence of the exo-type β -mannanase is as shown in SEQ ID NO 2, but with mutations at positions 188 and/or 296.

In a preferred embodiment, the amino acid at position 188 of the amino acid sequence of the excised β -mannanase is mutated from glutamic acid to alanine and/or amino acid 296 is mutated from glutamic acid to alanine.

In an eighth aspect, the present invention provides a mannan degradation product prepared by degrading mannan with the exo-type β -mannanase of the first aspect.

In a preferred embodiment, the mannan is a mannan containing sugar chain branches or a linear mannan without sugar chain branches; preferably, the mannan is a linear mannan without sugar chain branches.

In a preferred embodiment, the mannan is glucomannan, galactomannan, pure mannan; pure mannans are preferred.

In a preferred embodiment, the mannan is konjac-derived glucomannan (KGM), locust bean gum-derived galactomannan (LBG); preferably, the mannan is konjac-derived glucomannan.

In a ninth aspect, the invention also provides the application of the excision-type beta-mannase and the mutant enzyme thereof as tool enzymes in researching the catalytic mechanism of the mannase.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 is a diagram showing the result of analysis of the structure of circumscribed-type beta-mannase Man02066 functional modules;

FIG. 2 is a gel electrophoresis diagram of polyacrylamide gel with recombinant excised beta-mannase rMan02066 expression and purification; in the figure: m, protein molecular weight standard, wherein the size of the bands from top to bottom is 116kDa, 66.2kDa, 45kDa, 35kDa, 25kDa, 18.4kDa and 14.4 kDa; lane 1, control strain before cell wall breaking, loading 10. mu.L; lane 2, the recombinant bacteria before wall breaking, 2. mu.L of loading amount; lane 3, wall-broken supernatant of the recombinant bacteria, and 2. mu.L of sample loading amount; lane 4, rMan02066 purified by nickel column, loading 2 μ L;

FIG. 3 is a graph showing the effect of temperature on the activity of recombinant β -mannanase rMan02066 in degrading konjac glucomannan (A) and locust bean gum (B);

FIG. 4 is a graph showing the effect of pH on the stability of the recombinant β -mannanase rMan02066 in degrading konjac glucomannan (A) and locust bean gum (B);

FIG. 5 is a graph showing the effect of temperature on the stability of recombinant β -mannanase rMan02066 in degrading konjac glucomannan (A) and locust bean gum (B);

FIG. 6 is a graph showing the effect of pH on the activity and stability of recombinant β -mannanase rMan02066 in degrading konjac glucomannan (A) and locust bean gum (B);

FIG. 7 is a bar graph showing the effect of metal ions and chemical reagents on the activity of recombinant β -mannase rMan02066 in degrading konjac glucomannan (A) and locust bean gum (B);

FIG. 8 is a TLC analysis chart of oligosaccharide products in the process of degrading konjac glucomannan (A) and locust bean gum (B) by recombinant beta-mannase rMan 02066; wherein, the A picture: m, mannose-mannose hexaose in abscissa; 1, comparison; 2-10, respectively represent: 10s, 1min, 10min, 1h, 4h, 6h, 24h, 48h and 72 h; and B, drawing: m, mannose-mannose hexaose in abscissa; 1, comparison; 2 to 9, respectively represent: 10s, 1min, 10min, 1h, 6h, 24h, 48h and 72 h;

FIG. 9 shows oligosaccharide fragments prepared by completely degrading konjac glucomannan (A) and locust bean gum (B) with recombinant β -mannanase rMan02066¹H-NMR chart;

FIG. 10 is a TLC analysis chart of the oligosaccharide products of recombinant β -mannanase rMan02066 completely degrading the series of large and small mannooligosaccharides (M-M6); wherein, the A picture: in the abscissa: a: sample sequence: m: M1-M6; 1: M1 (-); 2: M1 (+); 3: M2 (-); 4: M2 (+); 5: M3 (-); m3 (+); 7: M4 (-); 8: M4 (+); 9: M5 (-); m5 (+); 11: M6 (-); m6 (+); wherein (-) is a negative control group and (+) is an experimental group; and B, drawing: in the abscissa: m, M1-M6; 1: comparison; 2: diluting the enzyme solution by 10 times and reacting for 10 s; 3-8:10s, 1min, 30min, 3h, 12h and 48 h; and (C) diagram: in the abscissa: m, M1-M6; 1, comparison; 2, diluting the enzyme solution by 10 times and reacting for 10 s; 3-5, 10s, 1min and 48 h;

FIG. 11 is a graph of the analysis of the final main product of degradation of 2 AB-labeled large and small series of mannooligosaccharides (2 AB-M. about.2AB-M6) with recombinant β -mannanase rMan02066 (A), HPLC analysis of the oligosaccharide products of degradation of 2 AB-labeled mannopentaose (2AB-M5) (B) and 2 AB-labeled mannohexaose (2AB-M6) (C) at different reaction times (fluorescence);

FIG. 12 is a multiple sequence alignment of the β -mannanase Man02066 with a GH26 family β -mannanase;

FIG. 13 is a relative activity analysis of the beta-mannase Man02066 mutant degrading konjac glucomannan (A) and locust bean gum (B), respectively.

Detailed Description

The inventors have made extensive and intensive studies and have unexpectedly obtained a disaccharide exo-type β -mannanase Man02066 from a Pyrobacterium bacterium (Flammeovirga yaeyamensis) MY 04; the enzyme can degrade konjac glucomannan and other polysaccharide substrates such as locust bean gum, and has certain thermal stability (0-40 ℃), so that the enzyme can be used for the specific preparation of mannobiose. The present invention has been completed based on this finding.

All scientific or technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For clarity, some terms or concepts used herein are defined or explained as follows. The following definitions or explanations are provided merely to better understand the spirit of the present invention for the skilled person and are not intended to show the scope of the present invention in any way.

Beta-mannanase

As used herein, "beta-mannanase" has the meaning conventionally understood by those skilled in the art and refers to a class of hydrolases capable of hydrolyzing mannooligosaccharides and mannans (including mannans, glucomannans, galactomannans, and galactomannans) containing beta-1, 4 mannosidic linkages.

