WO2021208519A1 - 一种内切型β-甘露聚糖水解酶Man01929及其突变成糖基转移酶的方法及应用 - Google Patents

一种内切型β-甘露聚糖水解酶Man01929及其突变成糖基转移酶的方法及应用 Download PDF

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WO2021208519A1
WO2021208519A1 PCT/CN2020/142062 CN2020142062W WO2021208519A1 WO 2021208519 A1 WO2021208519 A1 WO 2021208519A1 CN 2020142062 W CN2020142062 W CN 2020142062W WO 2021208519 A1 WO2021208519 A1 WO 2021208519A1
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man01929
mannanase
amino acid
endo
type
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French (fr)
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韩文君
程媛媛
宓延红
古静燕
李新
卫洁
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济南爱科替维生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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

Definitions

  • the invention relates to an endo-type ⁇ -mannan hydrolase Man01929 and its mutation into a glycosyltransferase method and application, belonging to the field of biotechnology, and related to protein improvement technology.
  • Mannan is a type of polysaccharide with a complex structure. According to the different structure of sugar unit, it can be divided into four categories: pure mannan, glucomannan, galactomannan ) And galactoglucomannan (galactoglucomannan). Due to its complex structure, mannan requires the synergistic effect of multiple enzymes for complete hydrolysis, such as ⁇ -mannanase (EC 3.2.1.78), ⁇ -mannosidase (EC 3.2.1.25), ⁇ -glucosidase (EC 3.2.1.21), acetylmannan esterase (EC 3.1.1.6), ⁇ -galactosidase (EC 3.2.1.22).
  • ⁇ -mannanase EC 3.2.1.78
  • ⁇ -mannosidase EC 3.2.1.25
  • ⁇ -glucosidase EC 3.2.1.21
  • acetylmannan esterase EC 3.1.1.6
  • ⁇ -galactosidase
  • ⁇ -Mannanase is a type of glycosyl hydrolase (Glycoside Hydrolase, GH), which plays a key role in the hydrolysis process. It can hydrolyze the ⁇ -1,4 glycosidic bonds inside the mannan backbone to produce mannose oligosaccharides. Fragment. Mannan oligosaccharides are made up of 2-10 sugar units linked by glycosidic bonds, and have a variety of physiological activities, such as anti-tumor, regulating immunity, and promoting cell division. Compared with chemical degradation method, the strategy of enzymatic degradation of mannose to prepare oligosaccharides has the advantages of mild reaction conditions, strong controllability, clear substrate selectivity and clear products.
  • ⁇ -mannanase has the potential to be popularized and applied. .
  • ⁇ -mannanase has become more and more widely used in various fields such as food, aquaculture, detergent industry, bioethanol and oil drilling.
  • the substrate selectivity of ⁇ -mannanase and its recognition mechanism are not clear. As a tool enzyme, both a clear substrate selectivity and a clear substrate selection mechanism are required. However, there are few existing studies on the factors that determine enzyme substrate selectivity. This may be caused by two reasons: First, the size or structure of the various sugar units in the mannan substrate is highly similar, which makes it difficult for the enzyme to recognize and degrade the substrate specifically, and most of the mannan polymer The broad spectrum of carbohydrases is an example. Second, ⁇ -mannanase itself has a flexible structure and can recognize and bind a variety of substrates. However, which glycosyl binding sites are specifically involved in the selective recognition and action process of substrates and other mechanisms have been seldom studied.
  • Some natural ⁇ -mannanases such as GH5 and GH113 family members, have both glycosylhydrolase activity and glycosyltransferase activity, which is also specific for the production of oligosaccharides by degrading polysaccharides.
  • the development of tool enzymes has caused difficulties. It is necessary to understand the mechanism of the same enzyme for the conversion and regulation of the two catalytic mechanisms of glycosylhydrolase and glycosyltransferase.
  • the present invention provides an endo-type ⁇ -mannan hydrolase Man01929 and a method and application for its mutation into glycosyltransferase.
  • the ⁇ -mannan hydrolase Man01929 is hereinafter referred to as ⁇ -mannanase Man01929 for short.
  • the encoding gene man01929 of an endo-type ⁇ -mannanase Man01929 the nucleotide sequence is shown in SEQ ID NO.1.
  • the endo-type ⁇ -mannanase Man01929 has an amino acid sequence as shown in SEQ ID NO.2.
  • a recombinant expression vector I contains the above-mentioned endo-type ⁇ -mannanase Man01929 encoding gene man01929.
  • a recombinant strain I contains the above-mentioned endo-type ⁇ -mannanase Man01929 encoding gene man01929.
  • the endo-type ⁇ -mannanase Man01929 is used in the degradation of glucomannan and galactomannan.
  • an endo-type ⁇ -mannanase Man01929 mutant enzyme the amino acid mutation site is the 118th, 119th, 124th, 221st, 268th, and 269th of the endo-type ⁇ -mannanase Man01929 amino acid sequence SEQ ID NO.2. , One or more than two amino acids at position 323.
  • the amino acid mutation site of the mutant enzyme is the amino acids 118, 119, 124, 221, 268, 269, 323 of the endo-type ⁇ -mannanase Man01929 amino acid sequence SEQ ID NO.2 One or more than two; the amino acids are:
  • amino acid at position 118 was mutated from aspartic acid to alanine, glutamic acid or tyrosine;
  • the amino acid at position 119 was mutated from methionine to valine
  • amino acid at position 124 was mutated from aspartic acid to alanine, glutamic acid or tyrosine;
  • the amino acid at position 221 was mutated from tyrosine to alanine
  • Amino acid at position 268 was mutated from glutamic acid to alanine
  • amino acid at position 269 was mutated from isoleucine to tyrosine;
  • the amino acid at position 323 was mutated from glutamic acid to alanine, aspartic acid or tyrosine.
  • the amino acid mutation site is that the amino acid at position 124 of the endo-type ⁇ -mannanase Man01929 amino acid sequence SEQ ID NO. 2 is mutated from aspartic acid to tyrosine.
  • the coding gene of the above-mentioned mutant enzyme is subjected to site-directed mutation on the coding gene man01929 of the above-mentioned endo-type ⁇ -mannanase Man01929 according to the mutation site of the above-mentioned amino acid.
  • a recombinant expression vector II which contains the coding gene of the above-mentioned mutant enzyme.
  • the endo-type ⁇ -mannanase Man01929 mutant enzyme is used in the degradation of glucomannan and galactomannan.
  • the endo-type ⁇ -mannanase Man01929 mutant enzyme is used in the degradation of manntetraose to produce mannpentaose.
  • the amino acid mutation site is that the amino acid at position 124 of the endo-type ⁇ -mannanase Man01929 amino acid sequence SEQ ID NO. 2 is mutated from aspartic acid to tyrosine.
  • the aforementioned endo-type ⁇ -mannanase Man01929 and the mutants of the enzyme are used as tool enzymes in revealing the conversion and regulation mechanism of GH5 family members for two catalytic mechanisms of glycosyl hydrolases and glycosyltransferases.
  • the mutant of the enzyme is the aforementioned endo-type ⁇ -mannanase Man01929 mutant enzyme.
  • the amino acid mutation site is that the amino acid at position 124 of the endo-type ⁇ -mannanase Man01929 amino acid sequence SEQ ID NO. 2 is mutated from aspartic acid to tyrosine .
  • the encoding gene man01929 of the endo-type ⁇ -mannanase Man01929 has a total length of 2799 bp, and the encoded protein contains 932 amino acids and has a molecular weight of 103.1 kDa.
  • the endo-type ⁇ -mannanase Man01929 has only glycosyl hydrolase activity but no glycosyl transferase activity.
  • the endo-type ⁇ -mannanase Man01929 degrades mannan from the reducing end.
  • the endo-type ⁇ -mannanase Man01929 can degrade both konjac-derived glucomannan and galactomannan derived from locust bean gum, and the most suitable polysaccharide substrate is konjac-derived glucomannan ; Therefore, compared with mannans containing sugar chain branches, endo-type ⁇ -mannanase Man01929 is more suitable for degrading linear mannans without branches.
  • the endo-type ⁇ -mannanase Man01929 contains 5 functional modules, of which the N-terminal contains a Glycoside hydrolase family 5 (GH5) functional module, and the 447-585 amino acids are the Coagulation factor 5/8 C-terminal domain functional module, Amino acids 595-766 contain two PKD/Chitinase domain functional modules, and the C-terminal contains two functional modules: Carbohydrate-binding module 64 (CBM64) and secretion system C-terminal sorting domain.
  • Glycoside hydrolase family 5 GH5
  • 447-585 amino acids are the Coagulation factor 5/8 C-terminal domain functional module
  • Amino acids 595-766 contain two PKD/Chitinase domain functional modules
  • the C-terminal contains two functional modules: Carbohydrate-binding module 64 (CBM64) and secretion system C-terminal sorting domain.
  • the optimal temperature for the endo-type ⁇ -mannanase Man01929 to degrade konjac glucomannan oligosaccharide (KGM) is 40°C, and the optimal pH is 8.0; when degrading locust bean gum (LBG), the optimal temperature is 50 °C, the optimum pH is 5.0.
