WO2021249503A1 - 一种二糖外切型β-甘露聚糖水解酶及其应用 - Google Patents

一种二糖外切型β-甘露聚糖水解酶及其应用 Download PDF

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WO2021249503A1
WO2021249503A1 PCT/CN2021/099490 CN2021099490W WO2021249503A1 WO 2021249503 A1 WO2021249503 A1 WO 2021249503A1 CN 2021099490 W CN2021099490 W CN 2021099490W WO 2021249503 A1 WO2021249503 A1 WO 2021249503A1
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mannanase
exo
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rman02066
mannan
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韩文君
宓延红
程媛媛
古静燕
胡玮
张真庆
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绿谷(上海)医药科技有限公司
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/2488—Mannanases
    • C12N9/2494—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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    • C12P19/00—Preparation of compounds containing saccharide radicals
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01078—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/011—Mannan 1,4-mannobiosidase (3.2.1.100)

Definitions

  • the present invention relates to the field of biotechnology; specifically, the present invention relates to a disaccharide exomannase and its coding gene and application.
  • Hemicellulose is a structural polysaccharide that constitutes the cell wall of plants, and is usually tightly combined with cellulose and lignin to form lignocellulosic biomass.
  • the two polysaccharides, mannan and xylan are closely related to people’s lives. They are mainly used in food, medicine, textiles, pulp and paper, and biofuels. And other industrial fields.
  • Mannan is the second highest content of hemicellulose in nature. It is a type of polysaccharide with a complex structure.
  • composition and structure of the sugar unit it can be divided into four categories: pure mannan, glucomannan (glucomannan), galactomannan (galactomannan) and galactoglucomannan (galactoglucomannan).
  • mannans in addition to the common characteristics of mannose units as the main component, the polysaccharide backbone structure also has the complex characteristics of random heteropolymerization of multiple sugar units, partial modularization and other complex features, and the side chain structure of mannans It also has similar characteristics. Therefore, in order to completely degrade these glycans, the synergistic action of multiple types of enzymes (enzyme systems) is required.
  • mannanase a tool enzyme for preparing oligosaccharides
  • ⁇ -mannanase has been widely used in various fields such as food, animal breeding, papermaking, biofuels and oil drilling.
  • ⁇ -mannanase is used as a feed additive to act on mannans in food to assist in the production of mannose-oligosaccharide prebiotics.
  • mannan In the detergent industry, many types of daily chemical products and foods, such as hair spray, shampoo, conditioner, toothpaste, ice cream and barbecue sauce, contain a certain amount of mannan as a thickener or stabilizer. Once these ingredients form stains, they are usually difficult to remove. If the mannans are hydrolyzed by ⁇ -mannanase for treatment, the poorly soluble polysaccharides will become oligosaccharide fragments that are easily soluble in water, making it easier to remove stains. In the paper industry, pulp and paper industry, the use of ⁇ -mannanase can promote the removal of lignin in the pulp, produce results equivalent to alkaline pretreatment, and can greatly reduce environmental pollution.
  • the present invention provides a disaccharide exo-type ⁇ -mannan hydrolase Man02066 and its coding gene and application.
  • the present invention provides an exo-type ⁇ -mannanase whose amino acid sequence is shown in SEQ ID NO: 2.
  • the exo-type ⁇ -mannanase degrades mannans containing sugar chain branches or linear mannans without sugar chain branches; preferably, the exo-type ⁇ -mannanase Carbohydrases degrade linear mannans without sugar chain branches.
  • the mannan is glucomannan, galactomannan, pure mannan; preferably pure mannan.
  • the exo-type ⁇ -mannanase can degrade glucomannan (KGM) derived from konjac and galactomannan (LBG) derived from locust bean gum; preferably, the external The polysaccharide substrate of cut-type ⁇ -mannanase is glucomannan derived from konjac.
  • KGM glucomannan
  • LBG galactomannan
  • the exo-type ⁇ -mannanase consists of only one Glycoside hydrolase family 26 domain module.
  • the reaction temperature for the exo-type ⁇ -mannanase to degrade mannan is 0-40°C, and the pH is 6.0-8.0.
  • the optimal temperature for the exo-type ⁇ -mannanase to degrade konjac glucomannan (KGM) and locust bean gum (LBG) is 40°C, and the optimal pH is 6.0.
  • the exo-type ⁇ -mannanase can also degrade mannose oligosaccharides, the smallest substrate that can be degraded is mannotriose (M3), and the smallest product is mannose (M).
  • the present invention provides the nucleotide sequence encoding the exo-type ⁇ -mannanase of the first aspect.
  • nucleotide sequence is shown in SEQ ID NO:1.
  • the present invention provides an expression vector comprising the nucleotide sequence described in the second aspect.
  • the present invention provides a host cell comprising the expression vector of the third aspect or the nucleotide sequence of the second aspect integrated into its genome.
  • the host cell is a host cell for producing the exo-type ⁇ -mannanase or for degrading ⁇ -mannan.
  • the present invention provides the use of the exo-type ⁇ -mannanase described in the first aspect in degrading mannan.
  • the mannan contains sugar chain branched mannan or linear mannan without sugar chain branch; preferably, the mannan is a linear mannan without sugar chain branch.
  • the mannan is glucomannan, galactomannan, pure mannan; preferably pure mannan.
  • the present invention provides the application of the exo-type ⁇ -mannanase described in the first aspect in analyzing and determining the number of moles of sugar chains of its original polysaccharide substrate and judging the odd-even status of the original substrate.
  • the present invention provides an exo-type ⁇ -mannanase, which is the exo-type ⁇ -mannanase mutant of the first aspect.
  • amino acid sequence of the exo-type ⁇ -mannanase is shown in SEQ ID NO: 2, but mutations occur at positions 188 and/or 296.
  • the 188th amino acid in the amino acid sequence of the exo-type ⁇ -mannanase is mutated from glutamic acid to alanine, and/or the 296th amino acid is mutated from glutamic acid to alanine. Acid.
  • the present invention provides a mannan degradation product prepared by degrading mannan by the exo-type ⁇ -mannanase described in the first aspect.
  • 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 .
  • the mannan is glucomannan, galactomannan, pure mannan; preferably pure mannan.
  • the mannan is konjac-derived glucomannan (KGM) and locust bean gum-derived galactomannan (LBG); preferably, the mannan is konjac-derived glucomannan (KGM). Mannose.
  • the present invention also provides the application of the exo-type ⁇ -mannanase and its mutant enzyme as a tool enzyme in the study of the catalytic mechanism of mannanase.
  • Figure 1 is the result of analysis of functional module composition of exo-type ⁇ -mannanase Man02066;
  • Figure 2 is a polyacrylamide gel electrophoresis diagram of recombinant exo- ⁇ -mannanase rMan02066 expression and purification;
  • M protein molecular weight standard, the size of the band from top to bottom is 116kDa, 66.2kDa, 45kDa, 35kDa, 25kDa, 18.4kDa, 14.4kDa;
  • Lane 1 control strain before broken cell, loading amount 10 ⁇ L;
  • Lane 2 Recombinant bacteria before broken cell, loading amount 2 ⁇ L
  • Lane 3 Broken recombinant bacteria After the supernatant, the loading volume was 2 ⁇ L;
  • Lane 4 rMan02066 purified by the nickel column, the loading volume was 2 ⁇ L;
  • Figure 3 is a graph showing the influence of temperature on the activity of recombinant ⁇ -mannanase rMan02066 to degrade konjac glucomannan (A) and locust bean gum (B);
  • Figure 4 is a graph showing the influence of pH on the stability of recombinant ⁇ -mannanase rMan02066 to degrade konjac glucomannan (A) and locust bean gum (B);
  • Figure 5 is a graph showing the influence of temperature on the stability of recombinant ⁇ -mannanase rMan02066 to degrade konjac glucomannan (A) and locust bean gum (B);
  • Figure 6 is a graph showing the influence of pH on the activity and stability of recombinant ⁇ -mannanase rMan02066 to degrade konjac glucomannan (A) and locust bean gum (B);
  • Figure 7 is a bar graph showing the effects of metal ions and chemical reagents on the activity of recombinant ⁇ -mannanase rMan02066 to degrade konjac glucomannan (A) and locust bean gum (B);
  • Figure 8 is a TLC analysis diagram of oligosaccharide products during the degradation of konjac glucomannan (A) and locust bean gum (B) by recombinant ⁇ -mannase rMan02066;
  • Figure A M in the abscissa, mannose-mannose Hexaose; 1, control; 2-10, respectively: 10s, 1min, 10min, 1h, 4h, 6h, 24h, 48h, 72h;
  • Figure B M in the abscissa, mannose-mannose; 1, control ; 2-9, respectively represent: 10s, 1min, 10min, 1h, 6h, 24h, 48h, 72h;
  • Figure 9 is a 1 H-NMR chart of oligosaccharide fragments prepared by using recombinant ⁇ -mannanase rMan02066 to completely degrade konjac glucomannan (A) and locust bean gum (B);
  • Figure 10 is the TLC analysis diagram of the oligosaccharide products of recombinant ⁇ -mannanase rMan02066 completely degrading series of large and small mannose oligosaccharides (M-M6); where A: in the abscissa: A: sample order: M: M1-M6; 1:M1(-); 2:M1(+); 3:M2(-); 4:M2(+); 5:M3(-); 6:M3(+); 7:M4(-); 8 :M4(+); 9: M5(-); 10: M5(+); 11: M6(-); 12: M6(+); where (-) is the negative control group, (+) is the experimental group; Panel B: In the abscissa: M, M1-M6; 1: Control; 2: Enzyme solution diluted 10 times and react for 10s; 3-8: 10s, 1min, 30min, 3h, 12h, 48h; Panel C: In
  • Figure 11 is the analysis of the final main product of the series of large and small mannose oligosaccharides (2AB-M ⁇ 2AB-M6) degraded by recombinant ⁇ -mannanase rMan02066 (2AB-M ⁇ 2AB-M6), and the degradation of 2AB-labeled mannpentaose ( 2AB-M5) (B) and 2AB-labeled mannose (2AB-M6) (C) HPLC analysis chart (fluorescence) of oligosaccharide products;
  • Figure 12 is a multiple sequence alignment of ⁇ -mannanase Man02066 and GH26 family ⁇ -mannanase;
  • Figure 13 shows the relative activity analysis of ⁇ -mannanase Man02066 mutant in degrading konjac glucomannan (A) and locust bean gum (B), respectively.