Depending on the characteristics of the substrate selectivity and the catalytic pattern of the enzyme, the β -mannanases can be further classified into endo- β -mannanases (EC 3.2.1.78) and exo- β -mannanases (EC 3.2.1.100). Wherein, the endo-type beta-mannase can hydrolyze beta-1, 4 glycosidic bonds in the mannan backbone to generate a series of mannose oligose fragments with different polymerization degrees; while exo-type β -mannanase is a product of sugar units, such as mannobiose, that act continuously from the non-reducing chain ends on the β -1,4 glycosidic bonds in the substrate sugar chains, cleaving and producing a fixed size. The beta-mannanase is widely present in natural organisms and comprises: animals, plants and microorganisms. Intensive research shows that microorganisms are the main source for producing the beta-mannanase, particularly bacteria, fungi, actinomycetes and the like. In contrast, animal and plant derived β -mannanases are less and are mainly distributed in the seeds of leguminous plants and in the digestive juices of mollusks.

At present, the research on exo-mannase at home and abroad is weak as a whole, compared with endo-mannase, the research literature or patent application number is less, and: (1) up to now, the endo-mannanases reported in the literature have hundreds, and are mainly classified into glycosyl hydrolase families GH5, GH26, GH113 or GH34 as recorded in the carbohydrate-active enzyme database (CAZy, http:// www.cazy.org /), while the exo-mannanases have few and are classified into GH 26. Among The members of The GH26 family, only 2 exo-type mannanohydrolases have been systematically identified, including BfMan26A (K. Kawaguchi, T. senoura, S. Ito, T. Taira, H. Ito, J. Wataki, S. Ito, The mango-forming exo-manna-in a new mannan carboxylic acid pathway in Bacillus fragrans, Arch. Microbiol.196(2014)17-23) from Bacteroides fragi NCTC 9343 and CjMan26C (K. Kawaguchi, T.Senoura, S.Ito, T.Tairi, H. Ito, J. sanmanni, S. bulking, S. nectar. 17-23) from Cellvibri japonica 107. In contrast, members of the GH26 family, still harbor endo-mannanohydrolase enzymes, such as CjMan26A (Ducros, V., Zechel, D.L., Murshudov, G., Gilbert, H.J., Szabo, L., Stoll, D., Withers, S.G., Davies, G.J, (2002) Angel Chem Int Engl 41:2824), Boman26B (Bagener, V., Wiemann, M., Reddy, S.K., Bhatcharya, A., Rosegren, A., D.T., Stalbrand, H.O. 919) Biol 20100, Bacillus subtilis, Kriginospora, Krigi, S1, Kligin, S.K., Bhattacharya, Hakkeri, Ka, Katsubak, K, Bacillus, Kliginospora, Hakkeri Uigun, K, Hakkeri, No. 21, Bacillus, Hakkeri, No. 21, No. 7, No.2, No. 7, No. 2. host, No. 7, No. 2. 7, No.2, No. 7, No.2, No. 7, No.2, a. Therefore, in order to obtain more tool enzymes, it is necessary to increase the development of new resources of the exo-mannanase. (2) Second, analysis of GH26 familyThe recorded literature on exo-mannanases (EC3.2.1.100) only currently knows that BfMan26A and CjMan26C are typical of disaccharide exo-mannanase enzymes that cleave disaccharide units continuously from the non-reducing end of the substrate sugar chain to completely degrade mannan, and that the final oligosaccharide main product is mainly disaccharide. In contrast, BoMan26A of Bacteroides ovatus can cleave a disaccharide or trisaccharide unit from the non-reducing end of the substrate sugar chain, and finally give a mannobiose main product (S) ((S)) V,Reddy S K,Bouraoui H,et al. Galactomannan Catabolism Conferred by a Polysaccharide Utilization Locus of Bacteroides ovatus ENZYME SYNERGY AND CRYSTAL STRUCTURE OF A β-MANNANASE[J]Journal of Biological Chemistry,2017,292(1): 229-243). However, from the analysis of two catalytic properties, a substrate degradation pattern and an oligosaccharide production property, BoMan26A does not belong to the strict disaccharide exo-mannanase, and referring to the results of the study of the endonuclease Man01929 by the inventors of the present patent application, it is presumed that BoMan26A should be a typical endo-mannanase having a variable substrate degradation pattern. Furthermore, investigation of polysaccharide degradation patterns of RsMan26H from Reiticulture sites has also found that oligosaccharides with high degree of polymerization (DP5-DP20) accumulate continuously over time (10-30 min) for a short period of time (10min), and that mannopentaose and mannohexaose accumulate continuously over time (10-30 min), and finally (after 30min of reaction) mannotriose, mannobiose and mannose accumulate continuously, so that RsMan26H is reported as endo-mannanase (Tsukugoshi H, Nakamura A, Ishida T, et al.the GH 26. beta. -mannase RsMan26H from a systematic promoter of the terminal Reiticulture sites end-biological hydrolysis, 2014 27. and 525 Biochemical reaction: cellulose degradation: 520: 2014. Biochemical degradation: 520). The literature is not clear about the catalytic pattern and oligosaccharide-forming properties of uncultured bacterioides derived GH26 i. Therefore, in order to obtain tools, it is necessary to enhance the analysis and explanation of the core application value and characteristics of enzymes such as the substrate degradation pattern and oligosaccharide production characteristics of the enzymes. (3) The above-mentioned 5 members of the GH26 family of mannohydrolase share the common feature of specifically recognizing the glycosidic bond in mannose-mannose (MM), but whether their recognition, binding and degradation to the substrate is affected by galactosyl modifications and the specific contribution of key active site residues remains to be studied intensively.

Beta-mannanase of the invention

In this context, the terms "exo-type β -mannanase of the invention", "β -mannanohydrolase of the invention", "β -mannanase Man 02066" and the like have the same meaning.

In a specific embodiment, the amino acid sequence of the exo-type β -mannanase of the present invention is shown in SEQ ID NO 2. The exo-type beta-mannase can degrade mannan containing sugar chain branches and can also degrade linear mannan without sugar chain branches; however, linear mannans without sugar chain branches are the best substrates for the exo-type β -mannanase of the invention.

The exo-type beta-mannase has certain temperature tolerance, and the reaction temperature for degrading mannan is 0-40 ℃, preferably 20-40 ℃; in addition, the pH condition of the exo-type beta-mannase for degrading mannan is 6.0-8.0.

In a specific embodiment, the optimum temperature of the exo-type beta-mannanase of the invention for degrading Konjac Glucomannan (KGM) and Locust Bean Gum (LBG) is 40 ℃, and the optimum pH is 6.0.

When degrading mannan substrate, the exo-type beta-mannase can continuously cut sugar chain from a non-reducing end to the end by taking mannobiose as a single position, so that the exo-type beta-mannase can be used for the specific preparation of mannobiose.