  • the substrate is pure mannose oligosaccharide
  • the smallest substrate of the enzyme is manntetraose (M4)
  • the smallest product is mannose (M).
  • the smallest substrate of the enzyme is manntetraose (2-AB-M4) whose reducing end is labeled with anthranilamide, and the smallest product is mannose (M) .
  • the present invention discloses for the first time that an endo-type ⁇ -mannanase Man01929 encoding gene man01929 is obtained from the genome of Flammeovirga yaeyamensis MY04, which can degrade konjac glucomannan. , Can also degrade many polysaccharide substrates such as locust bean gum; the enzyme has certain thermal stability (0-40°C), and wide pH tolerance (5.0-10.0), stable physical and chemical properties, and has industrial applications Potential.
  • Man01929 When the endo-type ⁇ -mannanase Man01929 involved in the present invention degrades a series of pure mannose oligosaccharide substrates or pure mannose oligosaccharide substrates whose reducing end is fluorescently labeled, it mainly cuts the sugar chain from the reducing end to produce less
  • the oligosaccharide product has a variable substrate degradation mode and can be applied to prepare mannose oligosaccharide products with different degrees of polymerization.
  • the present invention conducts site-directed mutagenesis on the endo-type ⁇ -mannanase Man01929, and confirms the endo-type ⁇ -mannase through comparative analysis.
  • the invention provides important clues for further revealing the substrate selective mechanism of endo-type ⁇ -mannanase Man01929, and at the same time provides a useful reference for revealing the substrate selective mechanism of other ⁇ -mannanase members of the GH5 family.
  • the present invention successfully mutated the 124th amino acid of endo-type ⁇ -mannanase Man01929 from aspartic acid to tyrosine by means of gene-directed mutation, the mutant enzyme not only still has glycosyl hydrolysis Enzyme activity, the activity of glycosyltransferase was also obtained, which exceeded the expectations of researchers. This is to reveal that GH5 family members, including endo-type ⁇ -mannanase Man01929, carry out glycosyl hydrolase and glycosyl transfer. The study and application of the mechanism of the conversion and regulation of the two catalytic mechanisms of enzymes provide an important basis.
  • M protein molecular weight standard, the size of the band from top to bottom is 170kDa, 130kDa, 100kDa, 70kDa, 55kDa, 40kDa, 35kDa, 25kDa, 15kDa, 10kDa; lane 1, control strain, pre-broken cell, loading sample The amount is 10 ⁇ L; Lane 2, the cell before the recombinant bacteria is broken, the loading amount is 2 ⁇ L; Lane 3, The supernatant after the recombinant bacteria is broken, the loading amount is 2 ⁇ L; Lane 4, rMan01929 purified by a nickel column, the loading amount is 2 ⁇ L ;
  • Figure 4 A graph showing the influence of pH on the stability of recombinant ⁇ -mannanase rMan01929 to degrade konjac glucomannan (A) and locust bean gum (B);
  • Figure 6 A graph showing the influence of pH on the activity and stability of recombinant ⁇ -mannanase rMan01929 to degrade konjac glucomannan (A) and locust bean gum (B);
  • Figure 7 Bar graph of the influence of metal ions and chemical reagents on the activity of recombinant ⁇ -mannanase rMan01929 to degrade konjac glucomannan (A) and locust bean gum (B);
  • FIG. 1 TLC analysis diagram of oligosaccharide products during the degradation of konjac glucomannan (A) and locust bean gum (B) by recombinant ⁇ -mannanase rMan01929;
  • FIG. 1 M in the abscissa, mannose-mannose; 1, control; 2-9, respectively: 10s, 1min, 10min, 1h, 6h, 24h, 48h, 72h;
  • Figure B In the abscissa, M, mannose-mannose; 1, control; 2-9, respectively represent: enzyme solution diluted 50 times for 10s, enzyme solution diluted 10 times for 10s, 10s, 30s, 1min, 30min, 48h, 72h;
  • Figure 9. 1 H-NMR chart of oligosaccharide fragments prepared by using recombinant ⁇ -mannase rMan01929 to completely degrade konjac glucomannan oligosaccharides (A) and locust bean gum (B);
  • Figure B In the abscissa: M, M1-M6; 1: Control; 2: Enzyme solution diluted 10-fold and react for 10s; 3:10s; 4:24h; 5,48h;
  • Figure C In the abscissa: M, M1-M6; 1, control; 2, enzyme solution diluted 10-fold and react for 10s; 3-8, 10s, 10min, 1h, 4h, 24h, 48h;
  • FIG. 11 Degradation of 2AB-labeled series of large and small mannose oligosaccharides (2AB-M ⁇ 2AB-M6) with recombinant ⁇ -mannase rMan01929 (A), degradation of 2AB-labeled mannpentasaccharides (A) 2AB-M5) (B) and 2AB-labeled mannose (2AB-M6) (C) HPLC analysis chart (fluorescence) of oligosaccharide products;
  • Figure 14 The relative activity analysis of degrading konjac glucomannan (A) and locust bean gum (B) with a series of mutants of recombinant enzyme ⁇ -mannanase rMan01929;
  • FIG. 15 TLC analysis diagram of oligosaccharide products of konjac glucomannan (A) and locust bean gum (B) degraded by a series of mutants of recombinant ⁇ -mannanase rMan01929;
  • M M1-M6; 1, (-); 2, Man01929; 3, D118A; 4, D118E; 5, D118Y; 6, D124A; 7, D124E; 8, D124H; 9, D124Y; 10, E323A; 11, E323A; 12, E323Y;
  • Figure 16 After the mutation of Asp 118 of ⁇ -mannase Man01929 to Ala or Glu, and the mutation of Asp 124 to Tyr, Ala or His, the degradation of mannohexaose (A), mannopentaose (B) or mannotetraose (C) ) TLC analysis;
  • M M1-M6; 1: negative control; 2: Man01929; 3: D124Y; 4: D124A; 5: D124H; 6: D118A; 7: D118E;
  • FIG. 1 M, M1-M6; 1, negative control; 2, 10min; 3, 1h; 4, 4h; 5, 6h; 6, 8h; 7, 24h; 8, Man01929;
  • Panel B M, M1-M6; 1, negative control; 2, 1min; 3, 30min; 4, 2h;; 5, 12h; 6, 24h; 7, 48h; 8, Man01929;
  • Panel C M, M1-M6; 1, negative control; 2, 3h; 3, 12h; 4, 24h; 5, 48h; 6, Man01929.
  • the description of the following embodiments is for the purpose of comprehensively disclosing some common techniques of how to implement the present invention, but not for limiting the application scope of the present invention.
  • the inventors have tried their best to ensure the accuracy of the parameters in the examples (such as amount, temperature, etc.), but some experimental errors and deviations should also be considered.
  • the molecular weight in the present invention refers to the weight average molecular weight.
  • the temperature is in degrees Celsius.
  • Flammeovirga yaeyamensis MY04 strain 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 , The deposit number is CGMCC NO.2777.
  • the above liquid medium YT04 has the following components per liter:
  • Example 1 The genomic DNA prepared in Example 1 was scanned and sequenced using pyrosequencing technology, which was completed by Shanghai Meiji Biological Company.
  • NCBI National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/
  • NCBI National Center for Biotechnology Information
  • the analysis software of the NCBI website used is Open Reading Frame Finder (ORF Finder, http://www.ncbi.nlm.nih.gov/gorf/gorf.html) and Basic Local Alignment Search Tool (BLAST, http:// blast.ncbi.nlm.nih.gov/Blast.cgi).
  • the results of analysis with the above biological software show that the genomic DNA of the MY04 strain of Chromobacterium pyogenes carries a ⁇ -mannanase encoding gene man01929.
  • the coding region of the gene man01929 is 2799 bp in length, and the nucleotide sequence is as shown in SEQ ID NO.1 Show.
  • the ⁇ -mannanase Man01929 encoded by the gene man01929 contains a total of 932 amino acids, and its amino acid sequence is shown in SEQ ID NO.2.
  • Man01929 has the greatest similarity to Man5A from Blue Mussel Mytilus edulis (that is, it is similar to the protein sequence of Man5A encoded in the blue mussel genome. Degree maximum), reaching 36.79%.
  • the comprehensive analysis function module of InterProScan shows that ⁇ -mannanase Man01929 is composed of five functional modules, including Glycoside hydrolase, family 5 (GH5 module), Coagulation factor 5/8 C-terminal domain, Two PKD/Chitinase domains, Carbohydrate-binding module 64 (CBM 64 module), Secretion system C-terminal sorting domain; analysis with the biology website ExPASY shows that the theoretical molecular weight of protein ⁇ -mannanase Man01929 is about 103.1kD ; Use signal peptide online prediction software SignalP 5.0 (http://www.cbs.dtu.dk/services/SignalP/) online analysis, the protein has no signal peptide.
  • SignalP 5.0 http://www.cbs.dtu.dk/services/SignalP/
  • the primer sequence is as follows:
  • the underline of the forward primer is the specific site of restriction enzyme Nde I
  • the underline of the 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-enzyme digested with restriction enzymes Nde I and Xho I, and the digested PCR product was recovered by agarose gel electrophoresis.