  • ⁇ -mannanase has the meaning conventionally understood by those skilled in the art, and refers to a class of mannooligosaccharides and mannan polysaccharides (including mannan, glucan, dextran) that can hydrolyze ⁇ -1,4-mannosidic bonds Mannan, galactomannan and galactomannan) hydrolase.
  • ⁇ -mannanase can be divided into endo-type ⁇ -mannanase (EC 3.2.1.78) and exo-type ⁇ -mannanase (EC 3.2.1.100).
  • the endo-type ⁇ -mannanase can hydrolyze the ⁇ -1,4 glycosidic bond inside the mannan backbone to produce a series of mannan-oligosaccharide fragments with different degrees of polymerization; and the exo-type ⁇ -mannanase It is the ⁇ -1,4 glycosidic bond that continuously acts on the sugar chain of the substrate from the end of the non-reducing chain to cut and produce a fixed-size sugar unit product, such as mannobiose.
  • ⁇ -Mannanase is widely present in natural organisms, including: animals, plants and microorganisms.
  • ⁇ -mannanase especially bacteria, fungi and actinomycetes.
  • ⁇ -mannanase derived from animals and plants is less, mainly distributed in the seeds of legumes and the digestive juices of mollusks.
  • GH26 family members are still mostly endomannan hydrolases, such as Cellvibrio japonicus Ueda107's CjMan26A (Ducros, V., Zechel, DL, Murshudov, G., Gilbert, HJ, Szabo, L., Stoll, D., Withers, SG, Davies, GJ (2002) Angew Chem Int Ed Engl 41:2824), BoMan26B of Bacteroides ovatus (Bagenholm, V., Wiemann, M., Reddy, SK, Bhattacharya, A., Rosengren , A., Logan, DT, Stalbrand, H.
  • CjMan26A Ducros, V., Zechel, DL, Murshudov, G., Gilbert, HJ, Szabo, L., Stoll, D., Withers, SG, Davies, GJ (2002) Angew Chem Int Ed Engl 41:2824
  • BoMan26B of Bacteroides ovatus Bogenhol
  • BoMan26A of Bacteroides ovatus can cleave the disaccharide or trisaccharide unit from the non-reducing end of the sugar chain of the substrate, and finally mannobiose is the main product ( V,Reddy SK, Bouraoui H,et al. Galactomannan Catabolism Conferred by a Polysaccharide Utilization Locus of Bacteroides ovatus ENZYME SYNERGY AND CRYSTAL STRUCTURE OF A ⁇ -MANNANASE[J].Journal of Biological Chemistry,2017,292(1):229-243 ).
  • BoMan26A is not a strict type of disaccharide exomannase. According to the research results, it is speculated that BoMan26A should be a typical endomannose hydrolase with variable substrate degradation mode.
  • the GH26 ⁇ -mannanase RsMan26H from a symbiotic protist of the termite Reticulitermes speratus is an endo-processive mannobiohydrolase: heterologous expression and characterization. Biochem Biophys Res Commun. 2014;452(3):520-525).
  • the literature has unclear conclusions on the catalytic mode and oligosaccharide production characteristics of GH26i derived from uncultured Bacteroidetes bacterium. Therefore, in order to obtain tools, it is necessary to strengthen the analysis and interpretation of the core application value and characteristics of enzymes such as the substrate degradation mode and oligosaccharide production characteristics of enzymes.
  • the amino acid sequence of the exo-type ⁇ -mannanase of the present invention is shown in SEQ ID NO: 2.
  • the exo-type ⁇ -mannanase of the present invention can degrade mannans containing sugar chain branches, and can also degrade linear mannans without sugar chain branches; but linear mannans without sugar chain branches are of the present invention The best substrate for exo-type ⁇ -mannanase.
  • the exo-type ⁇ -mannanase of the present invention has a certain temperature tolerance, and its reaction temperature for degrading mannan is 0-40°C, preferably 20-40°C; in addition, the exo-type ⁇ -mannase of the present invention
  • the pH condition of mannanase to degrade mannan is 6.0-8.0.
  • the optimal temperature for the exo-type ⁇ -mannanase of the present invention to degrade konjac glucomannan (KGM) and locust bean gum (LBG) is 40° C., and the optimal pH is 6.0.
  • the exo-type ⁇ -mannanase of the present invention degrades the mannan substrate, it can continuously cleave the sugar chain from the non-reducing end to the end with mannobiose as the unit, so it can be used for the specificity of mannobiose preparation.
  • exo-type ⁇ -mannanase of the present invention can also be used to analyze and determine the number of moles of sugar chains of its original polysaccharide substrate and determine the odd-even status of the original substrate.
  • the inventors further discovered that the conserved catalytic site of the exo-type ⁇ -mannanase can be used, thereby laying a foundation for further modification of the enzyme. .
  • the conservative catalytic sites of the exo-type ⁇ -mannanase of the present invention are at positions 188 and 296.
  • the 188th amino acid in the amino acid sequence of the exo-type ⁇ -mannanase is mutated from glutamic acid to alanine, and/or the 296th amino acid is mutated from glutamic acid to alanine.
  • Amino acid can obtain inactive mutants. Therefore, the exo-type ⁇ -mannanase and its mutant enzyme of the present invention can be used as tool enzymes to study the catalytic mechanism of mannanase.
  • the present invention discloses for the first time that the disaccharide exo-type ⁇ -mannanase Man02066 is obtained from Flammeovirga yaeyamensis MY04;
  • the enzyme Man02066 can degrade konjac glucomannan and other polysaccharide substrates such as locust bean gum;
  • the enzyme has a certain degree of thermal stability (0-40°C) and narrow pH tolerance (6.0-8.0);
  • the enzyme degrades a series of pure mannose oligosaccharide substrates or pure mannose oligosaccharide substrates whose reducing end is fluorescently labeled, it uses mannobiose as the unit to continuously cut the sugar chain from the non-reducing end to the end, so it can be used for mannose two Specific preparation of sugar;
  • the present 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 study of exo-type ⁇ -mannanase;
  • the enzyme When the enzyme is degrading a series of mannose oligosaccharide substrates labeled with anthranilamide at the reducing end, it can continuously cleave M2 sugars from the non-reducing end until the remaining mannobiose (2AB-M2) or mannose (2AB- M), and when the final product is 2AB-M2, the corresponding original substrate is an even-numbered sugar chain, and the number of moles is equal; when the final product is 2AB-M1, the corresponding original substrate is an odd-numbered sugar chain , And the number of moles is equal to it.
  • the enzyme or its recombinase combined with the fluorescent labeling and analysis of sugar chain substrates, can be used to analyze and determine the number of moles of sugar chains of its original polysaccharide substrate, that is, it can be used as a mole counter for polysaccharide substrates. You can also determine the parity status of the original substrate.
  • Flammeovirga yaeyamensis strain MY04 originated from the General Microbiology Center of China Microbial Culture Collection Management Committee, Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3 Beichen West Road, Chaoyang District, Beijing, preservation date November 27, 2008 , Deposit number CGMCC NO.2777.
  • lysozyme buffer (10 mM Tris-HCl, pH 8.0
  • the above-mentioned liquid medium YT04 has the following components per liter:
  • the coding region of the gene man02066 is 1137bp long, and the nucleotide sequence is as shown in SEQ ID NO:1 Show.
  • the ⁇ -mannanase Man02066 encoded by the gene man02066 contains a total of 378 amino acids, and its amino acid sequence is shown in SEQ ID NO: 2.
  • the primer sequence is as follows:
  • Man02066-F 5'-gcg CATATG GAAGATAAGGCAAAGACACC-3' (SEQ ID NO: 3);
  • Man02066-F 5'-gcg CTCGAG ATTCACTTTCATATTCATCAAATC-3' (SEQ ID NO: 4);
  • the underlined forward primer is the specific site of restriction enzyme Nde I
  • the underlined reverse primer is the specific site of restriction enzyme Xho I.
  • the high-fidelity DNA polymerase Prime STAR HS DNA Polymerase was purchased from Dalian Bao Biological Company, China, and the PCR reagents used were operated in accordance with the product instructions provided by the company.
  • the PCR product was double digested with restriction enzymes Nde I and Xho I, and the digested PCR product was recovered by agarose gel electrophoresis.
  • the product pET-30a(+) plasmid DNA purchased from Invitrogen in the United States was digested with Nde I and Xho I, and subjected to agarose gel electrophoresis, and the digested product fragments were recovered.
  • Restriction endonucleases Nde I and Xho I were purchased from Dalian Bao Biological Company, China. The reaction system, temperature and time of the enzyme and substrate used for digestion were operated in accordance with the product instructions provided by the company.
  • the PCR product that has been digested with Nde I and Xho I is ligated with the pET-30a(+) plasmid vector that has also been digested with double enzymes under the catalysis of DNA ligase; the ligation product is transformed into E.
  • the concentration of newly formed reducing sugar (OD 540 ) in each reaction system was measured by DNS-reducing sugar method, and the average value was calculated for deviation analysis.
  • the reaction temperature corresponding to the maximum absorption value is the optimum temperature of the recombinase, and the relative enzyme activity (RA) is defined as: the percentage of each absorption value to the maximum absorption value.
