WO2018086008A1 - 一种细菌β-1,3-葡聚糖酶及其编码基因与应用 - Google Patents

一种细菌β-1,3-葡聚糖酶及其编码基因与应用 Download PDF

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WO2018086008A1
WO2018086008A1 PCT/CN2016/105187 CN2016105187W WO2018086008A1 WO 2018086008 A1 WO2018086008 A1 WO 2018086008A1 CN 2016105187 W CN2016105187 W CN 2016105187W WO 2018086008 A1 WO2018086008 A1 WO 2018086008A1
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protein
glucanase
sequence
hydrolysis
glucan
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French (fr)
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江正强
秦臻
闫巧娟
游鑫
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中国农业大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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)
    • 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
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C12P19/08—Dextran

Definitions

  • the invention belongs to the field of food biotechnology, and particularly relates to a bacterial ⁇ -1,3-glucanase and a gene encoding the same and application thereof.
  • --1,3-glucan ( ⁇ -1,3-glucan) is a polymer polysaccharide widely distributed in nature, and its main chain is composed of glucose residues at the C1 and C3 positions through ⁇ -1. 3-glycosidic linkages are formed. --1,3-glucan is widely distributed in fungi, bacteria and plants and exerts a variety of physiological functions.
  • the ⁇ -1,3-glucan commonly found in nature includes laminarin, available polysaccharide, yeast glucan, lentinan, alfalfa polysaccharide, and schizophyllan.
  • ⁇ -1,3-glucan degradation product ⁇ -1,3-gluco oligosaccharide (also known as laminaria oligosaccharide) is a novel functional oligosaccharide that promotes the growth of intestinal probiotics, anti-tumor, Biological activities such as blood glucose lowering have broad application prospects in food, agriculture, medicine, etc. (Ann Microbiol, 2011, 62: 307-312; Appl Microbiol Biotechnol, 2012, 93: 525-531).
  • Endo- ⁇ -1,3-glucanase (EC 3.2.1.39; abbreviated as ⁇ -1,3-glucanase), also known as laminin, is a kind of An enzyme that specifically hydrolyzes the ⁇ -1,3-glycosidic linkage in ⁇ -1,3-glucan.
  • the ⁇ -1,3-glucanase hydrolyzes glycans from the inside of the sugar chain, and the product is a series of oligosaccharides of different sizes.
  • the ⁇ -1,3-glucanase has a strict substrate specificity, and the hydrolysis efficiency of ⁇ -1,3-1,4-glucan containing a ⁇ -1,4-glycosidic bond is low.
  • ⁇ -1,3-glucanase is widely distributed, mainly from fungi, bacteria, plants and some lower animals and viruses, and has various biological functions (Acta Cryst, 2013, D69, 2027-2038). According to the Carbohydrate-Active enZYmes Database (http://www.cazy.org) classification, the discovered ⁇ -1,3-glucanase can be attributed to the hydrolysis of 7 glycosides according to the sequence evolution relationship.
  • Microorganisms are a major source of beta-1,3-glucanase.
  • the bacterial ⁇ -1,3-glucanase mainly belongs to the glycoside hydrolase GH16 family.
  • the extracellular ⁇ -1,3-glucanase secreted by bacteria is mainly used to degrade the fungal cell wall or plant-derived ⁇ -1,3-glucan in its growth environment as an energy source for its growth and reproduction (J Bacteriol, 1995, 177: 6937–6945).
  • ⁇ -1,3-glucanase has also been found in some archaea or nematodes (Biochem J, 2005, 389: 117–125).
  • the fungal ⁇ -1,3-glucanase mainly belongs to the GH16 family and the GH81 family, and generally contains a signal peptide, which is localized to the cell membrane.
  • ⁇ -1,3-glucanases plays an important role in the process of partial digestion and remodeling of fungal cell walls.
  • the fungal ⁇ -1,3-glucanase synergizes with chitinase and transglycosidase to act on the ⁇ -1,3-glucan in the cell wall to complete the cell wall division and recasting process of the fungal cell. (Front Microbiol, 2013, 4:81).
  • the ⁇ -1,3-glucanase preparation can be widely used in the fields of beer industry, feed industry, biological control, and medicine (Publication No. [CN101565682B]; Appl Environ Microbiol, 2011, 77: 983-990; J Appl Microbiol, 2000, 88: 961–967).