The exo-type beta-mannanase of the present invention can also be used for analysis, determination of the number of moles of sugar chains of its original polysaccharide substrate and judgment of the parity of the original substrate.

On the basis of the exo-type beta-mannase, the inventor further finds out the conservative catalytic site of the exo-type beta-mannase, thereby laying a foundation for further modifying the enzyme.

In a specific embodiment, the conserved catalytic site of the exo-type β -mannanase of the invention is at positions 188 and 296.

In a preferred embodiment, the amino acid sequence of the excised β -mannanase has an amino acid mutation from glutamate to alanine at position 188 and/or from glutamate to alanine at position 296 to obtain an inactive mutant. Therefore, the exo-type beta-mannase and the mutant enzyme thereof can be used as tool enzymes to research the catalytic mechanism of the mannase.

The invention has the advantages of

1. The invention discloses a disaccharide excision-type beta-mannase Man02066 obtained by Flammeovirga yaeyamensis MY 04;

2. the enzyme Man02066 can degrade konjac glucomannan and other polysaccharide substrates such as locust bean gum;

3. the enzyme has certain thermal stability (0-40 ℃) and narrow pH tolerance (6.0-8.0);

3. when degrading a series of pure mannooligosaccharide substrates or pure mannooligosaccharide substrates with a reducing end being labeled by a fluorescent label, the enzyme takes mannobiose as a unit and continuously cuts a sugar chain from a non-reducing end until the sugar chain is finished, so the enzyme can be used for the specific preparation of the mannobiose;

4. the invention confirms that the catalytic site residues of the enzyme are Glu188 (acid/base catalysis) and Glu296 (nucleophilic catalysis), thereby laying a foundation for the research of the exo-type beta-mannase;

5. the enzyme can continuously cut M2 sugar from a non-reducing end until mannose (2AB-M2) or mannose (2AB-M) remains when degrading a series of mannooligosaccharide substrates of which the reducing ends are marked by anthranilamide, and when the final product is 2AB-M2, the corresponding original substrates are even number of sugar chains and the number of moles is equal to that of the original substrates; when the final product is 2AB-M1, the corresponding original substrate is an odd number of sugar chains and the number of moles is equal thereto. Therefore, theoretically, the enzyme or its recombinant enzyme can be used in combination with fluorescence labeling and analysis of sugar chain substrates to analyze and determine the number of moles of sugar chains of the original polysaccharide substrates, i.e., to serve as a molar counter for the polysaccharide substrates, and also to determine the parity of the original substrates.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures in the following examples, where specific conditions are not indicated, are generally carried out according to conventional conditions, or according to conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight.

Examples

Sources of Experimental materials

Strain pyrachrobacter (Flammeovirga yaeyamensis) MY04 is from the common microorganism center of the china committee for culture collection of microorganisms, address: the microbial research institute of China academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, with a preservation date of 2008, 11 months and 27 days, and a preservation number of CGMCC NO. 2777.

In the following examples, called T.pyralis MY04 for short.

The experimental materials referred to in the examples are not given a specific origin and are all common commercial products.

Example 1 extraction of genomic DNA of M.pyracantha MY04 Strain

Inoculating Flavobacterium furiosum MY04 into liquid medium YT04, culturing at 28 deg.C and 200rpm under shaking to 600nm absorbance (OD)₆₀₀) Is 1.2; taking 10mL of culture solution, centrifuging for 15min at 4 ℃ under the condition of 12,000 Xg, and collecting thalli precipitates; the cells were suspended in 10mL of lysozyme buffer (10mM Tris-HCl, pH 8.0), centrifuged at 12,000x g at 4 ℃ for 15min, and the pellet was collected.

The liquid culture medium YT04 comprises the following components per liter:

10g of tryptone, 5.0g of yeast extract and 30g of sodium chloride, and dissolving the components in water to obtain a constant volume of 1L and pH of 7.2.

Adding 6.0mL of lysozyme buffer solution into each tube of the thallus sediment to obtain about 7.0mL of bacterial liquid, and respectively adding 280 mu L of lysozyme solution with the concentration of 20mg/mL to ensure that the final concentration of lysozyme is 800 mu g/mL; placing in ice water bath for 1.0h, transferring to water bath at 37 ℃, and incubating for 2h until the reaction system is viscous; adding 0.41mL of 100mg/mL sodium hexadecylsulfonate solution and 30 μ L of 100mg/mL proteinase K solution, and incubating at 52 deg.C for 1.0 h; adding 7.5mL of Tris-balanced phenol/chloroform/isoamyl alcohol (volume ratio is 25: 24: 1), and mixing by gentle inversion; centrifuging at 4 deg.C for 10min at 10,000 Xg, collecting supernatant, adding 1.0mL NaAc-HAc (pH 5.2, 3.0M) buffer solution and 8.5mL anhydrous ethanol, and mixing well; picking out the filamentous DNA by using a gun head, transferring the filamentous DNA into a centrifugal tube of 1.5mL, washing for 2 times by using 70% ethanol (stored at-20 ℃), and discarding a supernatant after microcentrifugation; centrifuging at 10,000 Xg and 4 deg.C for 2min, and completely discarding supernatant; the DNA precipitate was air-dried in a sterile bench and then the genomic DNA was prepared by dissolving the DNA sample with sterile deionized water overnight at 4 ℃.

Example 2 scanning of the genome of strain My04 Pyrochrobacillus and sequence analysis thereof

The inventor finds a coding gene man02066 of beta-mannosidase in genomic DNA of the strain MY04 of the Flavobacterium sp prepared in example 1, the coding region of the gene man02066 is 1137bp in length, and the nucleotide sequence is shown in SEQ ID NO. 1. The beta-mannase Man02066 coded by the gene Man02066 contains 378 amino acids in total, and the amino acid sequence of the beta-mannase Man02066 is shown in SEQ ID NO. 2.

Example 3 recombinant expression of Gene man02066 in E.coli BL21(DE3) Strain

PCR amplification was performed using the genomic DNA prepared in example 1 as a template. The primer sequences are as follows:

forward primer for man02066 amplification

Man02066-F：5’-gcgCATATGGAAGATAAGGCAAAGACACC-3’(SEQ ID NO:3)；

Reverse primer for man02066 amplification

Man02066-F：5’-gcgCTCGAGATTCACTTTCATATTCATCAAATC-3’(SEQ ID NO:4)；

The forward primer is underlined the specific site for restriction endonuclease Nde I, and the reverse primer is underlined the specific site for restriction endonuclease Xho I.