  • Example 6 Analysis of temperature stability of recombinant ⁇ -mannanase rMan02066
  • Example 7 Analysis of pH stability of recombinant ⁇ -mannanase rMan02066
  • the recombinant ⁇ -mannanase rMan02066 enzyme solution prepared in Example 3 was pre-incubated for 2 hours in an ice water bath and different pH (pH 5-10) environments, and the mass volume concentration (g/mL) was 0.3%, the substrate solution of konjac glucomannan or locust bean gum pretreated at the optimum temperature for 1 hour is mixed in a ratio of 1:9 (volume ratio), and then the residual enzyme activity is measured at the optimum temperature to avoid The enzyme activity of the pretreated enzyme solution is defined as 100%, and the relative activity is calculated.
  • Example 8 The effect of metal ions and chemical reagents on the activity of recombinant ⁇ -mannanase rMan02066
  • the control group is the activity of rMan02066 without any metal ions (set as 100%).
  • Example 9 Determination of the enzyme activity of recombinant ⁇ -mannanase rMan02066 by DNS-reducing sugar method
  • the reaction product was heated in a boiling water bath for 10 min, transferred to an ice water bath for 5 min, centrifuged at 12,000 ⁇ g, 4 °C for 15 min, and the supernatant was collected; a certain volume of the supernatant and an equal volume of DNS (3,5-pair Nitroxylene)-reaction solution was mixed, heated in a boiling water bath for 10 minutes, and cooled to room temperature, and the absorbance was measured at 540nm. Using analytically pure mannose as a standard, the same method is used to draw a dose-effect relationship curve between the molar concentration of mannose and OD 540.
  • the protein content in the recombinant ⁇ -mannanase rMan02066 enzyme solution was determined with a protein quantification kit purchased from Shanghai Shenggong Bioengineering Co., Ltd.
  • the enzyme activity unit is calculated according to the international standard definition, that is, under standard conditions, the amount of enzyme required to produce 1 ⁇ mol product per minute is 1 IU.
  • the results show that the recombinant ⁇ -mannanase rMan02066 can use konjac glucomannan or locust bean gum as a substrate to enzymatically hydrolyze and produce reducing sugar products.
  • the enzyme activities are 30.67 ⁇ 1.2U/mg and 12.44 ⁇ 1.5U/ respectively. mg, but the enzyme can hardly degrade the guar polysaccharide used in the test.
  • Example 10 TLC analysis of oligosaccharide products of recombinant ⁇ -mannanase rMan02066 degrading konjac glucomannan and locust bean gum
  • the konjac glucomannan and locust bean gum substrates with a mass volume concentration (g/mL) of 0.3% were prepared with deionized water, heated and dissolved, and then placed in a water bath environment of 40°C and 50°C to cool down for 1 hour.
  • the reaction product was heated in a boiling water bath for 10 min, then transferred to an ice water bath and placed for 5 min; centrifuged at 12,000 ⁇ g and 4° C. for 15 min, and the supernatant was collected.
  • Recombinant ⁇ -mannan rMan02066 enzyme solution which was previously inactivated in a boiling water bath, was used as a negative control reaction.
  • TLC thin layer chromatography
  • Figure 8 is a TLC analysis diagram of oligosaccharide products during the degradation of konjac glucomannan (A) and locust bean gum (B) by recombinant ⁇ -mannanase rMan02066;
  • FIG. 1 M in the abscissa, mannose-mannose; 1, control; 2-10, respectively: 10s, 1min, 10min, 1h, 4h, 6h, 24h, 48h, 72h;
  • Panel B M in the abscissa, mannose-mannose; 1, control; 2-9, respectively: 10s, 1min, 10min, 1h, 6h, 24h, 48h, 72h.
  • the recombinase rMan02066 is in the exo-disaccharide mode when degrading KGM.
  • a series of oligosaccharides of different sizes will be produced during enzymatic hydrolysis.
  • the sugar product, and the oligosaccharide product when the recombinase rMan02066 degrades LBG has a certain difference in TLC analysis. Therefore, when the recombinase rMan02066 degrades different mannan substrates such as KGM and LBG, the composition and structure of the substrate, as well as the enzyme cleavage speed, will cause different substrate degradation behaviors.
  • Example 11 1 H-NMR identification of the oligosaccharide product of recombinant ⁇ -mannanase rMan02066 completely degrading konjac glucomannan and locust bean gum
  • Recombinant ⁇ -mannanase rMan02066 was used to completely degrade glucomannan and locust bean gum to obtain the enzymatic hydrolysate and then concentrate; the sample was separated and purified by high performance liquid gel chromatography; the detector was a refractive index detector (RID) ), the chromatographic column is Superdex TM 30Increase 10/300GL, the mobile phase is 0.2M ammonium bicarbonate, and the flow rate is 0.4 mL/min.
  • RID refractive index detector
  • the separated and purified samples were subjected to multiple freeze-drying to remove salt, and then dissolved in heavy water (D 2 O), freeze-dried to replace hydrogen and deuterium, and finally detected by 1 H-NMR.
  • the final main product of recombinase rMan02066 degraded KGM was separated by molecular gel chromatography column to obtain disaccharides, trisaccharides, and tetrasaccharide product fragments, which were respectively subjected to 1 H-NMR structural identification, as shown in Figure 9(A), 5.26ppm and The chemical shift value of 5.04ppm is the anomeric hydrogen signal of glucose and mannose corresponding to the reducing end of the oligosaccharide.
  • the main product disaccharide fragment in the enzymatic hydrolysis reaction is mainly MM, containing a small amount of MG (G is galactose)
  • G is galactose
  • the galactose content in the larger oligosaccharide product fragment is higher, which prevents the further degradation of these products by the recombinase rMan02066.
  • the recombinant ⁇ -mannanase rMan02066 was used to hydrolyze the sample of mannose oligosaccharide (M-M6), and 2uL was taken for detection. According to the color development conditions described in Example 10, the color was developed and then analyzed.
  • Figure 10 is a TLC analysis diagram of the oligosaccharide product of a series of large and small mannose oligosaccharides (M-M6) completely degraded by recombinant ⁇ -mannanase rMan02066;
  • Figure B In the abscissa: M, M1-M6; 1: Control; 2: Enzyme solution diluted 10-fold and react for 10s; 3-8: 10s, 1min, 30min, 3h, 12h, 48h;
  • Panel C In the abscissa: M, M1-M6; 1, control; 2, enzyme solution diluted 10-fold and react for 10s; 3-5, 10s, 1min, 48h.
  • the above-mentioned enzyme solution is diluted 10-fold and reacted for 10s, which means that the enzyme solution prepared in Example 3 is diluted 10-fold and then reacted for 10 seconds.
  • the rest is the enzyme solution prepared in Example 3 and reacted for 10s, 1min, 30min, 3h, 12h, 48h.
  • Figure 10(A) shows that M3-M6 sugars can be degraded, but M2 and M1 cannot be degraded; when M6 or M4 is degraded, the final product is M2, and when M5 or M3 is degraded, the final product is M2 and M1;
  • Figure 10(C) shows that when M6 is degraded, M4&M2 will be produced in a short time. As time goes by, M4 will gradually degrade into M2, and the final main product will be M2.
  • 2AB-M3 gradually decreases with time, and finally 2AB-M is produced, and its area integral is equal to the area integral of the substrate sugar chain (ie, the number of moles);
  • 2AB-M6 When the even-numbered sugar chain substrate 2AB-M6 is degraded , It produces 2AB-M4 sugar in a short time, and 2AB-M4 gradually decreases with time, and finally produces 2AB-M2, which is integrated with the same area of the substrate sugar chain (that is, the number of moles is equal).
  • the enzyme or its recombinase can be used to analyze and determine the number of moles of sugar chains of its original polysaccharide substrate, that is, it can be used as a mole counter for the polysaccharide substrate, and it can also judge the odd-even status of the original substrate.
  • Man02066 has the highest similarity with CjMan26A, CjMan26C, Mana-2, and Man26A respectively: 35.59%, 42.82%, 42.18%, 35.15%;
  • the recombinant plasmid pET30a-Man02066 prepared in Example 3 was used as a template for PCR amplification.
  • the amplification primers are shown in Table 1:
  • the site-directed mutagenesis kit used was purchased from Nanjing Novazan Biotechnology Co., Ltd., and the site-directed mutagenesis was performed according to the experimental operation steps provided in the company's kit, and the recombinant plasmid was obtained and sent to Shenggong Bioengineering (Shanghai) Co., Ltd. for sequencing verification Finally, the correctly constructed recombinant plasmids pET30a-Man02066-E188A and pET30a-Man02066-E296A were obtained.
  • Example 3 the mutant recombinant plasmid was induced and expressed, the bacteria were disrupted by ultrasound, centrifuged, and the supernatant was transferred to obtain crude enzyme solutions E188A and E296A of the mutant recombinase mutants.
  • the konjac glucomannan and locust bean gum substrates with a mass volume concentration (g/mL) of 0.3% were prepared with deionized water, heated and dissolved, and then placed in a water bath environment of 40°C and 50°C to cool down for 1 hour.
  • the volume is less than 200 ⁇ L, make up with sterile deionized water, mix well and continue the reaction for 12 hours.
  • the reaction product was heated in a boiling water bath for 10 minutes, transferred to an ice water bath for 5 minutes, centrifuged at 12,000 ⁇ g and 4°C for 15 minutes, and the supernatant was collected.
  • the concentration of newly formed reducing sugar (OD 540 ) in each reaction system was measured by DNS-reducing sugar method, and the average value was calculated for deviation analysis.
  • the control group is the activity of the recombinase rMan02066 (set to 100%) maximum absorption value, and the relative enzyme activity (RA) is defined as the percentage of the absorption value of each mutant oligosaccharide product to the maximum absorption value.