  • some ⁇ -1,3-glucans and some small molecule soluble ⁇ -1,3-glucose oligosaccharides are effective immune activators, and the biological activity of ⁇ -1,3-glucose oligosaccharides has been studied in recent years. There are also many reports.
  • the preparation methods of ⁇ -1,3-gluco oligosaccharides mainly include enzymatic, chemical, physical or multi-method coupling (Carbohyd Polym, 2008, 71: 277–286; Food Sci Biotechnol, 2014, 23: 799 – 806; Carbohyd Polym, 2011, 86: 574-580), in which enzymatic and chemical methods are more commonly used in the preparation of oligosaccharides. So far, there are still some bottlenecks in the enzymatic production of ⁇ -1,3-glucose oligosaccharide technology, such as low activity of endo- ⁇ -1,3-glucanase, long hydrolysis time, and difficult regulation of hydrolysis process.
  • Some patents disclose the preparation of beta-1,3-polyoligosaccharides.
  • the application method [201210136867.0] discloses a method for preparing ⁇ -1,3-glucose oligosaccharide, which is prepared by combining acid hydrolysis and enzymatic hydrolysis to prepare ⁇ -1,3-glucose oligosaccharide, but a large amount of trifluoroacetic acid is used in the method. , Ba(OH) 2 , ammonia and other reagents, the pollution is large, there are hidden dangers, the product is not suitable for applications in food, medicine and other fields.
  • [201510123040.X] discloses a method for directly producing ⁇ -1,3-gluco oligosaccharide by using a thermal gel fermentation broth.
  • the reaction system contains a fermentation broth, and the production process requires the use of chemical reagents such as HCl and NaOH. Conducive to the preparation and application of subsequent oligosaccharide pure products.
  • chemical reagents such as HCl and NaOH.
  • the polysaccharide is a microbial exopolysaccharide produced by Alcaligenes faecalis. It is a linear linkage of 400-500 D-glucose with ⁇ -1,3-glycosidic bond. It is the only linear ⁇ - in nature. 1,3-glucan. Polysaccharides are commonly used in food thickeners due to their unique gel properties. It is possible that the polysaccharide can be dissolved in a dilute alkali solution, but is insoluble in water, which limits its use in the pharmaceutical and food industries.
  • Degradation allows the polysaccharide to become a certain molecular weight of ⁇ -1,3-gluco oligosaccharide, which is a method to solve the poor solubility.
  • the protein provided by the present invention is a protein of the following a) or b) or c), which is named PbBgl64A,
  • amino acid sequence is a protein encoded by amino acid residues 29 to 449 of SEQ ID NO: 2;
  • the label shown in Table 1 may be attached to the amino terminus or carboxy terminus of the protein encoded by amino acid residues 29 to 449 of Sequence 2 in the Sequence Listing.
  • substitution and/or deletion and/or addition of the one or several amino acid residues is a substitution and/or deletion and/or addition of no more than 10 amino acid residues.
  • the protein in the above c) can be artificially synthesized, or the encoded gene can be synthesized first, and then obtained by biological expression.
  • the gene encoding the protein in the above c) can be obtained by deleting the codon of one or several amino acid residues in the DNA sequence shown in positions 85-1350 of the sequence 1, and/or performing one or several base pair errors.
  • Another object of the invention is to provide a biological material associated with the above proteins.
  • the biomaterial provided by the present invention is any one of the following B1)-B5):
  • B2 an expression cassette comprising the nucleic acid molecule of B1);
  • B3 a recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);
  • B4 a recombinant strain containing the nucleic acid molecule of B1) or a recombinant strain containing the expression cassette of B2) or a recombinant strain containing the recombinant vector of B3);
  • the nucleic acid molecule of B1) is a DNA molecule of the following 1) or 2) or 3):
  • nucleotide sequence is the DNA molecule shown in SEQ ID NO: 85-1350 of Sequence Listing 1;
  • the defined DNA sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least a DNA molecule having 98% or at least 99% homology and encoding the protein;
  • the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.
  • nucleotide sequence encoding PbBgl64A of the present invention can readily mutate the nucleotide sequence encoding PbBgl64A of the present invention using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having a nucleotide sequence encoding 75% or more of the nucleotide sequence of PbBgl64A, as long as they encode PbBgl64A and have the same function, are derived from the core of the present invention. The nucleotide sequence is identical to the sequence of the invention.