  • 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 was digested with Nde I and Xho I was ligated with the pET-30a(+) plasmid vector that was also digested with DNA ligase; the ligation product was transformed into E. coli DH5 ⁇ strain and coated On the solid plate of Luria-Bertani medium containing 50 ⁇ g/mL kanamycin, cultured at 37°C for 16h, pick a single clone; insert the single clone into liquid Luria-Bertani medium containing 50 ⁇ g/mL kanamycin The plasmid was cultured in medium, and the plasmid was extracted; the plasmid was verified by PCR with amplification primers, and the result was an amplified product with a size of 2.8kb, which preliminarily proved that the constructed recombinant plasmid was correct; then the recombinant plasmid was sequenced, and the results showed that in pET-30a The gene man0
  • the recombinant plasmid pE30a-Man01929 was transformed into E. coli strain BL21(DE3) (purchased from Invitrogen, USA), and then according to the operating steps provided by Invitrogen, the final concentration was 0.05mM isopropylthiogalactoside (IPTG) for recombination Induced expression of ⁇ -mannanase Man01929; centrifuged at 8,000 ⁇ g and 4°C for 15 minutes, collected the bacteria, and resuspended the bacteria in buffer A, and sonicated it in an ice-water bath.
  • IPTG isopropylthiogalactoside
  • 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.
  • the concentration of 50mM NaAc-HAC buffer, 50mM NaH 2 PO 4 -Na 2 HPO 4 buffer, 50mM Tris-HCl buffer, respectively, with konjac glucomannan and locust bean gum to prepare mass volume concentration ( g/mL) is 0.3% konjac glucomannan or locust bean gum substrate, the corresponding pH values are (5, 6), (6, 7, 8), (7, 8, 9, 10) In the three sections, each pH value adjusts the enzyme activity at the optimum temperature.
  • RA activity is defined as: the percentage of the average absorption value of each group to the maximum absorption value.
  • the pH corresponding to the maximum absorption value is the optimal pH of the recombinase.
  • the results are shown in Figure 4:
  • the optimal pH of the enzyme for degradation of konjac glucomannan is 8.0
  • the optimal pH for degradation of locust bean gum is 5.0. This result indicates that the optimal pH of ⁇ -mannanase Man01929 also changes due to the composition and structure of the substrate.
  • the enzyme solution of recombinant ⁇ -mannanase rMan01929 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 h is mixed in a ratio of 1:9 (volume ratio), and then the residual enzyme activity is measured at the optimum temperature to avoid pretreatment.
  • the enzyme activity of the treated enzyme solution is defined as 100%, and the relative activity is calculated.
  • the control group is the activity of rMan01929 without any metal ions (set as 100%). The results are shown in Figure 7.
  • 1mM Co 2+ and Cu 2+ significantly inhibit the activity of the recombinase rMan01929; (3) The other metal ions and chemical reagents all have different degrees of inhibition on the activity of the recombinase rMan01929.
  • the enzyme degrades locust bean gum, it is different from konjac glucomannan: (1) 1 mM Mg 2+ , Pb 2+ and 10 mM Mn 2+ and other divalent metal ions can promote the activity of the enzyme, and Cu 2+ and Co 2+ can significantly inhibit the activity of the enzyme; (2) 10mM DTT can significantly promote the activity of the recombinase rMan01929.
  • 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, 4°C for 15 minutes, and the supernatant was collected; a certain volume of the supernatant and an equal volume of DNS (3,5-pair Nitroxylene)-reaction liquid was mixed, heated in a boiling water bath for 10 minutes, and cooled to room temperature, and the absorbance was measured at 540nm.
  • DNS 3,5-pair Nitroxylene
  • the protein content in the recombinant ⁇ -mannanase rMan01929 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 rMan01929 can use konjac glucomannan or locust bean gum as a substrate to hydrolyze and produce reducing sugar products.
  • the enzyme activities are 552 ⁇ 2.1U/mg and 516 ⁇ 8.3U/mg, respectively. mg, but the enzyme can hardly degrade the guar polysaccharide tested.
  • the konjac glucomannan and locust bean gum substrates with a mass volume concentration (g/mL) of 0.3% were prepared with deionized water, heated to dissolve, 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 rMan01929 enzyme solution which was previously inactivated in a boiling water bath, was used as a negative control reaction.
  • TLC thin layer chromatography
  • FIG. 1 TLC analysis diagram of oligosaccharide products during the degradation of konjac glucomannan (A) and locust bean gum (B) by recombinant ⁇ -mannanase rMan01929;
  • FIG. 1 M in the abscissa, mannose-mannose; 1, control; 2-9, respectively: 10s, 1min, 10min, 1h, 6h, 24h, 48h, 72h;
  • Figure B In the abscissa, M, mannose-mannose; 1, control; 2-9, respectively represent: enzyme solution diluted 50 times for 10s, enzyme solution diluted 10 times for 10s, 10s, 30s, 1min, 30min, 48h, 72h;
  • the enzyme solution is diluted 50 times and reacted for 10s, which means that the enzyme solution prepared in Example 3 is diluted 50 times and then reacted for 10s.
  • the enzyme solution is diluted 10 times and reacted for 10s.
  • the enzyme solution is diluted 10 times and reacted for 10s.
  • Example 3 The prepared enzyme solution was reacted for 10s, 30s, 1min, 10min, 1h, 6h, 24h, 48h, 72h.
  • Recombinant ⁇ -mannanase rMan01929 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 freeze-dried several times to remove salt, then dissolved in heavy water (D 2 O), freeze-dried for hydrogen and deuterium replacement, and finally detected by 1 H-NMR;
  • the disaccharide structure in the oligosaccharide fragment produced by the recombinase rMan01929 degrading KGM is mainly XM (where X is mannose or glucose, and M is mannose), a small amount of XG, and the molar ratio of the two is calculated according to the area integration method: 4.27:1; the trisaccharide structure is mainly XXM; the tetrasaccharide structure is mainly XXXM.
  • the recombinase rMan01929 can be used to degrade konjac glucomannan and prepare a series of oligosaccharide product fragments whose reducing end is mainly mannose.
  • the final product of locust bean gum degraded by the recombinant enzyme rMan01929 was separated to obtain the disaccharide to heptasaccharide product fragments, and the structure was identified by 1 H-NMR; as shown in Figure 9(B), the 5.046ppm chemical shift value is the reduction end of the oligosaccharide
  • the anomeric hydrogen signal of mannose, the chemical shift value of 4.890ppm is the galactose anomeric hydrogen signal; the above signal peaks indicate: (1)
  • the structure of the disaccharide is MM, indicating that the main product of the enzymatic hydrolysis reaction-the disaccharide fragment-is mainly caused by MM is composed of; (2)
  • the proportion of galactose in the structure of the main product of the enzymatic hydrolysis reaction from trisaccharide to heptasaccharide product fragments gradually increases; (3) the main product of tetrasaccharide to heptasaccharide is rich in
  • the recombinant ⁇ -mannanase rMan01929 was used to hydrolyze the sample of mannooligosaccharide 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.
  • FIG. 1 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 and (+) is the experimental group.
  • Figure B In the abscissa: M, M1-M6; 1: Control; 2: Enzyme solution diluted 10-fold and react for 10s; 3:10s; 4:24h; 5,48h;
  • Figure C In the abscissa: M, M1-M6; 1, control; 2, enzyme solution diluted 10-fold and react for 10s; 3-8, 10s, 10min, 1h, 4h, 24h, 48h;
  • the above-mentioned enzyme solution is diluted 10 times and reacted for 10 seconds, which means that the enzyme solution prepared in Example 3 is diluted 10 times and then reacted for 10 seconds.
  • the rest is the enzyme solution prepared in Example 3 for reaction for 10s, 10min, 1h, 4h, 24h, 48h.
  • Figure 10(A) shows that it can degrade M4, M5, M6, but cannot degrade M3, M2, M; degrade M6 or M5 to produce M4, M3, M2, and a small amount of M; degrade M4 to produce M3, M2, and a small amount of M. And M4 cannot be completely degraded.
  • Figure 10(B) shows that when M5 is degraded, the generation mode can be divided into M&M4 or M2&M3;
  • FIG. 10(C) shows that when M6 is degraded, the generation modes are M&M5, M2&M4, and M3&M3, and M2&M4 is the main mode.
  • the time gradient of the degradation of 2AB-M6 and 2AB-M5 by the recombinase rMan01929 shows that the recombinase rMan01929 degrades 2AB-M6 or 2AB-M5, and 2AB-M2 is produced in a short time.
  • the 2AB-M2 product gradually accumulates over time, and a small amount of 2AB-M3 is also produced, and finally 2AB-M2 is the main product, and the molar ratio of the two is 6.5:1.
  • the three-dimensional structure of ⁇ -mannanase Man01929 described in this application is based on the SWISS-MODEL website using EfMan [4] (PDB No. 5y6t) as a template for homology modeling to obtain the three-dimensional structure of ⁇ -mannanase Man01929 .
  • Man01929 (Man01929 for short) protein is molecularly docked with the mannose M6 sugar:
  • Man01929 is a typical ( ⁇ / ⁇ ) 8 TIM barrel structure, belonging to the Clan-A superfamily;
  • Glu 172 acid-base catalysis
  • Glu 289 nucleophilic catalysis
  • Ledock software was used to molecularly dock the three-dimensional structure of ⁇ -mannanase Man01929 with the mannose (M6) sugar structure, and then PyMOL software was used to analyze the glycosyl binding sites of the protein.