The high fidelity DNA Polymerase Prime STAR HS DNA Polymerase was purchased from Dalibao, China, and the PCR reagents used were performed according to the product instructions provided by this company.

And (3) PCR reaction system:

2 × Primer star GC buffer 5. mu.L, amplified forward Primer 0.35. mu.L, amplified reverse Primer 0.35. mu.L, Template (1 ng/. mu.L) 1. mu.L, ddH₂O 3.3μL，polymerase 0.1μL，dNTP 0.8μL。

And (3) PCR reaction conditions:

pre-denaturation at 95 ℃ for 4 min; denaturation at 94 ℃ for 40s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 90s, and 35 cycles; extending for 10min at 72 ℃; stabilizing at 4 deg.C for 10 min.

The PCR product was digested with restriction enzymes Nde I and Xho I, and the digested PCR product was recovered by agarose gel electrophoresis. pET-30a (+) plasmid DNA, a product of Invitrogen, USA, was double-digested with Nde I and Xho I, subjected to agarose gel electrophoresis, and the product fragment after the digestion was recovered. Restriction enzymes Nde I and Xho I are commercially available from Dalibao biology, China, and the system, temperature and time for the reaction between the enzyme and the substrate used in the enzyme digestion are all operated according to the product specifications provided by the company.

Carrying out double enzyme digestion on the PCR product subjected to Nde I and Xho I and a pET-30a (+) plasmid vector subjected to double enzyme digestion in the same way, and carrying out grafting under the catalysis of DNA ligase; the ligation product is transformed into an Escherichia coli DH5 alpha strain, the strain is spread on a Luria-Bertani culture medium solid plate containing 50 mu g/mL kanamycin, after culture for 16h at 37 ℃, a single clone is picked; inoculating the single clone into a liquid Luria-Bertani culture medium containing 50 mu g/mL kanamycin for culture, and separating and purifying plasmid DNA; PCR verification is carried out on the plasmid by using an amplification primer, and an amplification product with the size of 2.8kb is obtained as a result, so that the constructed recombinant plasmid is proved to be correct preliminarily; the recombinant plasmid is then sequenced, and the result shows that the gene man02066 shown in SEQ ID NO.1 is inserted between Nde I and Xho I enzyme cutting sites of pET-30a (+) and the insertion direction is correct, thereby further proving that the constructed recombinant plasmid is correct.

The correct recombinant plasmid was designated pE30a-Man02066, transformed into e.coli strain BL21(DE3) (purchased from Invitrogen, usa) and then induced expression of recombinant β -mannanase Man02066 was performed using isopropyl thiogalactoside (IPTG) at a final concentration of 0.05mM according to the procedure provided by Invitrogen; centrifuging at 8,000 Xg and 4 deg.C for 15min, collecting thallus, resuspending thallus with buffer solution A, and ultrasonicating in ice water bath environment. Further centrifugation was carried out at 15,000 Xg at 4 ℃ for 30min, and water-soluble fractions were collected and adsorbed to the recombinant β -mannanase rMan02066 with Ni-Sepharose, respectively. Gradient elution was performed with buffer A containing imidazole at concentrations of 10, 50, 100, 250, 500mM, and the purification conditions were as per the manual of the gel. The purification of recombinant β -mannanase rMan02066 was examined by polyacrylamide gel electrophoresis. The results are shown in FIG. 2: after the recombinant plasmid pE30a-Man02066 is subjected to IPTG (isopropyl thiogalactoside) induction expression in an E.coli BL21(DE3) strain, a product is expressed in a water-soluble mode, and the recombinase rMan02066 purified by nickel column affinity chromatography is in a single band on an electrophoresis gel, and the position of the recombinase rMan02066 is identical with the predicted molecular weight; the purified recombinase rMan02066 sample is filled into a dialysis bag with the minimum molecular cut-off of 8-14kDa, and the buffer A is dialyzed in an environment at 4 ℃. The buffer solution A comprises 50mM Tris and 150mM NaCl, and has the pH value of 8.0, and the recombinant beta-mannase rMan02066 enzyme solution is prepared.

Example 4 determination of the optimum temperature of the recombinase beta-mannanase rMan02066

Preparing konjac glucomannan and locust bean gum with mass volume concentration of 0.3% (w/v) respectively with deionized water, heating to dissolve, and incubating in water bath environment of 0 deg.C, 10 deg.C, 20 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, and 80 deg.C for 1 h. 100 mu L of the diluted solution of the recombinant beta-mannase rMan02066 prepared in example 3 is added to each 900 mu L of the substrate solution, wherein the concentration of the diluted solution of the recombinant beta-mannase rMan02066 is 10 mu g/mL, the reaction is continued after the mixing, and the samples are taken at intervals. 3 parallel samples at each temperature were used as controls with a boiling water bath inactivated recombinase preparation.

The concentration (OD) of newly formed reducing sugar in each reaction system was measured by the DNS-reducing sugar method₅₄₀) And calculating the average value to perform deviation analysis. The reaction temperature corresponding to the maximum absorbance is the optimal temperature of the recombinase, and the relative enzyme activity (RA) is defined as: percentage of each absorption value to the maximum absorption value. The results are shown in FIG. 3: when the enzyme activity is measured by taking Konjac Glucomannan (KGM) and Locust Bean Gum (LBG) with equal mass volume concentration as substrates, the recombinant beta-mannase Man02066 achieves the maximum activity when KGM and LBG are degraded at 40 ℃, which shows that the optimal reaction temperature of the enzyme for degrading KGM and LBG is consistent and 40 ℃.

Example 5 determination of the optimum pH of recombinant β -mannanase rMan02066

Respectively using NaAc-HAC buffer solution with the concentration of 50mM and NaH with the concentration of 50mM₂PO₄-Na₂HPO₄Buffer solution and 50mM Tris-HCl buffer solution are respectively mixed with konjac glucomannan and locust bean gum to prepare a konjac glucomannan or locust bean gum substrate with the mass volume concentration (g/mL) of 0.3%, the corresponding pH values are respectively three sections of (5, 6), (6, 7, 8), (7, 8, 9 and 10), and the enzyme activity is measured at the optimum temperature for each pH value. Each substrate was incubated at the optimum temperature for 1h, then 100. mu.L of a dilution of the recombinant β -mannanase rMan02066 prepared in example 3 was added to each 900. mu.L of substrate, and after mixing, the reaction was started, and samples were taken at intervals. 3 replicates of each pH were treated with a boiling water bath inactivated recombinase preparation as a control. The concentration of newly formed reducing sugar (OD) in each reaction system was measured by the DNS-reducing sugar method₅₄₀) And the mean and deviation are calculated. Relative enzyme (RA) activity is defined as: percentage of the mean absorption value to the maximum absorption value for each group. The pH corresponding to the maximum absorbance is the optimum pH for the recombinase. The result is shown in fig. 4: the optimum reaction pH for the enzyme to degrade KGM and LBG was 6.0.