  • identity refers to sequence similarity to a native nucleic acid sequence. “Identity” includes 75% or more, or 85% or more, or 90% or more of the nucleotide sequence of a protein consisting of the amino acid sequence shown in positions 29-449 of the coding sequence 2 of the present invention. , or a nucleotide sequence of 95% or greater identity. Identity can be evaluated using the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed in percentage (%), which can be used to evaluate the identity between related sequences.
  • the above 75% or more of the identity may be 80%, 85%, 90% or 95% or more.
  • the expression cassette (PbBgl64A gene expression cassette) containing the nucleic acid molecule encoding PbBgl64A as described in A2) refers to a DNA capable of expressing PbBgl64A in a host cell, and the DNA may include not only a promoter which initiates transcription of PbBgl64A, but also a promoter. A terminator that terminates transcription of PbBgl64A can also be included. Further, the expression cassette may further comprise an enhancer sequence. Promoters useful in the present invention include, but are not limited to, constitutive promoters; tissues, organs, and development-specific promoters and inducible promoters.
  • promoters include, but are not limited to, constitutive promoter of cauliflower mosaic virus 35S: a wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester); tomatoes Protease inhibitor II promoter (PIN2) or LAP promoter (both induced by methyl jasmonate); heat shock promoter (U.S. Patent 5,187,267); tetracycline-inducible promoter (U.S.
  • Patent 5,057,422 seed-specific promoter
  • the millet seed-specific promoter pF128 CN101063139B (Chinese Patent 200710099169.7)
  • the seed storage protein-specific promoter for example, the promoters of Bean globulin, napin, oleosin and soybean beta conglycin (Beachy et al. (1985) EMBO) J.4: 3047-3053). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety.
  • Suitable transcription terminators include, but are not limited to, Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV35S terminator, tml terminator, pea rbcS E9 terminator, and nopaline and octopine Enzyme terminator (see, for example, Odell et al. (I 985 ) Nature 313: 810; Rosenberg et al. (1987) Gene, 56: 125; Guerineau et al. (1991) Mol. Gen. Genet, 262: 141; 1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5: 141; Mogen et al.
  • NOS terminator Agrobacterium nopaline synthase terminator
  • CaMV35S terminator cauliflower mosaic virus CaMV35S terminator
  • tml terminator tml terminator
  • pea rbcS E9 terminator pea rbcS E9 terminator
  • the vector may be a plasmid, a cosmid, a phage or a viral vector.
  • the microorganism may be yeast, bacteria, algae or fungi such as Agrobacterium.
  • Another object of the invention is to provide new uses of the above proteins.
  • the present invention provides the use of the above protein as a ⁇ -1,3-glucanase.
  • the invention also provides the use of the above protein for the hydrolysis of ⁇ -1,3-glucan.
  • the ⁇ -1,3-glucan is a obtainable polysaccharide, laminarin or yeast glucan.
  • the present invention also provides the use of the above protein for the preparation of ⁇ -1,3-glucose oligosaccharide and/or high polymerization degree ⁇ -1,3-gluco oligosaccharide.
  • the degree of polymerization is 2-21.
  • Another object of the invention is to provide a recombinant bacterium.
  • the recombinant strain provided by the present invention is a bacterium obtained by introducing the gene encoding the above protein into a host bacterium.
  • the gene encoding the protein is introduced into the host strain by a recombinant vector;
  • the recombinant vector is a vector obtained by inserting the gene encoding the above protein into a multiple cloning site of an expression vector.
  • the recombinant vector specifically inserts the DNA fragment shown in positions 85-1350 of Sequence Listing 1 between the Nhe I and Xho I sites of the vector pET-28a(+), and maintains the vector pET-28a(+) The other sequences are unchanged from the vector obtained.
  • the gene encoding the protein is the DNA molecule shown in SEQ ID NO: 85-1350 of Sequence Listing 1.
  • the host strain is Escherichia coli BL21 (DE3).
  • Still another object of the present invention is to provide a process for the preparation of a ⁇ -1,3-glucanase.
  • the preparation method of the ⁇ -1,3-glucanase provided by the present invention comprises the steps of inducing the culture of the above recombinant bacteria to obtain ⁇ -1,3-glucanase.
  • a final object of the present invention is to provide a process for producing a high degree of polymerization of ?-1,3-gluco oligosaccharides.
  • the method for producing a high polymerization degree ⁇ -1,3-gluco oligosaccharide comprises the steps of: hydrolyzing ⁇ -1,3-glucan with the above protein to obtain a high polymerization degree ⁇ -1,3-gluco oligosaccharide .