  • the amino acids are: Trp 33 , Phe 36 , Asp 118 , Met 119 , Asp 124 , Glu 172 , Phe 180 , Trp 182 , His 184 , Trp 218 , Ser 219 , Tyr 221 , His 260 , Tyr 261 , Tyr 262 , Asp 263 , Trp 264 , Glu 268 , Ile 269 , Ser 270 , Glu 289 , Trp 318 , Trp 320 , Glu 323, a total of 24 amino acid residues; among them, there are 12 potentially useful amino acids (Asp 118 , Met 119 , Asp 124 , Glu 172 , Trp 182 , Trp 218 , Ser 219 , Tyr 221 , Asp 263 , Glu 268 , Ile 269 , Glu 323 );
  • the residues at the -4 subsite include Asp 124 and Glu 323
  • the residues at the -3 subsite include Met 119 and Asp 118
  • the residues at the +2 subsite include Tyr 221 , Glu 268 , and Ile 269. .
  • the recombinant plasmid pET30a-Man01929 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.
  • Example 3 the mutant recombinant plasmid was induced to express, after ultrasonically disrupting the bacteria, centrifugation, and transferring the supernatant, the crude enzyme solution D118A, D118E, D118Y, M119V, D124A, D124E, D124Y of the mutant recombinase mutant was obtained. , E172A, Y221A, E268A, I269Y, E289A, E323A, E323D, E323Y.
  • the konjac glucomannan and locust bean gum substrates with a mass volume concentration (g/mL) of 0.3% were prepared with deionized water, heated to dissolve, and then placed in a water bath environment of 40°C and 50°C to cool down for 1 hour. Add 10-100 ⁇ L of the dilution of the mutant crude enzyme solution prepared in Example 16 to every 100 ⁇ L of the substrate. When the volume is less than 200 ⁇ L, make up with sterile deionized water. After mixing, 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.
  • g/mL mass volume concentration
  • 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 rMan01929 (set as 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.
  • ⁇ -mannanase Man01929 has a potential to recognize amino acid residues from the non-reducing end of the sugar chain, not only can bind to the substrate, but also has a very strong substrate recognition for the glucomannan substrate. But the recognition of galactomannan is weak.
  • the ⁇ -mannanase Man01929 series mutants were enzymatically hydrolyzed products of konjac mannose oligosaccharides and locust bean gum, and 2uL was taken for detection. According to the color development conditions described in Example 10, the color was developed and then analyzed.
  • Man01929 Analysis of the products of degrading mannose oligosaccharides by mutants at D124 and D118 of ⁇ -mannanase Man01929 (abbreviation: Man01929)
  • the supernatant was collected and used as the final main product of oligosaccharide degradation of the recombinant ⁇ -mannanase rMan01929 mutant.
  • the supernatant after TPTG induced expression was carried out with pET30a as a negative control reaction.
  • the crude enzyme solution of recombinant ⁇ -mannanase rMan01929 mutant was enzymatically hydrolyzed the final main product sample of mannose oligosaccharide (M-M6), and 2uL was taken for detection.
  • M-M6 mannose oligosaccharide
  • the color development analysis was performed, and then the analysis was performed.
  • Figure 16(A) shows that when D118A, D118E and D124Y degrade M6, the main products are M4 and M2;
  • D124A and D124H can degrade M6, M5 and M4, but compared with Man01929, the activity on M4 is significantly weakened;
  • the D124Y mutant enzyme solution was enzymatically hydrolyzed with a sample of mannose oligosaccharide (M-M6), and 2 uL was taken for detection.
  • M-M6 mannose oligosaccharide
  • the color development analysis was performed, and then the analysis was performed. As shown in Figure 17, when the D124Y mutant degrades mannose oligosaccharides:
  • Figure 17(A) shows that when M6 is degraded, the main products are M2 and M4, and only a small amount of M3 is produced;
  • Figure 17(B) shows that when M5 is degraded, the main products are M4 and M1, and a small amount of M2 and M3;
  • Figure 17(C) shows that when the M4 is degraded for 24 hours, a small amount of M5 sugar is produced; through the detection of fluorescent label-high performance liquid chromatography, the area integral calculation shows that it is only 3 ⁇ of the M4 sugar.
  • D124Y has glycosyltransferase activity. It is speculated that the mechanism by which the Asp 124 residue at the -4 subsite is involved in regulating the activities of glycosylhydrolase and glycosyltransferase is: when Asp 124 is mutated to Tyr 124 with a side chain of a benzene ring, the electron cloud density in this region is changed. Large, thereby increasing the attraction to the donor (oligosaccharide product M2), which facilitates the binding with the acceptor (oligosaccharide product M3), and finally makes the mutant Man01929-D124Y in the presence of significant glycosyl hydrolase function Based on it, it can exert weak glycosyltransferase activity.
  • the +2 subsite Trp residues can regulate the conversion of the two catalytic mechanisms of GH5 family mannanhydrolase-glycosyltransferase: one is that the position of the subsite is different, and the further The present invention relates to the Asp 124 mutation Tyr 124 at the -4 subsite; the second is the natural enzyme described in the literature, and the present invention is a mutant enzyme obtained by site-directed mutation after rational design.

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Abstract

提供了一种内切型β-甘露聚糖水解酶Man01929及其突变成糖基转移酶的方法及应用，Man01929的氨基酸序列如SEQ ID NO.2所示，编码Man01929的核苷酸序列如SEQ ID NO.1所示。对Man01929的氨基酸序列的第118、119、124、323位点进行了突变，还获得了具有转糖基能力的突变体D124Y。

Description

一种内切型β-甘露聚糖水解酶Man01929及其突变成糖基转移酶的方法及应用 技术领域
本发明涉及一种内切型β-甘露聚糖水解酶Man01929及其突变成糖基转移酶的方法及应用，属于生物技术领域，涉及蛋白质改进技术。
背景技术
甘露聚糖是一类具有复杂结构的多糖，根据糖单元组成结构的不同，可分为四类：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)。甘露聚糖因结构复杂，故彻底水解时需要多种酶的协同作用，如β-甘露聚糖酶(EC 3.2.1.78)、β-甘露糖苷酶(EC 3.2.1.25)、β-葡萄糖苷酶(EC 3.2.1.21)、乙酰甘露聚糖酯酶(EC 3.1.1.6)、α-半乳糖苷酶(EC 3.2.1.22)。β-甘露聚糖酶是一类糖基水解酶(Glycoside Hydrolase,GH)，在水解过程中起关键作用，它可以水解甘露聚糖主链内部的β-1,4糖苷键，产生甘露寡糖片段。甘露寡糖是由2-10个糖单元通过糖苷键链接而成的，具有多种生理活性，如抗肿瘤、调节免疫力、促进细胞分裂等。与化学方降解法相比，酶法降解甘露多糖制备寡糖的策略具有反应条件温和而可控性强，底物选择性清晰而产物明确等优势，因此β-甘露聚糖酶具有推广应用的潜质。随着研究的深入，β-甘露聚糖酶在食品、养殖业、洗涤剂行业、生物乙醇和石油钻探等各个领域的应用越来越广泛。
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，但主要集中在(1)β-甘露聚糖酶的产酶菌株及酶资源发掘；(2)优化异源表达的条件，从而提高酶的表达水平以应用于生产；(3)通过分子截短、定点突变以及构建糖基化修饰位点的手段，提高酶的催化效率、底物特异性、热稳定性以及耐酸碱的特性等。现有的研究仍然存在以下不足：
(1)β-甘露聚糖酶的底物选择性及其识别机制不明确。作为工具酶，既需要明确的底物选择性，也需要明确的底物选择机制，然而现有研究关于决定酶底物选择性的因素研究较少。这可能是由于两方面的原因造成的：第一，甘露聚糖底物中各种糖单元的大小或结构高度相似，给酶对底物的专一性识别、降解造成困难，大多数甘露聚糖酶具有底物广谱性恰恰是例证。第二，β-甘露聚糖酶本身具有柔性结构，可以识别、结合多种底物，但究竟是哪些糖基结合位点具体参与了底物的选择性识别与作用过程等机理研究甚少。仅少数文献报道了GH5家族β-甘露聚糖酶的-1亚位点和GH26家族β-甘露聚糖酶-2亚位点氨基酸残基参与半乳甘露聚糖的识别，是否存在其它类型活性位点残基也参与了酶对半乳甘露聚糖的识别与降解，未见报道。
(2)部分天然β-甘露聚糖酶，如GH5、GH113家族成员，既具有糖基水解酶的活性又有糖基转移酶的活性，这也对用于降解多糖制备寡糖的专一性工具酶的开发造成困难，需要了解同一个酶进行糖基水解酶、糖基转移酶两种催化机制转换调节的机理。
英文文献：Dilokpimol A,Nakai H,Gotfredsen C H,et al.Recombinant production and characterisation of two related GH5 endo-β-1,4-mannanases from Aspergillus nidulans FGSC A4 showing distinctly different transglycosylation capacity[J].Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics,2011,1814(12):1720-1729.