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Abstract

提供了一种外切型β-甘露聚糖水解酶及其突变体，所述外切型β-甘露聚糖水解酶的氨基酸序列如SEQ ID NO:2所示、编码基因的核苷酸序列如SEQ ID NO:1所示。所述外切型β-甘露聚糖水解酶能降解葡甘聚糖，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，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，具有分子计数器的功能。所述外切型β-甘露聚糖水解酶的突变体在SEQ ID NO:2的第188和/或296位发生突变。

Description

一种二糖外切型β-甘露聚糖水解酶及其应用 技术领域
本发明涉及生物技术领域；具体地说，本发明涉及一种二糖外切型甘露聚糖酶及其编码基因与应用。
背景技术
半纤维素是构成植物细胞壁的结构多糖，通常与纤维素和木质素紧密结合，形成木质纤维素生物质。在半纤维素的众多组成类型中，甘露聚糖(mannan)和木聚糖(xylan)这两种多糖与人们的生活密切相关，它们主要应用于食品、医药、纺织、制浆造纸以及生物燃料等工业领域。甘露聚糖是自然界中含量第二高的半纤维素，它是一类具有复杂结构的多糖，根据糖单元组成结构不同，可分四类：纯甘露聚糖(pure mannan)、葡甘聚糖(glucomannan)、半乳甘露聚糖(galactomannan)以及半乳葡甘聚糖(galactoglucomannan)。
这些甘露聚糖，除了以甘露糖单元为主成分的共性特征之外，其多糖骨架结构还具有多种糖单元随机杂聚、局部组成模块化等复杂性特征，且甘露聚糖的侧链结构也具有类似特征。因此，要想彻底地降解这些聚糖，就需要多种类型酶(酶系)的协同作用。
相比化学或物理学方法，酶法降解甘露多糖从而制备寡糖的策略，具有因反应条件温和而可控性强、因底物选择性清晰而产物明确等优点。因此，作为寡糖制备的工具酶甘露聚糖酶具有重要的研发价值和的经济价值。目前，β-甘露聚糖酶已广泛应用于食品、动物养殖、造纸、生物燃料和石油钻探等各个领域。在食品行业，β-甘露聚糖酶作为饲料添加剂，可作用于食物中的甘露聚糖，辅助产生甘露寡糖益生元。在洗涤剂行业中，许多类型的日化产品和食品，如发胶、洗发水、护发素、牙膏、冰淇淋和烧烤酱等，都含有一定量的甘露聚糖成分作为增稠剂或稳定剂，而这些成分一旦形成污渍，通常难以去除。如果通过β-甘露聚糖酶水解甘露聚糖进行处理，则从难溶多糖变成易溶于水的寡糖片段，从而更容易去除污渍。在造纸业、纸浆和纸的行业中，使用β-甘露聚糖酶可以促进纸浆中木质素的去除，产生与碱预处理相当的结果，且可以很大程度上减少环境污染。
目前，虽然关于β-甘露聚糖酶的专利申请及科研文献数量较多，但关于β-甘露聚糖酶的底物选择机制、多糖/寡糖底物的降解模式、寡糖产物的生成特性，及其与催化机制的内在联系等基础研究则相对较少。这种不足则造成两方面的行业困境：(1)目前虽然有一定数量的甘露聚糖酶产品已经实现了产业化和商品化销售，但可以精确应用的工具酶的数量和类型都极其稀少；(2)酶资源的深入开发与定向改造缺少指导性的理论和技术借鉴，这在一定程度上限制了工具酶的研发水平。
综上，为满足工具型甘露聚糖外切酶的重要应用需求，亟需从加大酶学新资源开发、加强酶的核心应用价值与应用特色分析，并探讨分子酶学改造所需新型指导理论与技术途径等多个角度和层次，全面地开展相关的研究与探索。
发明内容
针对现有技术的不同，本发明提供一种二糖外切型β-甘露聚糖水解酶Man02066及其编码基因与应用。
在第一方面，本发明提供一种外切型β-甘露聚糖酶，其氨基酸序列如SEQ ID NO:2所示。
在优选的实施方式中，所述外切型β-甘露聚糖酶降解含有糖链分支的甘露聚糖或没有糖链分支的线性甘露聚糖；优选地，所述外切型β-甘露聚糖酶降解没有糖链分支的线性甘露聚糖。
在优选的实施方式中，所述甘露聚糖是葡甘聚糖、半乳甘露聚糖、纯甘露聚糖；优选纯甘露聚糖。
在优选的实施方式中，所述外切型β-甘露聚糖酶能降解魔芋来源葡甘聚糖(KGM)、槐豆胶来源的半乳甘露聚糖(LBG)；优选地，所述外切型β-甘露聚糖酶的多糖底物是魔芋来源的葡甘聚糖。
在优选的实施方式中，所述外切型β-甘露聚糖酶仅由一个Glycoside hydrolase family 26 domain模块组成。
在优选的实施方式中，所述外切型β-甘露聚糖酶降解甘露聚糖的反应温度为0-40℃，pH为6.0-8.0。
在优选的实施方式中，所述外切型β-甘露聚糖酶降解魔芋葡甘露聚糖(KGM)和槐豆胶(LBG)的最适温度为40℃，最适pH为6.0。
在优选的实施方式中，所述外切型β-甘露聚糖酶还可以降解甘露寡糖，能降解的最小底物为甘露三糖(M3)，最小产物为甘露单糖(M)。
在第二方面，本发明提供第一方面所述的外切型β-甘露聚糖酶的编码核苷酸序列。
在具体的实施方式中，所述核苷酸序列如SEQ ID NO:1所示。
在第三方面，本发明提供一种表达载体，所述表达载体包含第二方面所述的核苷酸序列。
在第四方面，本发明提供一种宿主细胞，所述宿主细胞包含第三方面所述的表达载体或其基因组上整合有第二方面所述的核苷酸序列。
在优选的实施方式中，所述宿主细胞是用于生产所述外切型β-甘露聚糖酶或用于降解β-甘露聚糖的宿主细胞。
在第五方面，本发明提供第一方面所述的外切型β-甘露聚糖酶在降解甘露聚糖中的应用。
在优选的实施方式中，所述甘露聚糖含有糖链分支的甘露聚糖或没有糖链分支的线性甘露聚糖；优选地，所述甘露聚糖是没有糖链分支的线性甘露聚糖。
在优选的实施方式中，所述甘露聚糖是葡甘聚糖、半乳甘露聚糖、纯甘露聚糖；优选纯甘露聚糖。
在第六方面，本发明提供第一方面所述的外切型β-甘露聚糖酶在分析、测定其原始多糖底物糖链的摩尔数和判断原始底物的奇偶状况中的应用。
在第七方面，本发明提供一种外切型β-甘露聚糖酶，所述外切型β-甘露聚糖酶是第一方面所述的外切型β-甘露聚糖酶突变体。
在具体的实施方式中，所述外切型β-甘露聚糖酶的氨基酸序列如SEQ ID NO:2所示，但在第188和/或296位发生突变。
在优选的实施方式中，所述外切型β-甘露聚糖酶的氨基酸序列中第188位氨基酸由谷氨酸突变为丙氨酸，和/或第296位氨基酸由谷氨酸突变为丙氨酸。
在第八方面，本发明提供一种甘露聚糖降解产物，所述降解产物利用第一方面所述的外切型β-甘露聚糖酶降解甘露聚糖制得。
在优选的实施方式中，所述甘露聚糖是含有糖链分支的甘露聚糖或没有糖链分支的线性甘露聚糖；优选地，所述甘露聚糖是没有糖链分支的线性甘露聚糖。
在优选的实施方式中，所述甘露聚糖是葡甘聚糖、半乳甘露聚糖、纯甘露聚糖；优选纯甘露聚糖。
在优选的实施方式中，所述甘露聚糖是魔芋来源葡甘聚糖(KGM)、槐豆胶来源的半乳甘露聚糖(LBG)；优选地，所述甘露聚糖是魔芋来源的葡甘聚糖。
在第九方面，本发明还提供了所述外切型β-甘露聚糖酶及其突变酶作为工具酶在研究甘露聚糖酶的催化机制中的应用。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1为外切型β-甘露聚糖酶Man02066功能模块构成分析结果图；
图2为重组外切型β-甘露聚糖酶rMan02066表达与纯化的聚丙烯酰胺凝胶电泳图；图中：M、蛋白质分子量标准，条带自上至下大小为116kDa、66.2kDa、45kDa、35kDa、25kDa、18.4kDa、14.4kDa；泳道1、对照菌株破壁前菌体，上样量10μL；泳道2、重组菌破 壁前菌体，上样量2μL；泳道3、重组菌的破壁后上清，上样量2μL；泳道4、经镍柱纯化的rMan02066，上样量2μL；
图3为温度对重组β-甘露聚糖酶rMan02066降解魔芋葡甘聚糖(A)和槐豆胶(B)活性的影响曲线图；
图4为pH对重组β-甘露聚糖酶rMan02066降解魔芋葡甘聚糖(A)和槐豆胶(B)稳定性的影响曲线图；
图5为温度对重组β-甘露聚糖酶rMan02066降解魔芋葡甘聚糖(A)和槐豆胶(B)稳定性的影响曲线图；
图6为pH值对重组β-甘露聚糖酶rMan02066降解魔芋葡甘聚糖(A)和槐豆胶(B)活性和稳定性的影响曲线图；
图7为金属离子及化学试剂对重组β-甘露聚糖酶rMan02066降解魔芋葡甘聚糖(A)和槐豆胶(B)活性的影响柱形图；
图8为重组β-甘露聚糖酶rMan02066降解魔芋葡甘聚糖(A)和槐豆胶(B)过程中的寡糖产物TLC分析图；其中A图：横坐标中M，甘露糖-甘露六糖；1，对照；2-10，分别代表：10s、1min、10min、1h、4h、6h、24h、48h、72h；B图：横坐标中M，甘露糖-甘露六糖；1，对照；2-9，分别代表：10s、1min、10min、1h、6h、24h、48h、72h；
图9为用重组β-甘露聚糖酶rMan02066完全降解魔芋葡甘露聚糖(A)和槐豆胶(B)所制备寡糖片段的 1H-NMR图；
图10为重组β-甘露聚糖酶rMan02066完全降解系列大小甘露寡糖(M-M6)的寡糖产物TLC分析图；其中A图：横坐标中：A：样品顺序：M：M1-M6；1:M1(-)；2:M1(+)；3:M2(-)；4:M2(+)；5:M3(-)；6:M3(+)；7:M4(-)；8:M4(+)；9:M5(-)；10:M5(+)；11:M6(-)；12:M6(+)；其中(-)为阴性对照组，(+)为实验组；B图：横坐标中：M，M1-M6；1：对照；2：酶液稀释10倍反应10s；3-8：10s、1min、30min、3h、12h、48h；C图：横坐标中：M，M1-M6；1，对照；2，酶液稀释10倍反应10s；3-5，10s、1min、48h；