  • the ratio of the protein to the ⁇ -1,3-glucan is 1500 U: 1 kg.
  • the protein of claim 1 has a concentration of 0.06 U/mL.
  • the mass fraction of the ⁇ -1,3-glucan is 4%.
  • the hydrolysis condition is: pH 5.0, hydrolysis at 50-60 ° C for 4-6 h; the hydrolysis condition is specifically: pH 5.0, hydrolysis at 55 ° C for 5 h.
  • the ⁇ -1,3-glucan is a obtainable polysaccharide.
  • Figure 1 shows a purified map of E. coli recombinant PbBgl64A purified by Ni-IDA.
  • lane M represents a low molecular weight standard protein
  • lane 1 represents a supernatant after disruption
  • lane 2 represents a protein after affinity chromatography by Ni-IDA.
  • Figure 2 shows the effect of temperature and pH changes on PbBgl64A enzyme activity and stability.
  • A optimum pH
  • B pH stability
  • C optimum temperature
  • D temperature stability
  • buffer used McIlvaine buffer ( ⁇ ), acetic acid-sodium acetate buffer ( ⁇ ) and Tris–HCl buffer ( ⁇ ).
  • Figure 3 shows the hydrolysis characteristics of PbBgl64A for different substrates.
  • (A) is a result of a thin layer chromatography analysis of hydrolysis history of PbBgl64A hydrolyzed ⁇ -1,3-glucan (available polysaccharide).
  • (B) is the result of thin layer chromatography analysis of PbBgl64A hydrolyzed ⁇ -1,3-gluco oligosaccharide (Kumbu disaccharide, kelp triose, kelp tetrasaccharide, kelp pentasaccharide and kelp hexose) product, (B) In the analysis results of the group oligosaccharides, the left side is before the reaction and the right side is the reaction.
  • Figure 4 shows a MALDI-TOF MS analysis of the hydrolysis of PbBgl64A available polysaccharides. The figure shows that the degree of polymerization of the hydrolyzed product is in the range of 2-21, wherein the five-sugar abundance is the highest.
  • Figure 5 shows the effect of different initial pH conditions on the efficiency of PbBgl64A hydrolysis.
  • Figure 6 shows the effect of different initial temperature conditions on the efficiency of PbBgl64A hydrolysis.
  • Figure 7 shows the effect of different amounts of enzyme added on the efficiency of PbBgl64A hydrolysis.
  • Figure 8 shows the different substrate concentrations for the hydrolysis of PbBgl64A in the final hydrolysate of the polysaccharide. The effect of reducing sugar concentration.
  • Figure 9 is a graph showing the analysis of the PbBgl64A hydrolysis-derived polysaccharide product.
  • A is the result of the thin layer chromatography analysis of the hydrolysis history
  • B is the peak diagram of the ion chromatographic analysis of the final hydrolyzate component.
  • the Paeubacillus barengoltzii CAU904 in the following examples has been preserved in the China Collection of Microorganisms and Cultures (CGMCC, address: Datun Road, Chaoyang District, Beijing), and the deposit number is CGMCC No. 9530. It is disclosed in the document "Biotechnol Biofuel 2014, 7: 174".
  • the signal peptide fragment formed by amino acid residues 1-28 was removed, and the upstream primer PbBgl64A-up (5'-TGACT GCTAGC GCTGATTTCACTCAAGGAGCGG-3' was designed. Underlined Nhe I restriction site) and downstream primer PbBgl64A-down (5'-TGACT CTCGAG TTACCAGCCCACTCTGACGATG-3', underlined Xho I restriction site), and the genomic DNA of B. pallidum CAU904 is The template was subjected to PCR amplification to obtain a PCR amplification product.
  • the PCR amplification conditions were: pre-denaturation at 94 ° C for 5 min; denaturation at 94 ° C for 30 s, annealing at 54 ° C for 30 s, extension at 72 ° C for 90 s, 35 cycles; and finally extension at 72 ° C for 5 min.
  • the PCR product was recovered by 1% agarose gel electrophoresis and digested with Nhe I and Xho I.
  • the double-digested product was ligated with the same double-digested prokaryotic expression vector pET-28a (+) (Novagen, USA, product number: 69864-3) fragment by T4 DNA ligase to obtain a recombinant plasmid, and This was transformed into the host E. coli DH5 ⁇ .
  • Colony PCR (primer used for PCR and amplification conditions are the same as those described in the previous paragraph of this paragraph) was selected to verify that the transformants were positive.