Example 6 temperature stability analysis of recombinant β -mannanase rMan02066

The recombinant β -mannanase rMan02066 enzyme solution prepared in example 3 after heat treatment at 0 ℃, 10 ℃,20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃ for different times was mixed with konjac glucomannan or locust bean gum prepared with distilled water at a mass volume concentration (g/mL) of 0.3% in a ratio of 1:9 (volume ratio), and then the residual enzyme activity was measured at the optimum temperature, with the enzyme activity of the enzyme solution without heat treatment defined as 100% relative activity. The results are shown in FIG. 5: after the recombinase rMan02066 is subjected to warm bath at 0-40 ℃ for 24 hours, the residual activity is more than 75% when KGM is degraded; upon degradation of LBG, the residual activity was greater than 60%. The above results show that: the beta-mannase Man02066 is stable at 0-40 ℃.

Example 7 analysis of pH stability of recombinant β -mannanase rMan02066

The enzyme solution of the recombinant beta-mannase rMan02066 prepared in example 3 is preincubated for 2 hours in ice water bath and different pH (pH 5-10) environments respectively, then mixed with konjac glucomannan or locust bean gum substrate solution with mass volume concentration (g/mL) of 0.3% and pretreated for 1 hour at the optimal temperature according to the proportion of 1:9 (volume ratio), then the residual enzyme activity is determined at the optimal temperature, and the relative activity is calculated by defining the enzyme activity of the enzyme solution which is not subjected to pretreatment as 100%. The results are shown in FIG. 6: after 2h of pretreatment of recombinase rMan02066 in buffer solution at pH5.0-10.0, whether it degrades glucomannan (KGM) or Locust Bean Gum (LBG), the residual activity of recombinase rMan02066 is drastically reduced when the pH is less than 6.0 or more than 8.0. The above results show that: the beta-mannase Man02066 is not changed in acid and alkali resistance and is volatile and active.

Example 8 Effect of Metal ions and chemical reagents on recombinant β -mannanase rMan02066 Activity

After 0.3% by mass of konjac glucomannan or locust bean gum substrate prepared with deionized water, the recombinant β -mannanase rMan02066 prepared in example 3, and water were mixed in a ratio of 5:1:4 (by volume), different metal ions were added to the reaction system to a final concentration of 1mM or 10mM, and the reaction was carried out under optimum conditions, and the activity of the enzyme was measured by the DNS-reducing sugar method as described above. The control group is the activity of rMan02066 without any metal ions (set at 100%). The results are shown in FIG. 7: when the recombinase rMan02066 degrades KGM, (1)10mM Ag + can obviously inhibit the recombinase rMan02066The activity is only 7 percent relative to the enzyme activity; (2) remove Ca²⁺、Mg²⁺、Mn²⁺And also 1mM of Co²⁺、Fe²⁺In addition, the rest divalent and trivalent metal ions can inactivate recombinase rMan 02066; (3) EDTA at 10mM and SDS at 1mM or 10mM can significantly inhibit the activity of recombinase rMan02066 and even inactivate. The enzyme degrades LBG differently from KGM: (1) k of 10mM⁺Can obviously inhibit the activity of recombinase rMan 02066; (2) co²⁺The recombinase rMan02066 can be inactivated; (3) DTT at 10mM can remarkably inhibit the activity of recombinase rMan 02066.

Example 9 determination of enzymatic Activity of recombinant β -mannanase rMan02066 by DNS-reducing sugar method

Mixing 0.3% konjak glucomannan, locust bean gum and guar gum substrate prepared by deionized water, 10 mug/mL recombinant beta-mannase rMan02066 enzyme solution, an optimal buffer solution and water according to a ratio of 2:1:3:4 (volume ratio), and reacting at an optimal temperature. Heating the reaction product in boiling water bath for 10min, transferring into ice water bath for 5min, centrifuging at 12,000 Xg and 4 deg.C for 15min, and collecting supernatant; mixing a certain volume of supernatant with DNS (3, 5-p-nitroxylene) -reaction solution with the same volume, heating in boiling water bath for 10min, cooling to room temperature, and measuring the absorption value at 540 nm. Using analytically pure mannose as a standard, the same procedure was followed to plot the mannose molar concentration and OD₅₄₀The dose-effect relationship curve between. The protein content in the recombinant beta-mannase rMan02066 enzyme solution is measured by a protein quantitative kit purchased from Shanghai biological engineering Limited company. The units of enzyme activity were calculated according to the international standard definition, i.e. the amount of enzyme required to produce 1. mu. mol of product per minute under standard conditions was 1 IU. The results show that: the recombinant beta-mannase rMan02066 can take konjac glucomannan or locust bean gum as a substrate, carry out enzymolysis to generate reducing sugar products, and have the enzyme activity of 30.67 +/-1.2U/mg and 12.44 +/-1.5U/mg respectively, but the enzyme can hardly degrade guar gum polysaccharide used in the test.

Example 10 TLC analysis of oligosaccharide products of recombinant β -mannanase rMan02066 degradation of konjac glucomannan and locust Bean Gum

Preparing konjac glucomannan and locust bean gum substrates with the mass volume concentration (g/mL) of 0.3% by using deionized water, heating and dissolving, and then respectively placing in water bath environments of 40 ℃ and 50 ℃ for cooling for 1 h. Adding 1-100 μ L of the recombinase rMan02066 prepared in example 3 into 100 μ L of the substrate, and supplementing with sterile deionized water when the volume is less than 200 μ L; after mixing, the reaction is continued, and samples are taken at intervals. Heating the reaction product in boiling water bath for 10min, transferring into ice water bath, and standing for 5 min; the mixture was centrifuged at 12,000 Xg at 4 ℃ for 15min, and the supernatant was collected. The recombinant beta-mannan rMan02066 enzyme solution inactivated in a boiling water bath in advance is used as a negative control reaction.

The product was analyzed by Thin Layer Chromatography (TLC) and 4uL of the supernatant was applied to a chromatography plate (TLC Silica 60F 254, MERK, Germany) and stained for 10 seconds with a developing agent of n-butanol: ethanol: water in a volume ratio of 2:1:1 and a developer of diphenylamine: aniline: phosphoric acid: acetone of 1g:1mL:5mL:50mL, followed by development at 110 ℃ for 10min and photographing.