图11为用重组β-甘露聚糖酶rMan02066降解2AB标记的系列大小甘露寡糖(2AB-M～2AB-M6)最终主产物的分析(A)、不同反应时间降解2AB标记的甘露五糖(2AB-M5)(B)和2AB标记的甘露六糖(2AB-M6)(C)的寡糖产物的HPLC分析图(荧光)；
图12为β-甘露聚糖酶Man02066与GH26家族β-甘露聚糖酶多序列比对；
图13为β-甘露聚糖酶Man02066突变体分别降解魔芋葡甘聚糖(A)和槐豆胶(B)的相对活性分析。
具体实施方式
发明人经过广泛而深入的研究，出乎意料地从火色杆菌属细菌(Flammeovirga yaeyamensis)MY04中获得一种二糖外切型β-甘露聚糖酶Man02066；该酶既可以降解魔芋葡甘聚糖，也能降解槐豆胶等其它多种多糖底物，并且具有一定的热稳定性(0-40℃)，从而可用于甘露二糖的专一性制备。在此基础上完成了本发明。
本文利用的所有科学或技术术语具有本发明所属领域的普通技术人员理解的常规含义。为清晰起见，对本文用到的一些术语或概念定义或解释如下。但提供以下定义或解释只是为了技术人员更好地理解本发明的实质，并非要以任何方式显示本发明的保护范围。
β-甘露聚糖酶
本文所用的“β-甘露聚糖酶”具有本领域技术人员常规理解的含义，是指一类能够水解含有β-1,4甘露糖苷键的甘露寡糖和甘露多糖(包括甘露聚糖、葡甘聚糖、半乳甘露聚糖以及半乳葡甘聚糖)的水解酶。
根据酶的底物选择性和催化模式的特点，β-甘露聚糖酶又可分为内切型β-甘露聚糖酶(EC 3.2.1.78)和外切型β-甘露聚糖酶(EC 3.2.1.100)。其中，内切型β-甘露聚糖酶可以水解甘露聚糖主链内部的β-1,4糖苷键，产生一系列聚合度不同的甘露寡糖片段；而外切型β-甘露聚糖酶是从非还原链末端连续作用于底物糖链中的β-1,4糖苷键，切割并产生固定大小的糖单元产物，如甘露二糖。β-甘露聚糖酶广泛存在于自然界生物体内，包括：动物、植物和微生物。深入研究表明，微生物是生产β-甘露聚糖酶的主要来源，尤其细菌、真菌和放线菌等。相比之下，动、植物来源的β-甘露聚糖酶较少，主要分布与豆科植物的种子和软体动物的消化液中。
目前，国内外关于外切型甘露聚糖酶的研究整体较为薄弱，与内切酶相比，科研文献或专利申请的数量都较少，而且：(1)迄今为止，文献所报道的内切型甘露聚糖水解酶多达数十上百个，主要归类于碳水化合物活性酶数据库(CAZy，http://www.cazy.org/)所收录的糖基水解酶家族GH5、GH26、GH113或GH34家族，而外切型甘露聚糖酶只有少数几个，且归类于GH26家族。在GH26家族成员中，已系统进行鉴定的外切型甘露聚糖水解酶仅有2种，包括来自Bacteroides fragilis NCTC 9343的BfMan26A(K.Kawaguchi,T.Senoura,S.Ito,T.Taira,H.Ito,J.Wasaki,S.Ito,The mannobiose-forming exo-mannanase involved in a new mannan catabolic pathway in Bacteroides fragilis,Arch.Microbiol.196(2014)17-23)和来自Cellvibrio japonicus Ueda107的CjMan26C(K.Kawaguchi,T.Senoura,S.Ito,T.Taira,H.Ito,J.Wasaki,S.Ito,The mannobiose-forming exo-mannanase involved in a new mannan catabolic pathway in Bacteroides fragilis,Arch.Microbiol.196(2014)17-23)。相比之下，GH26家族成员中仍然以内切型甘露聚糖水解酶居多，如Cellvibrio japonicus Ueda107的CjMan26A(Ducros,V.,Zechel,D.L.,Murshudov,G.,Gilbert,H.J.,Szabo,L.,Stoll,D.,Withers,S.G.,Davies,G.J.(2002)Angew Chem Int Ed Engl 41:2824)、Bacteroides ovatus的BoMan26B(Bagenholm,V.,Wiemann,M.,Reddy,S.K.,Bhattacharya,A.,Rosengren,A.,Logan,D.T.,Stalbrand,H.(2019)J Biol Chem 294:9100-9117)，也有少量催化模式不明确的，如uncultured Bacteroidetes bacterium的GH26i(Mackenzie AK,Naas AE,Kracun SK,et al.A polysaccharide utilization locus from an uncultured bacteroidetes phylotype suggests ecological adaptation and substrate versatility.Appl Environ Microbiol.2015；81(1):187‐195)。因此，要想获得更多工具酶，亟需加大外切型甘露 聚糖酶新资源的发掘力度。(2)其次，分析GH26家族收录的外切型甘露聚糖酶(EC3.2.1.100)的文献，目前仅知BfMan26A和CjMan26C是严格从底物糖链的非还原端持续切割二糖单元从而彻底降解甘露聚糖的，且最终寡糖主产物以二糖为主，因而都是典型的二糖外切型甘露聚糖水解酶。与之不同的是，Bacteroides ovatus的BoMan26A可以从底物糖链非还原端切割二糖或者三糖单元，且最终以甘露二糖为主产物(
Figure PCTCN2021099490-appb-000001
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 Aβ-MANNANASE[J].Journal of Biological Chemistry,2017,292(1):229-243)。然而，从底物降解模式与寡糖生成特性这两点催化特性进行分析，BoMan26A并不属于严格型的二糖外切型甘露聚糖酶，且参照本专利申请的发明人对内切酶Man01929的研究结果，推测BoMan26A应该是一个典型的、具有可变的底物降解模式的内切型甘露糖水解酶。此外，对Reticulitermes speratus来自的RsMan26H多糖降解模式的研究还发现，短时间(10min)内以聚合度高的寡糖(DP5-DP20)为主，随着时间(10-30min)的延长甘露五糖和甘露六糖不断积累，最后(反应30min后)甘露三糖、甘露二糖、甘露糖不断积累，因而文献将RsMan26H报道为内切型甘露聚糖酶(Tsukagoshi H,Nakamura A,Ishida T,et al.The GH26β-mannanase RsMan26H from a symbiotic protist of the termite Reticulitermes speratus is an endo-processive mannobiohydrolase:heterologous expression and characterization.Biochem Biophys Res Commun.2014；452(3):520-525)。文献关于uncultured Bacteroidetes bacterium来源的GH26i的催化模式与寡糖生成特性的结论不清。因此，为获取工具，还需加强对酶的底物降解模式与寡糖生成特性等酶的核心应用价值与特色的分析与阐释。(3)上述GH26家族5个甘露聚糖水解酶成员的共同特征是，可以专一性的识别甘露糖-甘露糖(MM)内的糖苷键，但是它们对底物的识别、结合与降解是否会受到半乳糖基修饰的影响，且关键活性位点残基的具体贡献仍有待深入研究。
本发明的β-甘露聚糖酶
在本文中，“本发明的外切型β-甘露聚糖酶”、“本发明的β-甘露聚糖酶”、“本发明的β-甘露聚糖水解酶”、“β-甘露聚糖酶Man02066”、“β-甘露聚糖水解酶Man02066”等术语具有相同的含义。
在具体的实施方式中，本发明的外切型β-甘露聚糖酶的氨基酸序列如SEQ ID NO:2所示。本发明的外切型β-甘露聚糖酶可以降解含有糖链分支的甘露聚糖，也可以降解没有糖链分支的线性甘露聚糖；但没有糖链分支的线性甘露聚糖是本发明的外切型β-甘露聚糖酶的最佳底物。
本发明的外切型β-甘露聚糖酶具有一定的温度耐受性，其降解甘露聚糖的反应温度为0-40℃，优选20-40℃；此外，本发明的外切型β-甘露聚糖酶降解甘露聚糖的pH条件为6.0-8.0。
在具体的实施方式中，本发明的外切型β-甘露聚糖酶降解魔芋葡甘露聚糖(KGM)和槐豆胶(LBG)的最适温度为40℃，最适pH为6.0。
本发明的外切型β-甘露聚糖酶在降解甘露聚糖底物时，能够以甘露二糖为单位，从非 还原端连续切割糖链直至结束，因此可用于甘露二糖的专一性制备。
本发明的外切型β-甘露聚糖酶还可以用于分析、测定其原始多糖底物糖链的摩尔数和判断原始底物的奇偶状况。
在本发明的外切型β-甘露聚糖酶的基础上，本发明人进一步发现可以所述外切型β-甘露聚糖酶的保守性催化位点，从而为进一步改造该酶奠定了基础。