Rosengren A,
Figure PCTCN2020142062-appb-000001
P,Anderson L,et al.The role of subsite+2 of the Trichoderma reeseiβ-mannanase TrMan5A in hydrolysis and transglycosylation[J].Biocatalysis and Biotransformation,2012,30(3):338-352.
上述文献已报道这种转糖基的能力与β-甘露聚糖酶+2亚位点的色氨酸(Trp)有关，但是其它亚位点的氨基酸残基是否参与了这种调节机制，以及是否存在其它类型氨基酸残基的参与，未见报道。
发明内容
针对现有技术的不同，本发明提供一种内切型β-甘露聚糖水解酶Man01929及其突变成糖基转移酶的方法及应用。所述β-甘露聚糖水解酶Man01929下面简称为β-甘露聚糖酶Man01929。
一种内切型β-甘露聚糖酶Man01929的编码基因man01929，核苷酸序列如SEQ IDNO.1所示。
所述内切型β-甘露聚糖酶Man01929，氨基酸序列如SEQ ID NO.2所示。
一种重组表达载体I，包含上述内切型β-甘露聚糖酶Man01929的编码基因man01929。
一种重组菌株I，包含上述内切型β-甘露聚糖酶Man01929的编码基因man01929。
上述内切型β-甘露聚糖酶Man01929的编码基因man01929、重组表达载体、重组菌在制备内切型β-甘露聚糖酶Man01929的应用。
上述内切型β-甘露聚糖酶Man01929在降解甘露聚糖中的应用。
根据本发明优选的，所述内切型β-甘露聚糖酶Man01929在降解葡甘聚糖和半乳甘露聚糖中的应用。
一种内切型β-甘露聚糖酶Man01929突变酶，氨基酸突变位点为内切型β-甘露聚糖酶Man01929氨基酸序列SEQ ID NO.2的第118、119、124、221、268、269、323位氨基酸之一或两者以上。
根据本发明优选的，所述突变酶，氨基酸突变位点为内切型β-甘露聚糖酶Man01929氨基酸序列SEQ ID NO.2的第118、119、124、221、268、269、323位氨基酸之一或两者以上；所述氨基酸为：
第118位氨基酸由天冬氨酸突变为丙氨酸、谷氨酸或酪氨酸；
第119位氨基酸由蛋氨酸突变为缬氨酸；
第124位氨基酸由天冬氨酸突变为丙氨酸、谷氨酸或酪氨酸；
第221位氨基酸由酪氨酸突变为丙氨酸；
第268位氨基酸由谷氨酸突变为丙氨酸；
第269位氨基酸由异亮氨酸突变为酪氨酸；
第323位氨基酸由谷氨酸突变为丙氨酸、天冬氨酸或酪氨酸。
进一步优选的，所述突变酶，氨基酸突变位点为内切型β-甘露聚糖酶Man01929氨基酸序列SEQ ID NO.2的第124位氨基酸由天冬氨酸突变为酪氨酸。
上述突变酶的编码基因，根据上述氨基酸的突变位点在上述内切型β-甘露聚糖酶Man01929的编码基因man01929上进行定点突变。
一种重组表达载体II，包含上述突变酶的编码基因。
一种重组菌株II，包含上述突变酶的编码基因。
上述突变酶的编码基因、重组表达载体II、重组菌株II在制备上述切型β-甘露聚糖酶Man01929突变酶的应用。
上述内切型β-甘露聚糖酶Man01929突变酶在降解甘露聚糖中的应用。
根据本发明优选的，所述内切型β-甘露聚糖酶Man01929突变酶在降解葡甘聚糖和半乳甘露聚糖中的应用。
根据本发明优选的，所述内切型β-甘露聚糖酶Man01929突变酶在降解甘露四糖生成甘露五糖中的应用。
进一步优选的，所述突变酶，氨基酸突变位点为内切型β-甘露聚糖酶Man01929氨基酸序列SEQ ID NO.2的第124位氨基酸由天冬氨酸突变为酪氨酸。
上述内切型β-甘露聚糖酶Man01929及所述酶的突变体作为工具酶在揭示GH5家族成员底物选择性识别相关机制中的应用。
上述内切型β-甘露聚糖酶Man01929及所述酶的突变体作为工具酶在揭示GH5家族成员进行糖基水解酶、糖基转移酶两种催化机制的转换调节机理中的应用。
根据本发明优选的，所述酶的突变体为上述内切型β-甘露聚糖酶Man01929突变酶。
进一步优选的，所述作为工具酶的突变酶，氨基酸突变位点为内切型β-甘露聚糖酶Man01929氨基酸序列SEQ ID NO.2的第124位氨基酸由天冬氨酸突变为酪氨酸。
所述内切型β-甘露聚糖酶Man01929的编码基因man01929，全长共2799bp，所编码的蛋白质含有932个氨基酸，分子量大小为103.1kDa。
所述内切型β-甘露聚糖酶Man01929只有糖基水解酶活性、没有糖基转移酶活性。
所述内切型β-甘露聚糖酶Man01929是从还原端开始降解甘露聚糖。
所述内切型β-甘露聚糖酶Man01929既能降解魔芋来源葡甘聚糖，也能降解槐豆胶来源的半乳甘露聚糖，且最适多糖底物是魔芋来源的葡甘聚糖；因此，相比含有糖链分支的甘露聚糖，内切型β-甘露聚糖酶Man01929更适合降解没有分支的线性甘露聚糖。
所述内切型β-甘露聚糖酶Man01929含有5个功能模块，其中N端含一个Glycoside hydrolase family 5(GH5)功能模块，447-585位氨基酸为Coagulation factor 5/8C-terminal domain功能模块、595-766位氨基酸含有2个PKD/Chitinase domain功能模块、C端含有Carbohydrate-binding module 64(CBM64)和Secretion system C-terminal sorting domain两个功能模块。
所述内切型β-甘露聚糖酶Man01929在降解魔芋葡甘露寡糖(KGM)的最适温度为40℃，最适pH为8.0；降解槐豆胶(LBG)时，最适温度为50℃，最适pH为5.0。
所述内切型β-甘露聚糖酶Man01929在降解魔芋葡甘聚糖和槐豆胶时，均产生以二糖至四糖为主的寡糖终产物，且其中的二糖寡糖片段均以甘露糖-甘露糖(MM)为主。在降解槐豆 胶半乳甘露聚糖时，其寡糖最终主产物如五糖、六糖、七糖片段中的半乳糖含量呈现较大幅度增加。
所述底物为纯甘露寡糖时，该酶的最小底物为甘露四糖(M4)，最小产物为甘露单糖(M)。
所述底物为荧光标记的甘露寡糖时，该酶的最小底物为还原端被邻氨基苯甲酰胺标记的甘露四糖(2-AB-M4)，最小产物为甘露单糖(M)。
本发明技术方案的有益效果
1、本发明首次公开了由火色杆菌属细菌(Flammeovirga yaeyamensis)MY04的基因组中获得一种内切型β-甘露聚糖酶Man01929的编码基因man01929，该酶Man01929既可以降解魔芋葡甘聚糖，也能降解槐豆胶等多种多糖底物；该酶具有一定的热稳定性(0-40℃)，以及较宽的pH耐受性(5.0-10.0)，理化性质稳定，具备工业应用的潜质。
2、本发明涉及的内切型β-甘露聚糖酶Man01929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，主要从还原端切割糖链产生较小的寡糖产物，且具有可变的底物内切降解模式，可应用于制备不同聚合度的甘露寡糖产物。
3、本发明基于理性分析内切型β-甘露聚糖酶Man01929三维结构的基础上，对内切型β-甘露聚糖酶Man01929进行定点突变，还通过比较分析，确认了内切型β-甘露聚糖酶Man01929中与糖链底物非还原端识别并结合的关键位点残基D118、M119、D124、E323，以及这些残基位点对该酶的底物选择性的具体影响；本发明对于深入揭示内切型β-甘露聚糖酶Man01929的底物选择性机制提供了重要线索，同时对于揭示GH5家族其它β-甘露聚糖酶成员的底物选择性机理提供了有益的参考。
4、本发明通过基因定点突变的手段，成功的将内切型β-甘露聚糖酶Man01929的第124位氨基酸从天冬氨酸突变为酪氨酸后，突变体酶不仅仍然具有糖基水解酶活性，还获得了糖基转移酶的活性，这超出了科研人员的预料，这为揭示包括内切型β-甘露聚糖酶Man01929在内的GH5家族成员进行糖基水解酶、糖基转移酶两种催化机制转换调节的机理研究及应用提供了重要基础。
附图说明
图1、内切型β-甘露聚糖酶Man01929功能模块构成分析结果图；
图2、重组内切型β-甘露聚糖酶rMan01929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170kDa，130kDa，100kDa，70kDa，55kDa，40kDa，35kDa，25kDa，15kDa，10kDa；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rMan01929，上样量2μL；
图3、温度对重组β-甘露聚糖酶rMan01929降解魔芋葡甘聚糖(A)和槐豆胶(B)活性的影响曲线图；
图4、pH对重组β-甘露聚糖酶rMan01929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、温度对重组β-甘露聚糖酶rMan01929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、pH值对重组β-甘露聚糖酶rMan01929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、金属离子及化学试剂对重组β-甘露聚糖酶rMan01929降解魔芋葡甘聚糖(A)和槐豆胶(B)活性的影响柱形图；
图8、重组β-甘露聚糖酶rMan01929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；2-9，分别代表：10s、1min、10min、1h、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：酶液稀释50倍反应10s，酶液稀释10倍反应10s，10s、30s、1min、30min、48h、72h；
图9、用重组β-甘露聚糖酶rMan01929完全降解魔芋葡甘露寡糖(A)和槐豆胶(B)所制备寡糖片段的 1H-NMR图；
图10、重组β-甘露聚糖酶rMan01929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；3:10s；4:24h；5，48h；
C图：横坐标中：M，M1-M6；1，对照；2，酶液稀释10倍反应10s；3-8，10s、10min、1h、4h、24h、48h；
图11、用重组β-甘露聚糖酶rMan01929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、β-甘露聚糖酶Man01929与M6分子对接三维结构；
图13、β-甘露聚糖酶Man01929与M6糖配体
Figure PCTCN2020142062-appb-000002
以内的潜在作用位点分析图；
图14、用重组酶β-甘露聚糖酶rMan01929的系列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；