FIG. 8 is a TLC analysis chart of oligosaccharide products during degradation of konjac glucomannan (A) and locust bean gum (B) by recombinant β -mannanase rMan 02066;

wherein, the A picture: m, mannose-mannose hexaose in abscissa; 1, comparison; 2-10, respectively represent: 10s, 1min, 10min, 1h, 4h, 6h, 24h, 48h and 72 h;

and B, drawing: m, mannose-mannose hexaose in abscissa; 1, comparison; 2 to 9, respectively represent: 10s, 1min, 10min, 1h, 6h, 24h, 48h, 72 h.

As shown in FIG. 8(A), when recombinant enzyme rMan02066 degrades KGM, a series of oligosaccharides were produced in a short time, and as the time extended by 48 hours, tetrasaccharide to disaccharide became predominant. As also shown in fig. 8(B), the recombinase rMan02066 degrades LBG with greater diversity, i.e., the oligosaccharide primary product is a disaccharide for a short period of time, and over time, the disaccharide product gradually accumulates, eventually becoming predominantly disaccharide. Preliminary conjecture after comprehensive analysis: the recombinase rMan02066 is a disaccharide exo-excision mode when degrading KGM, but due to the low activity of rMan02066 to KGM and the existence of glucose units in a KGM main chain structure, a series of oligosaccharide products with different sizes can be generated in the enzymolysis process, and the oligosaccharide products have certain difference in TLC analysis compared with the oligosaccharide products when the recombinase rMan02066 degrades LBG. Therefore, when the recombinase rMan02066 degrades different mannan substrates such as KGM and LBG, the degradation behaviors of the substrates are different due to the composition and structure of the substrates, the size of the enzyme digestion speed and the like.

Example 11 recombinant β -mannanase rMan02066 degradation of oligosaccharide products of konjac glucomannan and locust bean gum completely¹H-NMR identification

Respectively and completely degrading glucomannan and locust bean gum by using the recombinant beta-mannase rMan02066 to obtain an enzymolysis product, and then concentrating; separating and purifying the sample by using high performance liquid gel chromatography; the detector is a differential Refractometer (RID) and the chromatographic column is Superdex^TM30 Increate 10/300GL, mobile phase 0.2M ammonium bicarbonate, flow rate 0.4 mL/min.

The separated and purified samples were freeze-dried and desalted several times, respectively, and then treated with heavy water (D)₂O) dissolving, freeze-drying for replacement of hydrogen and deuterium, and final¹H-NMR detection.

By passing¹H-NMR data analyzed the structural features of the oligosaccharide products of the above recombinant β -mannanase rMan02066 degradation of konjac glucomannan (FIG. 9A) and locust bean gum (FIG. 9B).

Separating the final main product of recombinase rMan02066 degradation KGM by a molecular gel chromatographic column to obtain disaccharide, trisaccharide and tetrasaccharide product fragments, and respectively carrying out¹H-NMR structural identification, as shown in FIG. 9(A), 5.26ppm and 5.04ppm chemical shift values are glucose and mannose anomeric hydrogen signals corresponding to the reducing end of the oligosaccharide, respectively. From the above results it is speculated that the recombinase rMan02066, similar to rMan01929, degrades KGM and then leans towards the production of a series of oligosaccharide end-product fragments with predominantly mannose reducing ends.

The final main product of the recombinant enzyme rMan02066 for degrading LBG is separated to obtain oligosaccharide fragments of disaccharide to tetrasaccharide, hexasaccharide to octasaccharide and the like, and a pentasaccharide product fragment cannot be obtained. By passing¹H-NMR measurement showed that the results are shown in FIG. 9(B), and it was considered that, when analyzed by the same method: (1) the disaccharide fragment of the main product in the enzymolysis reaction is mainly MM and contains a small amount of MG (G is galactose); (2) in the enzymatic LBG reaction, larger oligosaccharides are producedThe higher galactose content in the fragment represses the further degradation of these products by the recombinase rMan 02066. This indicates that during the degradation of LBG by Man02066, the disaccharide product MM is actively produced, whereas the higher molecular weight oligosaccharide product is passively made a component of the final main product of the oligosaccharide due to its galactose-rich content, which is not favoured for further deep enzymatic degradation.

Example 12 analysis of the end products of the recombinase rMan02066 degradation series of mannooligosaccharides

Taking a solution containing about 20 μ g of series mannooligosaccharides (M-M6), 150mmol/L NaH₂PO4-Na₂HPO₄(pH7.0) buffer solution, and the dilution of the recombinant β -mannanase rMan02066 prepared in example 3 were mixed in a volume ratio of 1:1:1, and reacted at 40 ℃ for 24 hours, respectively. The reaction system was placed in a boiling water bath for 10min, transferred to an ice water bath for 5min, and centrifuged at 12,000 Xg at 4 ℃ for at least 15 min. The supernatant was collected as the oligosaccharide degradation product of recombinant β -mannanase rMan 02066. The recombinant beta-mannan rMan02066 enzyme solution inactivated in boiling water bath in advance is used as a negative control reaction.

A sample of the recombinant β -mannanase rMan02066 enzyme-digested mannooligosaccharide (M-M6) was assayed at 2uL according to the development conditions described in example 10. The color was developed under the same color development conditions as in example 10, and then analyzed.

FIG. 10 is a TLC analysis chart of the oligosaccharide products of recombinant β -mannanase rMan02066 completely degrading the series of size mannooligosaccharides (M-M6);

wherein, the A picture: in the abscissa: a: sample sequence: m: M1-M6; 1: M1 (-); 2: M1 (+); 3: M2 (-); 4: M2 (+); 5: M3 (-); m3 (+); 7: M4 (-); 8: M4 (+); 9: M5 (-); m5 (+); 11: M6 (-); m6 (+); wherein (-) is a negative control group and (+) is an experimental group;

and B, drawing: in the abscissa: m, M1-M6; 1: comparison; 2, diluting the enzyme solution by 10 times and reacting for 10 s; 10s, 1min, 30min, 3h, 12h and 48h at the time of 3-8;

and (C) diagram: in the abscissa: m, M1-M6; 1, comparison; 2, diluting the enzyme solution by 10 times and reacting for 10 s; 3-5, 10s, 1min and 48 h.