  • the sequencing results showed that the recombinant plasmid was inserted into the DNA fragment shown in positions 85-1350 of Sequence 1 in the sequence of Nhe I and Xho I of the vector pET-28a(+).
  • the positive transformant contained the above recombination. Plasmid E. coli DH5 ⁇ .
  • gene PbBgl64A The gene shown in SEQ ID NO: 85-350 of Sequence Listing 1 was named as gene PbBgl64A, and the protein encoded by the gene was named as protein PbBgl64A, and its amino acid sequence is shown in positions 29-449 of Sequence Listing 2.
  • the recombinant plasmid of Example 1 was transformed and expressed into the host Escherichia coli BL21 (DE3) (Beijing Bomed Gene Technology Co., Ltd., product number: BC201-01) to obtain a recombinant strain, which was inoculated into 1 L LB liquid medium. (containing 50 ⁇ g mL -1 kanamycin), incubated at 37 ° C, 200rpm to an OD 600 between 0.6-0.8, adding IPTG (isopropyl- ⁇ -D-thiogalactoside) to the final concentration It was induced at 1 mM, 30 ° C overnight.
  • the crude enzyme solution contains the recombinant protein PbBgl64A, ie, recombinant ⁇ -1,3-glucanase ( PbBgl64A).
  • the recombinant protein PbBgl64A was purified using an agarose Ni-IDA affinity column (ie, the N-terminus of the amino acid sequence shown in sequence 2 of the sequence listing was linked to His- Tag tag sequence (HHHHHH) recombinant protein).
  • the specific purification steps are as follows:
  • the crude enzyme solution was applied to a Ni-IDA column for purification.
  • the specific procedure for purification is (flow rate is 1 mL min -1 ): first eluted with buffer A (20 mM Tris-Hcl flush, 0.5 M NaCl, 20 mM imidazole, pH 7.9) to an OD 280 of less than 0.05, then buffer B (20 mM Tris-Hcl flush, 0.5 M NaCl, 50 mM imidazole, pH 7.9) eluted to an OD 280 of less than 0.05 and finally washed with buffer C (20 mM Tris-Hcl flush, 0.5 M NaCl, 200 mM imidazole, pH 7.9) Take off. The fraction eluted with buffer C was collected to obtain a solution of purified recombinant ⁇ -1,3-glucanase (PbBgl64A).
  • Protein purity was determined by SDS-PAGE (Laemmli, U.K. Nature, 1970, 227 (5259): 680-685) (Fig. 1). The results showed that the recombinant protein PbBgl64A was purified by Ni-IDA affinity column in one step, and the molecular weight was about 45kDa.
  • the enzyme activity of the recombinant ⁇ -1,3-glucanase (PbBgl64A) prepared in the first step was 207.2 U mg -1 .
  • the method for determining the enzyme activity of ⁇ -1,3-glucanase is as follows: 350 ⁇ L of colloid is added to a 1.5 mL centrifuge tube to obtain a polysaccharide substrate and 50 ⁇ L of an appropriately diluted enzyme solution, and reacted at 55 ° C for 20 min, followed by The DNS method measures the reducing sugar content in the reaction solution.
  • the enzyme activity unit (1 U) is defined as the amount of enzyme required to produce 1 ⁇ mol of glucose per minute under the above reaction conditions.
  • the colloid can be obtained as a polysaccharide preparation method: 1.2 g of the obtainable polysaccharide is resuspended in 30 mL of a buffer solution (50 mM acetic acid-sodium acetate buffer pH 5.0, 100 mL of NaCl) to form a 4% (w/v) polysaccharide. Suspension. The suspension was placed in a magnetic stirring water bath and heated at 55 ° C for 4 h. Through this treatment, it is found that the polysaccharide suspension forms a uniform colloid.
  • a buffer solution 50 mM acetic acid-sodium acetate buffer pH 5.0, 100 mL of NaCl
  • the optimum pH of the enzyme was determined by the following different pH buffer systems: McIlvaine buffer (pH 3.0-7.0), acetic acid-sodium acetate buffer (pH 4.0-5.5), and Tris-HCl buffer (7.0-9.0). . Then, the enzyme activity of the purified recombinant ⁇ -1,3-glucanase prepared in the first step at each pH was measured at 55 ° C according to the method in step 1, and the highest enzyme activity was taken as 100%, and the result is shown in FIG. 2A. Shown. Figure 2A shows that the optimum pH of the recombinant ⁇ -1,3-glucanase (PbBgl64A) is pH 5.5 (acetic acid-sodium acetate buffer).