图15、用重组β-甘露聚糖酶rMan01929的系列突变体分别降解魔芋葡甘聚糖(A)和槐豆胶(B)的寡糖产物TLC分析图；
其中，横坐标中：M，M1-M6；1，(-)；2，Man01929；3，D118A；4，D118E；5，D118Y；6，D124A；7，D124E；8，D124H；9，D124Y；10，E323A；11，E323A；12，E323Y；
图16、用β-甘露聚糖酶Man01929的Asp 118突变为Ala或Glu、Asp 124突变为Tyr、Ala或His后降解甘露六糖(A)、甘露五糖(B)或甘露四糖(C)的TLC分析；
其中：M：M1-M6；1：阴性对照；2：Man01929；3：D124Y；4：D124A；5：D124H；6：D118A；7：D118E；
图17、重组酶β-甘露聚糖酶rMan01929-D124Y降解甘露六糖(A)、甘露五糖(B)和甘露四糖(C)过程中间隔时取样后产物的TLC分析；
其中A图：M，M1-M6；1，阴性对照；2，10min；3，1h；4，4h；5，6h；6，8h；7，24h；8，Man01929；
B图：M，M1-M6；1，阴性对照；2，1min；3，30min；4，2h；；5，12h；6，24h；7，48h；8，Man01929；
C图：M，M1-M6；1，阴性对照；2，3h；3，12h；4，24h；5，48h；6，Man01929。
具体实施方式
以下实施例的阐述，是为了全面公开本发明如何实施的一些常用技术，而不是为了限制本发明的应用范围。发明人已经尽最大努力确保实施例中个参数的准确性(例如量，温度，等等)，但是一些实验误差和偏差也应该予以考虑，除非另有说明，本发明中分子量是指重均分子量，温度是摄氏度。
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，振荡培养至其600nm吸光值(OD 600)为1.2；取培养菌液10mL，在12,000×g(g，地球引力常数)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
将实施例1制得的基因组DNA，采用焦磷酸测序技术进行基因组的扫描测序，由上海美吉生物公司完成。用NCBI(National Center for Biotechnology Information,http://www.ncbi.nlm.nih.gov/)网站的在线软件对DNA测序结果进行分析。所用到的NCBI网站的分析软件是Open Reading Frame Finder(ORF Finder,http://www.ncbi.nlm.nih.gov/gorf/gorf.html)和Basic Local Alignment Search Tool(BLAST,http://blast.ncbi.nlm.nih.gov/Blast.cgi)。
用上述生物学软件分析的结果显示，火色杆菌MY04菌株基因组DNA上携带一个β-甘露聚糖酶的编码基因man01929，基因man01929的编码区长2799bp，核苷酸序列如SEQ ID NO.1所示。基因man01929所编码的β-甘露聚糖酶Man01929共含有932个氨基酸，其氨基酸序列如SEQ ID NO.2所示。
BLASTp在线分析与CAZy数据库、PDB数据库检索表明，在已见文献报导的酶中，Man01929与来自Blue Mussel Mytilus edulis的Man5A相似度最大(即与来自蓝色贻贝基因组中所编码Man5A的蛋白质序列相似度最大)，达到36.79％。如图1所示，由InterProScan综合分析功能模块可知，β-甘露聚糖酶Man01929是由五个功能模块组成，包括Glycoside hydrolase,family 5(GH5 module)、Coagulation factor 5/8 C-terminal domain、2个PKD/Chitinase domain、Carbohydrate-binding module 64(CBM 64 module)、Secretion system C-terminal sorting domain；用生物学网站ExPASY进行分析，显示蛋白质β-甘露聚糖酶Man01929的理论分子量约为103.1kD；用信号肽在线预测软件SignalP 5.0(http://www.cbs.dtu.dk/services/SignalP/)在线分析，该蛋白无信号肽。
实施例3
基因man01929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：
man01929扩增的正向引物
Man01929-F：5’-gcg CATATGGCACTTTTTGCTCATGC-3’；
man01929扩增的反向引物
Man01929-F：5’-gcg CTCGAGTTGCTTGTAGATTCTCCTAAC-3’；
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180s，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，提取质粒；将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man01929，且插入方向正确，所以进一步证明构建的重组质粒正确，将该重组质粒命名为pE30a-Man01929。将重组质粒pE30a-Man01929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Man01929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rMan01929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rMan01929的纯化情况。结果如图2所示：重组质粒pE30a-Man01929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rMan01929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rMan01929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 7.9，制得重组β-甘露聚糖酶rMan01929酶液。
实施例4
重组酶β-甘露聚糖酶rMan01929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℃、90℃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rMan01929的稀释液100μL，其中重组β-甘露聚糖酶rMan01929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Man01929降解魔芋葡甘聚糖在40℃反应时达到最大活力，而降解槐豆胶在50℃达到最大反应活力，这表明该酶降解魔芋葡甘聚糖和槐豆胶的最适反应温度不同，分别是40℃和50℃。该结果还表明β-甘露聚糖酶Man01929的最适温度会因为底物的成分及结构不同而变化。
实施例5
重组β-甘露聚糖酶rMan01929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值均在最适温度下调定酶活。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rMan01929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：该酶降解魔芋葡甘聚糖的最适反应pH为8.0，降解槐豆胶的最适pH为5.0。该结果表明，β-甘露聚糖酶Man01929的最适pH也会因为底物的成分及结构不同而变化。
实施例6
重组β-甘露聚糖酶rMan01929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rMan01929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rMan01929在0-40℃温育24h后，降解魔芋葡甘聚糖时，残余活性>75％以上；当降解槐豆胶时，当重组酶rMan01929在0-20℃温育24h，残余活性为80％以上，而在30-40℃下温育大于4h后，残余活性>59％，表明：该酶在0-40℃下具有一定的热稳定性。
实施例7
重组β-甘露聚糖酶rMan01929的pH稳定性分析
将实施例3制得的重组β-甘露聚糖酶rMan01929的酶液，在冰水浴、不同的pH(pH5～10)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rMan01929在pH5.0-10.0的缓冲溶液中预处理2h后，酶降解葡甘聚糖或槐豆胶的残余活性均大于80％，受pH影响较小。这表明：该酶具有广泛的pH耐受性。
实施例8
金属离子及化学试剂对重组β-甘露聚糖酶rMan01929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rMan01929、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rMan01929的活性(设定为100％)。结果如图7所示，该酶在降解魔芋葡甘聚糖时(1)在1mM或10mM的Na +、K +、Li +等一价金属试剂对重组酶rMan01929的活性没有显著影响，仅Ag +对重组酶rMan1929具有微弱抑制作用；(2)10mM的Co 2+、Cu 2+、Mn 2+二价金属离子对重组酶rMan01929的活性具有促进作用且最高可将活性提高到145％，而1mM的Co 2+、Cu 2+显著抑制重组酶rMan01929的活性；(3)其余金属离子及化学试剂均对重组酶rMan01929的活性有不同程度的抑制作用。该酶降解槐豆胶时，与魔芋葡甘聚糖不同的是：(1)1mM的Mg 2+、Pb 2+与10mM的Mn 2+等二价金属离子对酶的活性具有促进作用，而Cu 2+、Co 2+对酶的活性有显著抑制作用；(2)10mM的DTT对重组酶rMan01929的活性有显著促进作用。
实施例9
DNS-还原糖法测定重组β-甘露聚糖酶rMan01929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rMan01929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rMan01929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rMan01929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552±2.1U/mg和516±8.3U/mg，但是该酶几乎不能降解所测试瓜尔胶多糖。