The 10-fold dilution of the enzyme solution for 10 seconds represents that the enzyme solution prepared in the example 3 is diluted by 10 times and then reacted for 10 seconds, and the rest of the enzyme solution prepared in the example 3 is reacted for 10 seconds, 1min, 30min, 3h, 12h and 48 h.

The results are shown in fig. 10, recombinant β -mannanase rMan02066 described herein:

(1) FIG. 10(A) shows that the M3-M6 sugar can be degraded, and M2 and M1 cannot be degraded; the final product is M2 when degrading M6 or M4, and the final product is M2 and M1 when degrading M5 or M3;

(2) FIG. 10(B) shows that M3& M2 is produced in a short time when M5 is degraded, and M3 is gradually degraded with time to produce M2& M1;

(3) fig. 10(C) shows that M4& M2 are produced in a short time when M6 is degraded, and M4 is gradually degraded into M2 with time, and the final main product is M2.

These results indicate that, when recombinase rMan02066 degrades mannooligosaccharides: (1) the minimum substrate is M3, the minimum product is M1; (2) when odd sugars are substrates, the final main products are M2 and M1; when even sugar is used as a substrate, the final main product is M2; (3) when rMan02066 degraded mannooligosaccharides, it was assumed that disaccharide continues exo-mode.

Example 13 fluorescence-High Performance Liquid Chromatography (HPLC) analysis of recombinant β -mannanase rMan02066 cleavage pattern

A series of solutions containing 10. mu.g of mannooligosaccharide (M-M6) were separately rotary evaporated to dryness. Adding dimethyl sulfoxide (DMSO) solution containing excessive o-aminobenzamide (2-AB) and sodium cyanoborohydride, mixing uniformly, and incubating in water bath at 60 deg.C for 2 h. And (3) performing rotary evaporation to dryness, adding deionized water to dissolve a sample, co-oscillating the sample and chloroform, centrifuging, and collecting a supernatant. Repeatedly extracting with chloroform for no less than 7 times to obtain mannose hexaose (2AB-M6), mannose pentaose (2AB-M5), mannose tetraose (2AB-M4), mannose trisaccharide (2AB-M3), mannose disaccharide (2AB-M2), mannose (2AB-M) and other series of substrate sugar chains with reducing ends respectively labeled by 2-AB.

Taking the products of 2AB-M6, 2AB-M5, 2AB-M3, 2AB-M2 and 2AB-M, the diluent of the recombinant beta-mannanase rMan02066 prepared in example 3 and 150mmol/L NaH₂PO4-Na₂HPO₄(pH7.0) buffer solution and water are mixed evenly according to the volume ratio of 2:1:3:4 and put into a water bath at 40 ℃ for reaction. Placing the reaction system in boiling water bath for 10min, transferring to ice water bath for 5min, and centrifuging at 4 deg.C under 12,000 Xg for at least 15 min. The supernatant was collected. Negative control reaction was performed with recombinant β -mannase rMan02066 enzyme solution that was previously heated in boiling water bath for 10min and then inactivated in ice water bath for 10 min.

With NH at a concentration of 0.20mol/L₄HCO₃Solution, equilibrium Superdex^TM30 Increate 10/300GL (GE, general electric) molecular gel chromatography column with a flow rate of 0.40mL/min for at least 2 beds. And (3) loading 20-200ng of samples of the fluorescence-labeled series of mannooligosaccharides at different enzymolysis times by using an automatic sample injector, and detecting the samples under the conditions of unchanged other conditions, excitation wavelength of 330nm and emission wavelength of 420 nm. The integrated area of each oligosaccharide component was analyzed by HPLC operating software, and the relative molar concentration was calculated in combination with the theoretical molecular weight.

As shown in FIG. 11(A), it was found that when the recombinase rMan02066 degrades mannose whose reducing end is labeled with 2 AB: (1) when the even sugar chain substrate 2AB-M6 or 2AB-M4 is degraded, the final product is mainly 2AB-M2& M2; (2) when the odd-numbered sugar chain substrate 2AB-M5 is degraded, the final product is mainly 2AB-M & M & M2; (3) when 2AB-M3 is degraded, the final product is mainly 2AB-M & M2; (4) the enzyme cannot degrade 2AB-M2 and 2 AB-M; (5) furthermore, it was found by the area integration method that the number of moles of 2AB-M2 produced was equal to the number of moles of the substrate sugar chains when 2AB-M4 was degraded, and the number of moles of 2AB-M produced was equal to the number of moles of the substrate sugar chains when 2AB-M3 was degraded.

As shown in FIGS. 11(B) and (C), the time gradient of degradation of 2AB-M5 and 2AB-M6 by recombinase rMan02066 is known: (1) degradation of the odd-numbered sugar chain substrate 2AB-M5 produced 2AB-M3 in a short time, 2AB-M3 gradually decreased with the lapse of time, and finally produced 2AB-M, and the area integral thereof was equal to the area integral (i.e., the number of moles) of the sugar chain of the substrate; (2) when the even-numbered sugar chain substrate 2AB-M6 was degraded, 2AB-M4 sugar was produced in a short time, 2AB-M4 was gradually decreased with the lapse of time, and finally 2AB-M2 was produced, and it was integrated in an equal area (i.e., in equal number of moles) to the substrate sugar chain.

The above results show that: when the recombinase rMan02066 degrades the fluorescence-labeled mannan oligosaccharide, (1) the minimum substrate is 2AB-M3, and the minimum product is M; (2) m2 was cleaved continuously from the non-reducing end of the substrate sugar chain substrate and exhibited a disaccharide exo-mode; (3) when the final product containing the fluorescent label is 2AB-M2, the corresponding original substrate is an even number of sugar chains and the number of moles of the original substrate is equal to that of the original substrate; when the final product containing the fluorescent label is 2AB-M1, the corresponding original substrate is an odd number of sugar chains and the number of moles is equal to that. Therefore, theoretically, the enzyme or its recombinant enzyme can be used for analyzing and determining the number of moles of sugar chains of the original polysaccharide substrate, i.e., as a molar counter for the polysaccharide substrate, and can also determine the parity of the original substrate.

Example 14 multiple sequence alignment analysis of Man02066

Alignment of protein sequences of Man02066 and the identified mannanase of the GH26 family was performed using DNAMAN software.

As shown in fig. 12, the β -mannanase Man02066 (Man 02066 for short) protein sequence was aligned with the protein sequence of the mannanase of the GH26 family:

(1) among the identified enzymes, the highest similarity of Man02066 to CjMan26A, CjMan26C, Mana-2, Man26A by Blast P analysis was: 35.59%, 42.82%, 42.18%, 35.15%;

(2) through DNAMAN 8.0, multiple sequence alignment analysis shows that: the conserved catalytic site residue in the Man02066 sequence is Glu¹⁸⁸(acid/base catalysis) and Glu²⁹⁶(nucleophilic catalysis).