  • the purified recombinant ⁇ -1,3-glucanase (PbBgl64A) solution was diluted with the following three different pH buffers: McIlvaine buffer (pH 3.0-7.0), acetic acid-sodium acetate buffer (pH 4.0- 5.5) and Tris-HCl buffer (7.0-9.0), the diluted enzyme solution was incubated in a constant temperature water bath at 30 ° C for 30 min, then cooled in an ice water bath for 30 min, and finally at step 5 at 55 ° C and pH 5.5.
  • the method of the present invention measures the enzyme activity of the purified recombinant ⁇ -1,3-glucanase prepared in the first step, and uses the enzyme activity of the untreated enzyme solution as a control to calculate the enzyme solution after different acid-base conditions. Residual enzyme activity. Relative enzyme activity was calculated as the percentage of residual enzyme activity as a percentage of control enzyme activity.
  • Figure 2B shows that the recombinant ⁇ -1,3-glucanase (PbBgl64A) has a broad pH stability range, and when the pH is pH 4.5-8.5, the residual enzyme activity is mostly above 80%.
  • the solution of the purified recombinant ⁇ -1,3-glucanase prepared in the first step was appropriately diluted in 50 mM acetic acid-sodium acetate buffer (pH 5.5), and then at different temperatures: 30 ° C, 40 ° C, respectively.
  • the enzyme activities of ⁇ -1,3-glucanase were measured at 50 ° C, 60 ° C, 70 ° C and 80 ° C according to the method in Step 1, and the highest value of the enzyme activity was taken as 100%, and the results are shown in Fig. 2C.
  • Figure 2C shows that the optimum temperature of the recombinant ⁇ -1,3-glucanase (PbBgl64A) is 70 °C.
  • the solution of the purified recombinant ⁇ -1,3-glucanase prepared in the first step was appropriately diluted in 50 mM acetic acid-sodium acetate buffer (pH 5.5), and then at different temperatures: 30 ° C, 40 ° C, respectively. , 50 ° C, 60 ° C, 70 ° C and 80 ° C, heat for 30 min, then cooled in an ice water bath for 30 min, and finally at 70 ° C and pH 5.5 according to the method in step 1 to determine ⁇ -1,3-glucanase
  • the enzyme activity was calculated by using the unheated enzyme solution as a control to calculate the residual enzyme activity of the enzyme solution after different heat treatment.
  • Relative enzyme activity was calculated as the percentage of residual enzyme activity as a percentage of control enzyme activity. The result is shown in Figure 2D.
  • Figure 2D shows that the recombinant ⁇ -1,3-glucanase (PbBgl64A) is stable below 65 °C and the enzyme activity can be maintained above 80%.
  • the silica gel plate was developed twice with a spreading agent, and after drying, the developer was uniformly used on the surface of the developer: sulfuric acid: methanol (5:95, v/ v) The solution was wetted and then baked in an oven at 130 ° C for 5 min to develop color.
  • PbBgl64A This feature of PbBgl64A is similar to the previously reported GH64 family of ⁇ -1,3-glucanase, a laminaria pentasaccharide ⁇ -1,3-glucanase (laminaripentaose-producing ⁇ -1,3-glucanase). ) (J Ferment Bioeng 1998, 85: 459–464). The hydrolysis of different ⁇ -1,3-gluco oligosaccharides by PbBgl64A is shown in Fig. 3B.
  • PbBgl64A has no hydrolysis ability for the tested kelp disaccharide, kelp triose, kelp tetrasaccharide, kelp pentasaccharide and kelp hexaose. There is also no transglycoside product formation, which is different from the hydrolysis characteristics of other family ⁇ -1,3-glucanases that have been reported.
  • the hydrolysis product of PbBgl64A can be identified by MALDI-TOF MS. It is found that the oligosaccharide content is extremely rich, and the degree of oligosaccharide polymerization can be detected from 2 to 21 continuously, and the content of pentose is the highest, and it is detected by TLC. The result is the same ( Figure 4).
  • the polysaccharide is a linear, unbranched, water-insoluble ⁇ -1,3-glucan which, as a bacterial extracellular polysaccharide, is usually secreted by Agrobacterium sp. Due to its good gel-forming properties, polysaccharides are widely used as a thickener in the food industry. Therefore, the polysaccharide is also a safe and reliable raw material for the preparation of ⁇ -1,3-gluco oligosaccharides.