在具体的实施方式中，本发明的外切型β-甘露聚糖酶的保守性催化位点在第188和296位。
在优选的实施方式中，所述外切型β-甘露聚糖酶的氨基酸序列中第188位氨基酸由谷氨酸突变为丙氨酸，和/或第296位氨基酸由谷氨酸突变为丙氨酸可以获得无活性的突变体。因此，本发明的外切型β-甘露聚糖酶及其突变酶可以作为工具酶研究甘露聚糖酶的催化机制。
本发明的有益效果
1.本发明首次公开了由火色杆菌属细菌(Flammeovirga yaeyamensis)MY04获得二糖外切型β-甘露聚糖酶Man02066；
2.该酶Man02066既可以降解魔芋葡甘聚糖，也能降解槐豆胶等其它多种多糖底物；
3.该酶具有一定的热稳定性(0-40℃)，以及较窄的pH耐受性(6.0-8.0)；
3.该酶在降解系列纯甘露寡糖底物或还原端被荧光标的纯甘露寡糖底物时，以甘露二糖为单位，从非还原端连续切割糖链直至结束，因此可用于甘露二糖的专一性制备；
4.本发明确认了该酶的催化位点残基为Glu188(酸/碱催化)与Glu296(亲核催化)，从而为外切型β-甘露聚糖酶的研究奠定了基础；
5.该酶在降解还原端被邻氨基苯甲酰胺标记的系列甘露寡糖底物时，可以从非还原端连续切割M2糖，直至剩余甘露二糖(2AB-M2)或甘露糖(2AB-M)，且当终产物是2AB-M2时，所对应的原始底物是偶数糖链，且与之摩尔数相等；而终产物是2AB-M1时，所对应的原始底物是奇数糖链，且摩尔数与之相等。因此，理论上该酶或其重组酶，与糖链底物的荧光标记及分析相结合，可用于分析、测定其原始多糖底物糖链的摩尔数，即用作多糖底物的摩尔计数器，还可以判断原始底物的奇偶状况。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例
实验材料来源
火色杆菌(Flammeovirga yaeyamensis)MY04菌株来源于中国微生物菌种保藏管理委员 会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏日期2008年11月27日，保藏编号CGMCC NO.2777。
在以下实施例中简称火色杆菌MY04。
实施例中涉及的实验材料未给出具体来源的，均为普通市售产品。
实施例1.火色杆菌MY04菌株基因组DNA的提取
将火色杆菌MY04接种至液体培养基YT04中，在28℃、200rpm的条件下，振荡培养至600nm吸光值(OD 600)为1.2；取培养菌液10mL，在12,000×g条件下，4℃离心15min，收集菌体沉淀；用10mL的溶菌酶缓冲液(10mM Tris-HCl，pH 8.0)悬浮菌体，在12,000x g，4℃条件下离心15min，收集菌体沉淀。
上述液体培养基YT04，每升组分如下：
胰蛋白胨10g、酵母提取物5.0g、氯化钠30g，用水溶解并定容至1L，pH 7.2。
向上述菌体沉淀中，每管加入溶菌酶缓冲液6.0mL，得到约7.0mL的菌液，分别加入浓度为20mg/mL的溶菌酶溶液各280μL，使溶菌酶终浓度为800μg/mL；置于冰水浴中1.0h，然后转移至37℃水浴中，温育2h，至反应体系粘稠；加入浓度为100mg/mL的十六烷基磺酸钠溶液0.41mL、100mg/mL的蛋白酶K溶液30μL，在52℃温育1.0h；加入Tris-平衡过的酚/氯仿/异戊醇(体积比25：24：1)溶液7.5mL，轻轻颠倒混匀；在10,000×g、4℃条件下离心10min，收集上清，并加入1.0mL的NaAc-HAc(pH 5.2，3.0M)缓冲液，以及8.5mL的无水乙醇，充分混匀；用枪头挑出丝状DNA，转移至1.5mL的离心管中，以70％乙醇(贮于-20℃)，洗涤2次，微离心后弃掉上清；在10,000×g、4℃条件下离心2min，彻底弃掉上清；将DNA沉淀于无菌工作台中风吹干燥，然后用无菌去离子水在4℃过夜溶解DNA样品，制得基因组DNA。
实施例2.火色杆菌MY04菌株基因组的扫描及其序列分析
本发明人在实施例1制得的火色杆菌MY04菌株基因组DNA中发现一β-甘露聚糖酶的编码基因man02066，基因man02066的编码区长1137bp，核苷酸序列如SEQ ID NO:1所示。基因man02066所编码的β-甘露聚糖酶Man02066共含有378个氨基酸，其氨基酸序列如SEQ ID NO:2所示。
实施例3.基因man02066在大肠杆菌BL21(DE3)菌株中的重组表达
以实施例1制得的基因组DNA为模板，进行PCR扩增。引物序列如下：
man02066扩增的正向引物
Man02066-F：5’-gcg CATATGGAAGATAAGGCAAAGACACC-3’(SEQ ID NO:3)；
man02066扩增的反向引物
Man02066-F：5’-gcg CTCGAGATTCACTTTCATATTCATCAAATC-3’(SEQ ID NO:4)；
正向引物下划线标注的是限制性内切酶Nde I的专一性位点，反向引物下划线标注的是限制性内切酶Xho I的专一性位点。
所用高保真DNA聚合酶Prime STAR HS DNA Polymerase购自中国大连宝生物公司，所用PCR反应试剂按照该公司提供的产品说明进行操作。
PCR反应体系：
2×Primer star GC buffer 5μL，扩增的正向引物0.35μL，扩增的反向引物0.35μL，Template(1ng/μL)1μL，ddH 2O 3.3μL，polymerase 0.1μL，dNTP 0.8μL。
PCR反应条件：
95℃预变性4min；94℃变性40s，60℃退火30s，72℃延伸90s，35个循环；72℃延伸10min；4℃稳定10min。
将PCR产物用限制性内切酶Nde I和Xho I进行双酶切，通过琼脂糖凝胶电泳回收酶切后的PCR产物。将购于美国Invitrogen公司的产品pET-30a(+)质粒DNA，用Nde I和Xho I双酶切，进行琼脂糖凝胶电泳并回收酶切后的产物片段。限制性内切酶Nde I和Xho I均购于中国大连宝生物公司，酶切所用到的酶与底物反应的体系、温度和时间，均按照该公司提供的产品说明操作。
将经过Nde I和Xho I双酶切的PCR产物，与同样经过双酶切的pET-30a(+)质粒载体，在DNA连接酶的催化下进行接；连接产物转化大肠杆菌DH5α菌株，涂布于含有50μg/mL卡那霉素的Luria-Bertani培养基固体平板上，37℃培养16h后，挑取单克隆；将单克隆接入含有50μg/mL卡那霉素的液体Luria-Bertani培养基中培养，分离纯化质粒DNA；将该质粒用扩增引物进行PCR验证，结果得到大小为2.8kb的扩增产物，初步证明构建的重组质粒正确；接着将该重组质粒进行测序，结果表明，在pET-30a(+)的Nde I和Xho I酶切位点之间插入SEQ ID NO.1所示的基因man02066，且插入方向正确，从而进一步证明构建的重组质粒正确。
将正确的重组质粒命名为pE30a-Man02066，转化大肠杆菌菌株BL21(DE3)(购自美国Invitrogen公司)，然后按照Invitrogen公司提供的操作步骤，使用终浓度为0.05mM异丙基硫代半乳糖苷(IPTG)进行重组β-甘露聚糖酶Man02066的诱导表达；在8,000×g、4℃条件下离心15min，收集菌体，并用缓冲液A重悬菌体，冰水浴环境中超声破碎。在15,000×g、4℃条件下进一步离心30min，收集水溶性组分，并用Ni-琼脂糖凝胶对重组β-甘露聚糖酶rMan02066分别进行吸附。用含有咪唑浓度为10、50、100、250、500mM的缓冲液A进行梯度洗脱，纯化条件按照凝胶的产品手册操作。用聚丙烯酰胺凝变性胶电泳检测重组β-甘露聚糖酶rMan02066的纯化情况。结果如图2所示：重组质粒pE30a-Man02066在E.coli BL21(DE3)菌株中经IPTG诱导表达后，产物呈水溶性表达，经镍柱亲和层析纯化后的重组酶rMan02066在电泳胶上呈单一条带，且位置与预测的分子量相吻合；将纯化后的重组酶rMan02066样品装入最小分子截留量为8-14kDa的透析袋，在4℃环境中对缓冲液A进行透析。所说缓冲液A的成分是50mM Tris、150mM NaCl，pH 8.0，制得重组β-甘露聚糖酶rMan02066酶液。
实施例4.重组酶β-甘露聚糖酶rMan02066最适温度的测定
用去离子水分别配制质量体积浓度为0.3％(w/v)的魔芋葡甘聚糖和槐豆胶，加热溶解后，分别置于0℃、10℃、20℃、30℃、35℃、40℃、45℃、50℃、60℃、70℃、80℃水浴环境中温育1h。分别向每900μL底物溶液中添加实施例3制得的重组β-甘露聚糖酶rMan02066的稀释液100μL，其中重组β-甘露聚糖酶rMan02066的稀释液浓度为10μg/mL，混匀后继续反应，隔时取样。每个温度条件下3个平行样品，以沸水浴灭活的重组酶制剂为对照组。
用DNS-还原糖法测定各反应体系中新生成还原糖的浓度(OD 540)，并计算平均值，进行偏差分析。最大吸收值对应的反应温度为重组酶的最适温度，相对酶活(RA)定义为：各吸收值与最大吸收值的百分比。结果如图3所示：以如上所述分别以等质量体积浓度的魔芋葡甘聚糖(简称KGM)和槐豆胶(简称LBG)为底物测定酶活时，重组β-甘露聚糖酶Man02066降解KGM和LBG时均在40℃反应时达到最大活力，这表明该酶降解KGM和LBG的最适反应温度一致，均为40℃。