实施例10
重组β-甘露聚糖酶rMan01929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rMan01929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rMan01929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、重组β-甘露聚糖酶rMan01929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；2-9，分别代表：10s、1min、10min、1h、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：酶液稀释50倍反应10s，酶液稀释10倍反应10s，10s、30s、1min、30min、48h、72h；
上述其中酶液稀释50倍反应10s代表将实施例3制得酶液稀释50倍后反应10s，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、30s、1min、10min、1h、6h、24h、48h、72h。
如图8所示，在上述条件下，重组酶rMan01929降解魔芋葡甘聚糖(图8A)时，短时间内生成三糖及更大的寡糖；随着时间的延长，五糖及更大寡糖产物逐步被降解；在48h以后，最终以四糖-二糖为主产物。该结果初步表明，在本申请研究条件下，重组酶rMan01929在降解魔芋葡甘聚糖时为内切模式。重组酶rMan01929降解槐豆胶(图8B)时，在反应10s时，产物稳定。TLC分析显示的变化规律也表明重组酶rMan01929降解半乳甘露聚糖时为内切模式。
实施例11
重组β-甘露聚糖酶rMan01929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rMan01929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rMan01929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。
用重组酶rMan01929降解魔芋葡甘聚糖的最终产物经分子凝胶色谱柱分离获得二糖、三糖、四糖产物片段后，分别进行 1H-NMR分析，结果如图9(A)所示：5.266ppm和5.050ppm化学位移值分别为寡糖还原端的葡萄糖和甘露糖的异头氢信号，4.421ppm和4.778ppm化学位移值分别为寡糖非还原端的葡萄糖和甘露糖的异头氢信号。上述结果表明，重组酶rMan01929降解KGM产生的寡糖片段中二糖结构主要为XM(其中X为甘露糖或者葡萄糖，M为甘露糖)，少量XG，根据面积积分法计算得到二者摩尔比：4.27:1；三糖结构主要为XXM；四糖结构主要为XXXM。这表明：重组酶rMan01929可用于降解魔芋葡甘聚糖并制备还原端主要为甘露 糖的系列寡糖产物片段
重组酶rMan01929降解槐豆胶的最终产物经分离，获得二糖～七糖产物片段，通过 1H-NMR进行结构鉴定；如图9(B)所示，5.046ppm化学位移值为寡糖还原端的甘露糖异头氢信号，4.890ppm化学位移值为半乳糖异头氢信号；以上信号峰表明：(1)二糖的结构为MM，表明酶解反应的主产物-二糖片段-主要是由MM构成的；(2)酶解反应的主产物三糖至七糖产物片段的结构中，半乳糖的比例逐渐增加；(3)四糖至七糖主产物中富含半乳糖成分。这还表明，在Man01929降解LBG的过程中，较小的寡糖产物(如二糖、三糖等)属于主动生成的，而更高分子量的寡糖产物(如四至七糖等片段)又因富含半乳糖，不利于深度酶学降解而被动成为寡糖主产物的主要成分。
实施例12
重组酶rMan01929降解甘露寡糖产物分析
取约含20μg系列甘露寡糖(M-M6)的溶液，150mmol/L的NaH 2PO4-Na 2HPO4(pH7.0)缓冲溶液、实施例3制得的重组β-甘露聚糖酶rMan01929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rMan01929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rMan01929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rMan01929酶解甘露寡糖寡糖(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、重组β-甘露聚糖酶rMan01929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；3:10s；4:24h；5，48h；
C图：横坐标中：M，M1-M6；1，对照；2，酶液稀释10倍反应10s；3-8，10s、10min、1h、4h、24h、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，10min，1h，4h，24h，48h。
结果如图10所示，本申请所述的重组β-甘露聚糖酶rMan01929：
(1)图10(A)可知，可以降解M4、M5、M6，不能降解M3、M2、M；降解M6或M5产生M4、M3、M2以及少量M；降解M4产生M3、M2以及少量M，且M4不能被彻底降解。
(2)图10(B)可知，降解M5时，生成模式可分为M&M4或者M2&M3；
(3)图10(C)可知，降解M6时，生成模式为M&M5、M2&M4以及M3&M3，且以M2&M4这种模式为主。
这些结果表明，重组酶rMan01929降解甘露寡糖时：最小底物是M4，最小产物M1。上述结果还表明，随着底物的增大，寡糖最小产物相应增大，因此甘露聚糖酶Man01929具有典型的可变的底物内切模式。
实施例13
重组β-甘露聚糖酶rMan01929酶切模式的荧光-高效液相色谱(HPLC)分析
取含10μg系列甘露寡糖(M-M6)底物溶液，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500μL去离子水溶解样品，将样品与200μL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，得到还原性末端被荧光标记了的甘露六糖(2AB-M6)、荧光标记的甘露五糖(2AB-M5)、荧光标记的甘露四糖(2AB-M4)、荧光标记的甘露三糖(2AB-M3)、荧光标记的甘露二糖(2AB-M2)、荧光标记的甘露糖(2AB-M)。
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rMan01929的稀释液、150mmol/L的NaH 2PO4-Na 2HPO4(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的重组β-甘露聚糖酶rMan01929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。
如图11(A)所示，在上述条件下，重组酶rMan01929降解2AB-M6或2AB-M5时，最终主产物均以2AB-M2为主，含有少量2AB-M3；重组酶rMan01929仅能微弱降解2AB-M4，产生2AB-M2；重组酶rMan01929不能降解2AB-M3和2AB-M2。
如图11(B)和(C)所示，由重组酶rMan01929降解2AB-M6、2AB-M5的时间梯度可知：重组酶rMan01929降解2AB-M6或2AB-M5，短时间内即产生2AB-M2产物，随着时间的延长2AB-M2产物逐渐积累，还产生少量2AB-M3，且最终以2AB-M2为主，其中二者摩尔比为6.5:1。
这些结果表明，重组酶rMan01929降解荧光标记的甘露寡糖时：(1)最小底物为2AB-M4，最小产物为M1；(2)从还原端切割糖链，产生2AB-M2&M4；(3)降解模式表现为二糖外切。
实施例14
β-甘露聚糖酶Man01929的三维结构模拟
运用SWISS-MODEL在线网站以PDB号为5y6t的蛋白为模板对β-甘露聚糖酶Man01929进行在线同源建模，分析β-甘露聚糖酶Man01929的催化腔结构。
本申请所述的β-甘露聚糖酶Man01929的三维结构是通过SWISS-MODEL网站以EfMan [4](PDB号为5y6t)为模板进行同源建模得到β-甘露聚糖酶Man01929的三维结构。
如图12所示，将β-甘露聚糖酶Man01929(简称Man01929)蛋白与甘露六糖M6糖进行分子对接：
(1)Man01929是典型的(β/α) 8TIM桶状结构，属于Clan-A超家族；
(2)Man01929催化腔长度约
Figure PCTCN2020142062-appb-000003
(3)一个甘露糖分子长度约
Figure PCTCN2020142062-appb-000004
(4)推测Man01929催化腔可容纳5-6个甘露糖单元；
(5)Man01929催化位点为Glu 172(酸碱催化)和Glu 289(亲核催化)是严格保守的。
实施例15
β-甘露聚糖酶Man01929(简称Man01929)与甘露六糖(M6)分子对接
利用Ledock软件将β-甘露聚糖酶Man01929的三维结构与甘露六糖(M6)糖结构进行分子对接，然后通过PyMOL软件分析蛋白质的糖基结合为位点。
如图13所示，按照上述要求，通过PyMOL软件分析Man01929与M6糖配体的潜在作用位点可知：
(1)Man01929与M6配体相距
Figure PCTCN2020142062-appb-000005
的氨基酸有：Trp 33、Phe 36、Asp 118、Met 119、Asp 124、Glu 172、Phe 180、Trp 182、His 184、Trp 218、Ser 219、Tyr 221、His 260、Tyr 261、Tyr 262、Asp 263、Trp 264、Glu 268、Ile 269、Ser 270、Glu 289、Trp 318、Trp 320、Glu 323总计24个氨基酸残基；其中具有潜在的作用的氨基酸有12个(Asp 118、Met 119、Asp 124、Glu 172、Trp 182、Trp 218、Ser 219、Tyr 221、Asp 263、Glu 268、Ile 269、Glu 323)；
(2)Man01929与M6采用-4至+2的方式与底物结合；
(3)-4亚位点的残基有Asp 124、Glu 323，-3亚位点的残基有Met 119、Asp 118，+2亚位点的残基有Tyr 221、Glu 268、Ile 269。
实施例16
β-甘露聚糖酶Man01929的定点突变