  • PbBgl64A hydrolysis can be obtained from the initial conditions of the polysaccharide reaction with reference to the optimum reaction conditions of the enzyme: pH 5.0, 60 ° C, substrate concentration 2%, enzyme amount 0.05 U / mL, hydrolysis time 8 h.
  • the volume of the hydrolyzate was 200 mL, and it was shaken and hydrolyzed at 80 rpm/min in a constant temperature shaker. After the reaction, it was kept in a boiling water bath. The reaction was stopped at a temperature of 10 min. Subsequent reduction of sugar concentration and TLC analysis were performed. The reducing sugar concentration of the reaction solution was determined by the DNS method, and the standard curve was determined using glucose as a standard.
  • the TLC analysis procedure was the same as in Example 2. Optimization of enzymatic conditions mainly includes initial pH, enzyme addition amount, enzymatic hydrolysis temperature, enzymatic hydrolysis time and substrate concentration. The specific experimental results are as follows:
  • pH has a certain effect on the hydrolysis efficiency of ⁇ -1,3-glucanase, which not only affects the protein conformation of ⁇ -1,3-glucanase, but also may affect ⁇ -1,3-glucan.
  • the pH at the time of enzymatic hydrolysis has a certain influence on the reducing sugar concentration of the reaction solution.
  • the concentration of reducing sugar increases first and then decreases at the same time of enzymatic hydrolysis, and the enzymatic hydrolysis effect at pH 5.0.
  • the concentration of reducing sugar (in terms of glucose) after hydrolysis for 8 h reaches 4.68 mg/mL, which is the same as the optimum pH of the enzyme.
  • the reaction temperature is an important condition for determining the final sugar concentration of the final reaction solution.
  • the enzymatic hydrolysis efficiency was significantly improved, and the yield of reducing sugar was also greatly increased.
  • the enzymatic hydrolysis temperature was 55 °C, the peak was reached, and the reducing sugar concentration was 3.97 mg/mL.
  • the reaction temperature has a great influence on the hydrolysis reaction. Under the reaction conditions of 30-45 ° C, the hydrolysis reaction proceeds slowly, but when the reaction temperature rises to 50 ° C, the reaction rate sharply increases.
  • the gel formed by the polysaccharide can be classified into a low gel and a high gel according to its properties (low gel: heating the water dispersion of the gel to about 55-65 ° C, the heat formed Reversible gel; high gel: and when the lower gel is heated above 80 ° C, a thermally irreversible gel is formed).
  • low gel heating the water dispersion of the gel to about 55-65 ° C, the heat formed Reversible gel
  • high gel and when the lower gel is heated above 80 ° C, a thermally irreversible gel is formed.
  • the hydrolysis reaction temperature reaches 50 ° C, the gel can be swollen to form a low gel.
  • the enzyme molecules can be more fully. It binds to the ⁇ -1,3-glucan sugar chain, so the hydrolysis reaction rate is effectively increased.
  • the enzyme stability decreased inactivation, resulting in reduced enzymatic efficiency.
  • Substrate concentration is one of the key factors affecting the efficiency of enzymatic hydrolysis and the final oligosaccharide yield. Higher substrate concentration can improve the enzymatic efficiency and reduce the cost of enzymatic hydrolysis and oligosaccharide subsequent treatment, but too high substrate concentration may form product inhibition, reduce enzymatic efficiency, and final product yield, while prolonging enzymatic hydrolysis time. .
  • the PbBgl64A hydrolysis was determined at a substrate concentration of 2-8% to determine the reducing sugar concentration and soluble oligosaccharide product yield in the final hydrolysate of the polysaccharide (Fig. 8, Table 1).
  • Soluble oligosaccharide product yield The soluble oligosaccharide obtained by centrifugation of the hydrolyzate was freeze-dried and weighed, and the mass of the buffer inorganic salt was subtracted. The experimental results show that with the increase of substrate concentration, the concentration of reducing sugar in the hydrolyzate gradually increases, and the concentration of reducing sugar in the hydrolysate reaches the peak at about 4h.
  • the soluble oligosaccharide yields were 80.9%, 75.4%, 68.0% and 69.7% at different substrate concentrations of 2%, 4%, 6% and 8%, respectively.
  • 4% substrate concentration and hydrolysis 4-6h were selected as the optimal conditions.