实施例5.重组β-甘露聚糖酶rMan02066最适pH的测定
分别用浓度为50mM的NaAc-HAC缓冲液、50mM的NaH 2PO 4-Na 2HPO 4缓冲液、50mM的Tris-HCl缓冲液，分别与魔芋葡甘聚糖和槐豆胶配制质量体积浓度(g/mL)为0.3％的魔芋葡甘聚糖或槐豆胶底物，所对应的pH值分别为(5、6)，(6、7、8)，(7、8、9、10)三个区段，各pH值均在最适温度下测定酶活。将各底物置于最适温度中孵育1h，然后向每900μL底物中添加实施例3制得的重组β-甘露聚糖酶rMan02066的稀释液100μL，混匀后开始反应，隔时取样。每个pH条件下3个平行样品，以沸水浴灭活的重组酶制剂为对照组。用DNS-还原糖法测定各反应体系中新生成的还原糖浓度(OD 540)，并计算平均值和偏差。相对酶(RA)活定义为：各组平均吸收值与最大吸收值的百分比。最大吸收值对应的pH为重组酶的最适pH。结果如图4所示：该酶降解KGM和LBG时的最适反应pH均为6.0。
实施例6.重组β-甘露聚糖酶rMan02066的温度稳定性分析
将在0℃、10℃、20℃、30℃、40℃、50℃、60℃、70℃、80℃下热处理不同时间后的实施例3制得的重组β-甘露聚糖酶rMan02066酶液，分别与用蒸馏水配置的质量体积浓度(g/mL)为0.3％的魔芋葡甘聚糖或槐豆胶，按1:9(体积比)的比例混合，然后在最适温度下测定残余酶活，以不经过热处理的酶液酶活定义为100％相对活力。结果如图5所示：重组酶rMan02066在0-40℃温浴24h后，降解KGM时，残余活性大于75％；降解LBG时，残余活性大于60％。上述结果表明：β-甘露聚糖酶Man02066在0-40℃下较为稳定。
实施例7.重组β-甘露聚糖酶rMan02066的pH稳定性分析
将实施例3制得的重组β-甘露聚糖酶rMan02066的酶液，在冰水浴、不同的pH(pH 5～10)环境中分别预孵育2h后，与质量体积浓度(g/mL)为0.3％、在最适温度下预处理1h的魔芋葡甘聚糖或槐豆胶底物溶液按1:9(体积比)的比例混合，然后在最适温度下测定残余酶活，以不经过预处理的酶液的酶活定义为100％，计算相对活力。结果如图6所示：重组酶rMan02066在pH5.0-10.0的缓冲溶液中预处理2h后，无论是降解葡甘露糖(KGM)还是槐豆胶(LBG)，当pH小于6.0或大于8.0时均导致重组酶rMan02066的残余活性急剧下降。上述结果表明：β-甘露聚糖酶Man02066不耐酸碱变化，易失活。
实施例8.金属离子及化学试剂对重组β-甘露聚糖酶rMan02066活性的影响
将用去离子水配置的质量浓度为0.3％的魔芋葡甘聚糖或槐豆胶底物、实施例3制得的重组β-甘露聚糖酶rMan02066、以及水按5:1:4(体积比)的比例混合后，接着向反应体系中添加不同的金属离子，添加的离子终浓度为1mM或10mM，然后在最适条件下反应，按前述的DNS-还原糖法测酶的活力。对照组为不加任何金属离子时rMan02066的活性(设定为100％)。结果如图7所示：重组酶rMan02066降解KGM时，(1)10mM的Ag+能显著抑制重组酶rMan02066的活性，相对酶活仅有7％；(2)除Ca 2+、Mg 2+、Mn 2+还有1mM的Co 2+、Fe 2+外，其余二价和三价金属离子均可以使重组酶rMan02066失活；(3)10mM的EDTA以及1mM或10mM的SDS可以显著抑制重组酶rMan02066的活性甚至失活。该酶降解LBG时与KGM不同的是：(1)10mM的K +可以显著抑制重组酶rMan02066的活性；(2)Co 2+可使重组酶rMan02066失活；(3)10mM的DTT可以显著抑制重组酶rMan02066的活性。
实施例9.DNS-还原糖法测定重组β-甘露聚糖酶rMan02066的酶活
将用去离子水配置的质量浓度为0.3％的魔芋葡甘聚糖、槐豆胶、瓜尔胶底物，浓度为10μg/mL的重组β-甘露聚糖酶rMan02066酶液、最适缓冲液以及水按2:1:3:4(体积比)的比例混合后，最适温度下反应。将反应产物在沸水浴中加热10min，转入冰水浴中5min,在12,000×g、4℃条件下离心15min，收集上清；将一定体积的上清与等体积的DNS(3,5-对硝基二甲苯)-反应液混匀，在沸水浴中加热10min，降至室温，在540nm测定吸收值。用分析纯的甘露糖作标准品，同样方法操作，绘制甘露糖的摩尔浓度与OD 540之间的量效关系曲线。用购于上海生工生物工程有限公司的蛋白质定量试剂盒测定重组β-甘露聚糖酶rMan02066酶液中的蛋白质含量。按照国际标准定义计算酶的活力单位，即在标准条件下，每分钟内产生1μmol产物所需的酶量为1个IU。结果表明：重组β-甘露聚糖酶rMan02066能以魔芋葡甘聚糖或槐豆胶为底物，酶解并产生还原糖产物，酶活分别为30.67±1.2U/mg和12.44±1.5U/mg，但是该酶几乎不能降解测试所用的瓜尔胶多糖。
实施例10.重组β-甘露聚糖酶rMan02066降解魔芋葡甘聚糖和槐豆胶的寡糖产物的TLC分析
用去离子水配制质量体积浓度(g/mL)为0.3％的魔芋葡甘聚糖和槐豆胶底物，加热溶解后，分别置于40℃和50℃水浴环境中降温1h。向每100μL底物中添加实施例3制得的重组酶rMan02066的稀释液1-100μL，不足200μL体积时，用无菌去离子水补足；混匀后继续反应，隔时取样。将反应产物在沸水浴中加热10min，转入冰水浴中放置5min；在12,000×g、4℃条件下离心15min，收集上清。预先在沸水浴中灭活的重组β-甘露聚糖rMan02066酶液，做阴性对照反应。
使用薄层色谱(TLC)的方法分析产物，取4uL上清于层析板(TLC Silica 60 F254，德国MERK)，展开剂为正丁醇:乙醇:水体积比为2:1:1，显色剂为二苯胺:苯胺:磷酸:丙酮＝1g:1mL:5mL:50mL的条件下染色10s，然后在110℃条件下显色10min，拍照。
图8是重组β-甘露聚糖酶rMan02066降解魔芋葡甘聚糖(A)和槐豆胶(B)过程中的寡糖产物TLC分析图；
其中A图：横坐标中M，甘露糖-甘露六糖；1，对照；2-10，分别代表：10s、1min、10min、1h、4h、6h、24h、48h、72h；
B图：横坐标中M，甘露糖-甘露六糖；1，对照；2-9，分别代表：10s、1min、10min、1h、6h、24h、48h、72h。
结果如图8(A)所示，重组酶rMan02066降解KGM时，短时间内生成一系列寡糖，随着时间延长48h后，最终以四糖至二糖为主。又如图8(B)所示，重组酶rMan02066降解LBG时，差异较大，即短时间内寡糖主产物是二糖，且随着时间延长，二糖产物逐渐积累，最终显著地以二糖为主。综合分析后初步推测：重组酶rMan02066降解KGM时是二糖外切模式，但由于rMan02066对KGM的活性低以及KGM主链结构中葡萄糖单元存在，造成酶解过程中会产生一系列不同大小的寡糖产物，而与重组酶rMan02066降解LBG时的寡糖产物在TLC分析中有一定差异。因此，重组酶rMan02066在降解KGM、LBG等不同甘露聚糖底物时，底物的组成与结构、酶切速度大小等会造成底物降解行为的不同。
实施例11.重组β-甘露聚糖酶rMan02066彻底降解魔芋葡甘聚糖和槐豆胶的寡糖产物的 1H-NMR鉴定
用重组β-甘露聚糖酶rMan02066分别完全降解葡甘聚糖和槐豆胶，得到酶解产物后浓缩；使用高效液相凝胶色谱法对样品分离纯化；检测器为示差折光检测器(RID)，色谱柱为Superdex TM 30Increase 10/300GL，流动相为0.2M碳酸氢铵，流速为0.4mL/min。
分离纯化样品分别进行多次冷冻干燥除盐，然后用重水(D 2O)溶解，冷冻干燥进行氢氘置换，最后 1H-NMR检测。
通过 1H-NMR数据分析上述重组β-甘露聚糖酶rMan02066降解魔芋葡甘聚糖(图9A)和槐豆胶(图9B)的寡糖产物的结构特征。
将重组酶rMan02066降解KGM的最终主产物经分子凝胶色谱柱分离获得二糖、三糖、四糖产物片段，分别进行 1H-NMR结构鉴定，如图9(A)所示，5.26ppm和5.04ppm化学位移值分别为对应寡糖还原端的葡萄糖和甘露糖异头氢信号。从上述结果推测重组酶 rMan02066与rMan01929类似，降解KGM后倾向于产生还原端为甘露糖为主的系列寡糖终产物片段。
重组酶rMan02066降解LBG的最终主产物经分离，获得二糖～四糖、六糖-八糖等寡糖片段，未能获得五糖产物片段。通过 1H-NMR测试，结果如图9(B)所示，同样方法分析后，认为：(1)酶解反应中的主产物二糖片段主要为MM，含少量MG(G为半乳糖)；(2)在酶解LBG的反应中，更大寡糖产物片段中半乳糖含量较高，阻遏了重组酶rMan02066对这些产物的进一步降解。这表明，在Man02066降解LBG的过程中，二糖产物MM属于主动生成的，而更高分子量的寡糖产物因富含半乳糖，不利于进一步被深度酶学降解而被动成为了寡糖最终主产物的成分。
实施例12.重组酶rMan02066降解系列甘露寡糖的终产物分析
取约含20μg系列甘露寡糖(M-M6)的溶液，150mmol/L的NaH 2PO4-Na 2HPO 4(pH 7.0)缓冲溶液、实施例3制得的重组β-甘露聚糖酶rMan02066的稀释液，按照体积比1:1:1混匀，分别在40℃反应24h。将反应体系置沸水浴中10min，转至冰水浴5min，在12,000×g、4℃条件下离心至少15min。收集上清，作为重组β-甘露聚糖酶rMan02066的寡糖降解产物。以预先在沸水浴中灭活的重组β-甘露聚糖rMan02066酶液，做阴性对照反应。