以实施例3制得的重组质粒pET30a-Man01929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 PCTCN2020142062-appb-000006
Figure PCTCN2020142062-appb-000007
Figure PCTCN2020142062-appb-000008
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-Man01929-D118A、pET30a-Man01929-D118E、pET30a-Man01929-D118Y、pET30a-Man01929-M119V、pET30a-Man01929-D124A、pET30a-Man01929-D124E、pET30a-Man01929-D124Y、pET30a-Man01929-E172A、pET30a-Man01929-Y221A、pET30a-Man01929-E268A、pET30a-Man01929-I269Y、pET30a-Man01929-E289A、pET30a-Man01929-E323A、pET30a-Man01929-E323D、pET30a-Man01929-E323Y。
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D118A、D118E、D118Y、M119V、D124A、D124E、D124Y、E172A、Y221A、E268A、I269Y、E289A、E323A、E323D、E323Y。
实施例17
β-甘露聚糖酶Man01929(简称Man01929)系列突变体的酶活分析
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rMan01929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。
Man01929的系列突变体降解魔芋葡甘聚糖(图14A)和槐豆胶(图14B)时，如图所示：当-4，-3以及+2亚位点的氨基酸残基突变后发现，-4和-3亚位点氨基酸残基(Asp 118、Met 119、Asp 124、Glu 323)的突变，导致对LBG的活性明显减弱甚至失活，但重组酶突变体对KGM的酶活无明显变化。上述结果表明，β-甘露聚糖酶Man01929与糖链的非还原端具有潜在识别作用的氨基酸残基，不仅可以与底物结合，而且对葡甘聚糖底物具有非常强的底物识别，但对半乳甘露聚糖的识别微弱。
实施例18
β-甘露聚糖酶Man01929系列突变体降解魔芋葡甘聚糖和槐豆胶的产物分析
取实施案例17中β-甘露聚糖酶Man01929系列突变体降解魔芋葡甘聚糖和槐豆胶的产物进行TLC分析。
按照实施例10所述的展开条件，将β-甘露聚糖酶Man01929系列突变体酶解魔芋甘露寡糖和槐豆胶的产物，取2uL检测。按照实施例10所述显色条件，进行显色，然后分析。
结果如图15所示，本申请所述的β-甘露聚糖酶Man01929系列突变体降解魔芋葡甘聚糖(A)和槐豆胶(B)的产物分析可知：下述野生型为未突变的原始酶
(1)与野生型相比，D118A、D118E以及D124Y突变体无论降解KMG还是LBG时，均导致产物的聚合度发生变化，产物的平均分子量变大且主产物变为以四糖以及更大寡糖为主；
(2)突变体D124A、D124E、D124H、E323A、E323D、E323Y降解KGM、LBG的主产物，与野生型相比无明显变化。
上述结果表明，本发明中Man01929的-4亚位点的Asp 118和Asp 124残基参与了底物的定位并影响或决定了寡糖主产物的大小。
实施例19
β-甘露聚糖酶Man01929(简称：Man01929)的D124、D118位点系列突变体降解甘露寡糖的产物分析
取约含20μg系列甘露寡糖(M4-M6)的溶液，150mmol/L的NaH 2PO 4-Na 2HPO 4(pH7.0)缓冲溶液、实施例16制得的重组β-甘露聚糖酶rMan01929突变体粗酶的稀释液，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rMan01929突变体的寡糖降解最终主产物。以pET30a进行TPTG诱导表达后的上清，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rMan01929突变体粗酶液酶解甘露寡糖(M-M6)的最终主产物样品，取2uL检测。按照实施例10所述显色条件，进行显色分析，然后分析。
结果如图16所示，D124、D118位点突变体降解甘露寡糖时，
(1)图16(A)可知，D118A、D118E和D124Y降解M6时，主产物为M4和M2；
(2)图16(B)可知，D118A、D118E和D124Y降解M5时，主产物为M4和M1；
(3)图16(C)可知，D118A、D118E不能降解M4，D124Y仅能微弱降解M4；
(4)D124A和D124H可以降解M6、M5和M4，但是与Man01929相比，对M4的活性明显减弱；
这些结果表明，Asp 118或Asp 124位点突变为Ala 118、Glu 118或Tyr 124后，导致-4亚位点与底物的非还原端结合更加牢靠，不利于对四糖片段的降解，最终导致寡糖主产物发生成分变化。
实施例20
突变体D124Y糖基转移酶活性的分析
取约含20μg甘露寡糖(M4-M6)的溶液，150mmol/L的NaH 2PO 4-Na 2HPO 4(7.0)缓冲溶液、实施例16制得的D124Y突变酶的稀释液，按照体积比1:1:1混匀，分别在40℃反应，隔时取样。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D124Y突变体的寡糖降解产物。以pET30a进行TPTG诱导表达后的上清，做阴性对照反应。
按照实施例10所述的展开条件，将D124Y突变体酶液酶解甘露寡糖(M-M6)的样品，取2uL检测。按照实施例10所述显色条件，进行显色分析，然后分析。如图17所示，D124Y突变体降解甘露寡糖时：
(1)图17(A)可知，降解M6时，主产物为M2和M4，仅少量的M3产生；
(2)图17(B)可知，降解M5时，主产物为M4和M1，少量M2和M3；
(3)图17(C)可知，仅能微弱降解M4，产生少量的M3、M2和M1；
(4)图17(C)可知，降解M4反应24h时，有微量的M5糖产生；通过荧光标记-高效液相色谱检测，进行面积积分计算，表明仅为M4糖的3‰。
这些结果表明：D124Y有糖基转移酶活性。推测其中-4亚位点Asp 124残基参与调节糖基水解酶和糖基转移酶活性的机制为：当Asp 124突变为含苯环侧链的Tyr 124后，导致该区域的电子云密度变大，从而增加了对供体(寡糖产物M2)的吸引力，从而使之利于与受体(寡糖产物M3)的结合，最终使突变体Man01929-D124Y在具有显著糖基水解酶功能的基础上，可发挥 微弱糖基转移酶的活性。这与文献已报告的+2亚位点Trp残基可调节转换GH5家族甘露聚糖水解酶-糖基转移酶两种催化机制的机理有所不同：一是亚位点的位置不同，进一步的本发明涉及的是-4亚位点上的Asp 124突变Tyr 124；二是文献所说为天然酶，而本发明是通过理性设计后通过定点突变获得的突变体酶。

Claims (10)

  1. 一种内切型β-甘露聚糖酶Man01929的编码基因man01929，其特征在于，核苷酸序列如SEQ IDNO.1所示。
  2. 一种内切型β-甘露聚糖酶Man01929，其特征在于，氨基酸序列如SEQ ID NO.2所示。
  3. 一种重组表达载体I，其特征在于，包含权利要求1所述内切型β-甘露聚糖酶Man01929的编码基因man01929。
  4. 一种重组菌株I，其特征在于，包含权利要求1所述内切型β-甘露聚糖酶Man01929的编码基因man01929。
  5. 权利要求1、3、4任一项在制备内切型β-甘露聚糖酶Man01929的应用。
  6. 权利要求2所述内切型β-甘露聚糖酶Man01929在降解甘露聚糖中的应用；
    优选的，所述内切型β-甘露聚糖酶Man01929在降解葡甘聚糖和半乳甘露聚糖中的应用。
  7. 一种内切型β-甘露聚糖酶Man01929突变酶，其特征在于，氨基酸突变位点为内切型β-甘露聚糖酶Man01929氨基酸序列SEQ ID NO.2的第118、119、124、221、268、269、323位氨基酸之一或两者以上；
    优选的，所述突变酶，氨基酸突变位点为内切型β-甘露聚糖酶Man01929氨基酸序列SEQ ID NO.2的第118、119、124、221、268、269、323位氨基酸之一或两者以上；所述氨基酸为：
    第118位氨基酸由天冬氨酸突变为丙氨酸、谷氨酸或酪氨酸；
    第119位氨基酸由蛋氨酸突变为缬氨酸；
    第124位氨基酸由天冬氨酸突变为丙氨酸、谷氨酸或酪氨酸；
    第221位氨基酸由酪氨酸突变为丙氨酸；
    第268位氨基酸由谷氨酸突变为丙氨酸；
    第269位氨基酸由异亮氨酸突变为酪氨酸；
    第323位氨基酸由谷氨酸突变为丙氨酸、天冬氨酸或酪氨酸；
    进一步优选的，所述突变酶，氨基酸突变位点为内切型β-甘露聚糖酶Man01929氨基酸序列SEQ ID NO.2的第124位氨基酸由天冬氨酸突变为酪氨酸。
  8. 权利要求7所述突变酶的编码基因，其特征在于，所述编码基因为根据权利要求7所述氨基酸的突变位点在权利要求1中所述编码基因man01929上进行定点突变后的基因；
    优选的，一种重组表达载体II，包含所述突变酶的编码基因；
    优选的，一种重组菌株II，包含所述突变酶的编码基因。
  9. 权利要求8所述突变酶的编码基因、重组表达载体II、重组菌株II任一项在制备权利要求7切型β-甘露聚糖酶Man01929突变酶的应用。
  10. 权利要求7所述内切型β-甘露聚糖酶Man01929突变酶在降解甘露聚糖中的应用；
    优选的，所述突变酶在降解葡甘聚糖和半乳甘露聚糖中的应用；
    优选的，所述突变酶在降解甘露四糖生成甘露五糖中的应用；
    进一步优选的，所述突变酶，氨基酸突变位点为内切型β-甘露聚糖酶Man01929氨基酸 序列SEQ ID NO.2的第124位氨基酸由天冬氨酸突变为酪氨酸；
    优选的，权利要求2所述内切型β-甘露聚糖酶Man01929及权利要求7所述的突变酶作为工具酶在揭示GH5家族成员底物选择性识别相关机制中的应用；
    优选的，权利要求2所述内切型β-甘露聚糖酶Man01929及权利要求7所述的突变酶作为工具酶在揭示GH5家族成员进行糖基水解酶、糖基转移酶两种催化机制的转换调节机理中的应用；
    优选的，作为工具酶的突变酶，氨基酸突变位点为内切型β-甘露聚糖酶Man01929氨基酸序列SEQ ID NO.2的第124位氨基酸由天冬氨酸突变为酪氨酸。
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