Example 15 Gene mutation of Man02066

PCR amplification was carried out using the recombinant plasmid pET30a-Man02066 obtained in example 3 as a template, and the amplification primers are shown in Table 1:

TABLE 1 primers used for the mutations

The site-directed mutagenesis kit is purchased from Nanjing Novozam biotech GmbH, and the site-directed mutagenesis is carried out according to the experimental operation steps provided by the kit of the company, so as to obtain recombinant plasmids which are sent to the biological engineering (Shanghai) GmbH for sequencing verification, and finally obtain the recombinant plasmids pET30a-Man02066-E188A and pET30a-Man02066-E296A with correct construction.

The mutant recombinant plasmids were induced and expressed according to example 3, and after cell disruption by sonication, the cells were centrifuged and the supernatant was transferred to obtain crude enzyme solutions E188A and E296A of the mutant recombinant enzyme.

Example 17 analysis of enzyme Activity of rMan02066 (abbreviated Man02066) mutant

Preparing konjac glucomannan and locust bean gum substrates with the mass volume concentration (g/mL) of 0.3% by using deionized water, heating and dissolving, and then respectively placing in water bath environments of 40 ℃ and 50 ℃ for cooling for 1 h. To 100. mu.L of substrate was added 10 to 100. mu.L of the diluted solution of the crude enzyme solution of the mutant prepared in example 16, and when the volume was less than 200. mu.L, the mixture was supplemented with sterile deionized water, and the reaction was continued for 12 hours after mixing. Heating the reaction product in boiling water bath for 10min, transferring into ice water bath for 5min, centrifuging at 12,000 Xg and 4 deg.C for 15min, and collecting supernatant.

The concentration (OD) of newly formed reducing sugar in each reaction system was measured by the DNS-reducing sugar method₅₄₀) And calculating an average value for deviation analysis. The control group is the maximal absorbance of the recombinase rMan02066 (set at 100%), and the relative enzyme activity (RA) is defined as: the percentage of absorbance to maximal absorbance for each mutant oligosaccharide product.

Results the series of mutants of Man02066, when degrading KGM (fig. 13A) and LBG (fig. 13B), as shown in the figure: after mutation of residues E188 and E296, the enzyme mutant is inactivated, so that the two glutamic acid residues are proved to be conserved catalytic site residues of Man 02066.

All documents referred to herein are incorporated by reference into this application as if each had been individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined in the appended claims.

Sequence listing

<110> Shanghai Green grain pharmaceutical Co., Ltd

<120> disaccharide excision-type beta-mannase hydrolase and application thereof

<130> P2020-1101

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<170> SIPOSequenceListing 1.0

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<213> Flammeovirga yaeyamensis

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gcagtgactg acgatcagtt ttttaccacc aagttattgg acaagctaaa tcacaatgaa 960

tggacaaaga aagccgctta tgtgatgctt tggagaaacg ccaactatca aaaagaacag 1020

agagatcatt tttatgtgcc ttacaaaggt cactcttctg ccaaagactt tatcaaattc 1080

aaagaagatc caagcattct ctttgagtct gatttgatga atatgaaagt gaattaa 1137

<210> 2

<211> 378

<212> PRT

<213> Flammeovirga yaeyamensis

<400> 2

Met Arg Lys Arg Leu Val Ala Ala Ser Phe Leu Ile Gly Leu Leu Thr

1 5 10 15

Leu Asn Phe Gly Thr Ala Glu Asp Lys Ala Lys Thr Pro Lys Leu Asn

20 25 30

Asp Lys Lys Ala Thr Lys Glu Thr Val Ala Leu Tyr Glu Gln Leu His

35 40 45

Gln Val Ser Val Ser Gly Lys Thr Ile Phe Gly His Gln Asp Asp Leu

50 55 60

Ala Tyr Gly Tyr His Trp Trp Gly Asp Gly Ser Asp Val Lys Asn Val

65 70 75 80

Thr Gly Asp Tyr Pro Gly Leu Tyr Gly Trp Asp Met Gly His Ile Gly

85 90 95

Glu Glu Lys Asn Leu Asp Gly Val Pro Phe Glu Asp Ile Lys Arg Tyr

100 105 110

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

115 120 125

Met Ile Asn Leu Lys Glu Lys Ser Ser Ser Trp Asp Thr Thr Arg Val

130 135 140

Leu His Glu Met Met Glu Gly Gly Lys Tyr His Gln Asp Phe Ile Lys

145 150 155 160

Lys Leu Asp Leu Phe Ala Glu Phe Val Asp Asp Leu Glu Leu Glu Gly

165 170 175

Lys Lys Ile Pro Val Leu Phe Arg Pro Trp His Glu His Asn Gly Ser

180 185 190

Trp Phe Trp Trp Gly Gly Lys Asn Val Glu Ile Lys Asp Tyr Lys Thr

195 200 205

Leu Trp Gln Phe Thr Val Glu Tyr Leu Arg Asp Lys Lys Gly Ile His

210 215 220

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

225 230 235 240

Tyr Leu Glu Arg Tyr Pro Gly Asp Lys Tyr Val Asp Val Leu Gly Phe

245 250 255

Asp Asp Tyr Gly Ala Tyr Arg Ala Asn Ala Thr Pro Glu Arg Glu Gln

260 265 270

Trp Ile Ile Arg Glu Leu Glu Thr Val Ala Lys Leu Ala Gln Gln Lys

275 280 285

Asn Lys Ile Cys Ala Phe Thr Glu Ser Gly Leu Glu Ala Val Thr Asp

290 295 300

Asp Gln Phe Phe Thr Thr Lys Leu Leu Asp Lys Leu Asn His Asn Glu

305 310 315 320

Trp Thr Lys Lys Ala Ala Tyr Val Met Leu Trp Arg Asn Ala Asn Tyr

325 330 335

Gln Lys Glu Gln Arg Asp His Phe Tyr Val Pro Tyr Lys Gly His Ser

340 345 350

Ser Ala Lys Asp Phe Ile Lys Phe Lys Glu Asp Pro Ser Ile Leu Phe

355 360 365

Glu Ser Asp Leu Met Asn Met Lys Val Asn

370 375

<210> 3

<211> 29

<212> DNA