按照实施例10所述的展开条件，将重组β-甘露聚糖酶rMan02066酶解甘露寡糖(M-M6)的样品，取2uL检测。按照实施例10所述显色条件，进行显色，然后分析。
图10是重组β-甘露聚糖酶rMan02066完全降解系列大小甘露寡糖(M-M6)的寡糖产物TLC分析图；
其中A图：横坐标中：A：样品顺序：M：M1-M6；1:M1(-)；2:M1(+)；3:M2(-)；4:M2(+)；5:M3(-)；6:M3(+)；7:M4(-)；8:M4(+)；9:M5(-)；10:M5(+)；11:M6(-)；12:M6(+)；其中(-)为阴性对照组，(+)为实验组；
B图：横坐标中：M，M1-M6；1：对照；2:酶液稀释10倍反应10s；3-8:10s、1min、30min、3h、12h、48h；
C图：横坐标中：M，M1-M6；1，对照；2，酶液稀释10倍反应10s；3-5，10s、1min、48h。
上述其中酶液稀释10倍反应10s代表将实施例3制得酶液稀释10倍后反应10s，其余为实施例3制得酶液进行反应10s，1min，30min，3h，12h，48h。
结果如图10所示，本申请所述的重组β-甘露聚糖酶rMan02066：
(1)图10(A)可知，可以降解M3-M6糖，不能降解M2以及M1；降解M6或者M4时最终产物为M2，降解M5或者M3时，最终产物为M2和M1；
(2)图10(B)可知，降解M5时，短时间内产生M3&M2，随着时间的延长，M3逐渐降解，产生M2&M1；
(3)图10(C)可知，降解M6时，短时间内产生M4&M2，随着时间延长，M4逐渐降解为M2，最终主产物为M2。
这些结果表明，重组酶rMan02066降解甘露寡糖时：(1)最小底物为M3，最小产物为M1；(2)当奇数糖为底物时，最终主产物为M2和M1；偶数糖为底物时，最终主产物为M2；(3)rMan02066降解甘露寡糖时，推测为二糖持续外切模式。
实施例13.重组β-甘露聚糖酶rMan02066酶切模式的荧光-高效液相色谱(HPLC)分析
取一系列含10μg甘露寡糖(M-M6)的溶液，分别旋转蒸干。加入含过量邻氨基苯甲酰胺(2-AB)、氰基硼氢化钠sodium cyanoborohydride的二甲亚砜(DMSO)溶液，混匀后置60℃水浴中温育2h。旋转蒸干，加入去离子水溶解样品，将样品与氯仿共振荡，离心，收集上清。继续用氯仿反复抽提，不少于7次，得到还原性末端分别被2-AB标记了的甘露六糖(2AB-M6)、甘露五糖(2AB-M5)、甘露四糖(2AB-M4)、甘露三糖(2AB-M3)、甘露二糖(2AB-M2)、甘露糖(2AB-M)等系列底物糖链。
取上述2AB-M6、2AB-M5、2AB-M3、2AB-M2、2AB-M产物、实施例3制得的重组β-甘露聚糖酶rMan02066的稀释液、150mmol/L的NaH 2PO4-Na 2HPO 4(pH7.0)缓冲液及水，按照体积比2:1:3:4混匀，置于40℃水浴中反应。将反应体系置沸水浴中10min，转至冰水浴5min，在12,000×g、4℃条件下离心至少15min。收集上清。以预先在沸水浴中加热10min，然后冰水浴10min失活的重组β-甘露聚糖酶rMan02066酶液，做阴性对照反应。
用浓度为0.20mol/L的NH 4HCO 3溶液，平衡Superdex TM 30Increase 10/300GL(GE公司，即美国通用电气公司)分子凝胶色谱柱，流速0.40mL/min，至少2柱床。将上述荧光标记的系列甘露寡糖的不同酶解时间的样品，以自动进样器加载20-200ng/样品，其它条件不变，330nm激发波长，420nm发射波长检测。用HPLC操作软件，分析各寡糖组分的积分面积，结合理论分子量大小，计算相对摩尔浓度。
结果如图11(A)所示，可知重组酶rMan02066降解还原端被2AB标记的甘露寡糖时：(1)降解偶数糖链底物2AB-M6或2AB-M4时，终产物以2AB-M2&M2为主；(2)降解奇数糖链底物2AB-M5时，终产物以2AB-M&M&M2为主；(3)降解2AB-M3时，终产物以2AB-M&M2为主；(4)该酶不能降解2AB-M2和2AB-M；(5)此外，经面积积分法得知，降解2AB-M4时，产生的2AB-M2的摩尔数与底物糖链的摩尔数相等，而降解2AB-M3时，产生的2AB-M的摩尔数与底物糖链的摩尔数相等。
如图11(B)和(C)所示，重组酶rMan02066降解2AB-M5和2AB-M6的时间梯度知：(1)降解奇数糖链底物2AB-M5时，短时间内产生2AB-M3，随着时间的延长2AB-M3逐渐减少，最终产生2AB-M，且其面积积分与底物糖链的面积积分(即摩尔数)相等；(2)降解偶数糖链底物2AB-M6时，短时间内产生2AB-M4糖，随着时间的延长2AB-M4逐渐减少，最终产生2AB-M2，且其与底物糖链等面积积分(即摩尔数相等)。
上述结果表明：重组酶rMan02066降解荧光标记的甘露寡糖时(1)最小底物为2AB-M3，最小产物为M；(2)从底物糖链底物的非还原端持续切割M2，并表现为二糖外切模式；(3)当含荧光标记的终产物是2AB-M2时，所对应的原始底物是偶数糖链，且与之摩尔数相等；而含荧光标记的终产物是2AB-M1时，所对应的原始底物是奇数糖链，且摩尔数 与之相等。因此，理论上该酶或其重组酶可用于分析、测定其原始多糖底物糖链的摩尔数，即用作多糖底物的摩尔计数器，还可以判断原始底物的奇偶状况。
实施例14.Man02066的多序列比对分析
使用利用DNAMAN软件对Man02066和已鉴定的GH26家族的甘露聚糖酶的蛋白质序列比对。
如图12所示，将β-甘露聚糖酶Man02066(简称Man02066)蛋白序列与GH26家族的甘露聚糖酶的蛋白质序列比对：
(1)通过Blast P分析，在已鉴定的酶中，Man02066与CjMan26A、CjMan26C、Mana-2、Man26A的相似度最高分别为：35.59％、42.82％、42.18％、35.15％；
(2)通过DNAMAN 8.0进行多序列比对分析可知：Man02066序列中保守性的催化位点残基为Glu 188(酸/碱催化)和Glu 296(亲核催化)。
实施例15.Man02066的基因突变
以实施例3制得的重组质粒pET30a-Man02066为模板，进行PCR扩增，扩增引物如表1所示：
表1.突变所用的引物
Figure PCTCN2021099490-appb-000002
所用定点突变试剂盒购自南京诺唯赞生物科技有限公司，按照该公司试剂盒中所提供的实验操作步骤，进行定点突变，获得重组质粒送至生工生物工程(上海)股份有限公司测序验证，最后得到构建正确的重组质粒pET30a-Man02066-E188A和pET30a-Man02066-E296A。
按照实施例3，对突变重组质粒进行诱导表达，超声破碎菌体后，离心，转移上清，获得突变后的重组酶突变体的粗酶液E188A和E296A。
实施例17.rMan02066(简称Man02066)突变体的酶活分析
用去离子水配制质量体积浓度(g/mL)为0.3％的魔芋葡甘聚糖和槐豆胶底物，加热溶解后，分别置于40℃和50℃水浴环境中降温1h。向每100μL底物中添加实施例16制得的突变体粗酶液的稀释液10-100μL，不足200μL体积时，用无菌去离子水补足，混匀后继续反应，反应12h。将反应产物在沸水浴中加热10min，转入冰水浴中5min，在12,000×g、4℃条件下离心15min，收集上清。
用DNS-还原糖法测定各反应体系中新生成还原糖的浓度(OD 540)，并计算平均值，进行偏差分析。对照组为重组酶rMan02066的活性(设定为100％)最大吸收值,相对酶活(RA)定义为：各突变体寡糖产物的吸收值与最大吸收值的百分比。
结果Man02066的系列突变体在降解KGM(图14A)和LBG(图14B)时，如图所示：E188、E296残基突变后，酶突变体均失活，因此证实该两个谷氨酸残基为Man02066的保守催化位点残基。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种外切型β-甘露聚糖酶，其氨基酸序列如SEQ ID NO:2所示。
  2. 权利要求1所述的外切型β-甘露聚糖酶的编码核苷酸序列。
  3. 如权利要求2所述的核苷酸序列，其特征在于，所述核苷酸序列如SEQ ID NO:1所示。
  4. 一种表达载体，所述表达载体包含权利要求2或3所述的核苷酸序列。
  5. 一种宿主细胞，所述宿主细胞包含权利要求4所述的表达载体或其基因组上整合有权利要求2或3所述的核苷酸序列。
  6. 如权利要求1所述的外切型β-甘露聚糖酶在降解甘露聚糖中的应用。
  7. 如权利要求1所述的外切型β-甘露聚糖酶在分析、测定其原始多糖底物糖链的摩尔数和判断原始底物的奇偶状况中的应用。
  8. 一种外切型β-甘露聚糖酶，其特征在于，所述外切型β-甘露聚糖酶是权利要求1所述的外切型β-甘露聚糖酶突变体。
  9. 如权利要求8所述的外切型β-甘露聚糖酶，其氨基酸序列如SEQ ID NO:2所示，但在第188和/或296位发生突变。
  10. 一种甘露聚糖降解产物，所述降解产物利用权利要求1所述的外切型β-甘露聚糖酶降解甘露聚糖制得。
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