  • the final PbBgl64A hydrolysis can be obtained as follows: pH 5.0, 55 ° C, substrate concentration 4%, enzyme amount 0.06 U/mL, hydrolysis time 4-6 h. Under the hydrolysis conditions, the final soluble oligosaccharide yield was 75.4% or more.
  • the hydrolyzate was subjected to TLC detection.
  • the specific steps are as follows: Samples were taken from the hydrolyzate at 1, 2, 3, 4, and 6h, respectively, and the sample was kept in a boiling water bath for 10 min to inactivate the enzyme, and then the supernatant was centrifuged, and the hydrolyzed product was compared by TLC analysis.
  • the final reaction liquid oligosaccharide composition was quantified by ion chromatography. Specific steps are as follows: The reaction mixture was centrifuged at 10000 ⁇ g 1min, and the supernatant was diluted 100 times with 100mM NaOH solution, using HPAEC-PAD (ICS-5000 + , Thermo, USA) combined CarboaPac TM PA1 (4 ⁇ 250mm, Thermo, USA Anion exchange column detects the sample. The loading amount was 25 ⁇ L.
  • the elution conditions 100 mM NaOH solution containing 0-350 mM sodium acetate linear elution, elution time 20 min, flow rate 1 mL/min, column temperature 30 °C.
  • the ⁇ -1,3-gluco oligosaccharide hydrolyzate prepared in the second step was filtered through a frame (200-400 mesh, pressure 0.3 MPa) or centrifuged (3000-5000 rpm) to remove the insoluble matter.
  • the soluble oligosaccharide hydrolyzate is collected, and then the feed liquid is pumped into the ion exchange column, using an anion-cation exchange resin (macroporous weakly basic styrene anion exchange resin (D301 type); strong acid styrene cation exchange resin (001) ⁇ 7 type))
  • the ions in the feed liquid are removed.
  • the solution after deionization is concentrated by rotary evaporation (70-80 ° C) to The sugar liquid solid content is 30-60%, and spray-dried to obtain a ⁇ -1,3-gluco oligosaccharide powder.
  • the ⁇ -1,3-gluco oligosaccharide hydrolyzate prepared in the second step was filtered through a frame (200-400 mesh, pressure 0.3 MPa) or centrifuged (3000-5000 rpm) to remove the insoluble matter.
  • the soluble oligosaccharide hydrolyzate was collected, and the membrane liquid filtration system (nanofiltration membrane: interception amount 150; operating pressure 0.4-0.8 mP) was used to concentrate the liquid to 20-30% of the solid content of the sugar liquid, during which the liquid was removed. Most of the ions give oligosaccharide syrup.
  • the oligosaccharide syrup is spray dried to obtain a ⁇ -1,3-gluco oligosaccharide powder.
  • the protein provided by the present invention (specifically, the recombinant protein PbBgl64A in the examples) has ⁇ -1,3-glucanase activity, has a specific enzyme activity of 207.2 U mg -1 , and the optimum reaction pH is 5.5, and It is stable in a wide pH range (4.5-8.5); the optimum reaction temperature is 70 ° C, and maintains high enzyme activity below 65 ° C, and has good heat resistance; the protein acts as ⁇ -1,3
  • the glycanase can be efficiently hydrolyzed to obtain a high polymerization degree ⁇ -1,3-glucose oligosaccharide, and the obtained oligosaccharide polymerization degree is distributed in 2-21, and the yield of the oligosaccharide obtained by hydrolysis is 80% or more.
  • the protein provided by the invention has good enzymatic properties of ⁇ -1,3-glucanase, and has good application value in oligosaccharide preparation and food, feed and

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Abstract

本发明提供了一种β-1,3-葡聚糖酶及其编码基因与应用。所述β-1,3-葡聚糖酶是如下a）或b）或c）：a）氨基酸序列是序列表中序列2第29-449位的蛋白质；b）在序列表中序列2第29-449位所示的蛋白质的N端和/或C端连接标签得到的融合蛋白质；c）将序列表中序列2第29-449位所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的且具有相同功能的蛋白质。还提供了所述β-1,3-葡聚糖酶在制备β-1,3-葡寡糖和/或高聚合度β-1,3-葡寡糖中的应用，以及一种生产高聚合度β-1,3-葡寡糖的方法。

Description

一种细菌β-1,3-葡聚糖酶及其编码基因与应用 技术领域
本发明属于食品生物技术领域，具体涉及一种细菌β-1,3-葡聚糖酶及其编码基因与应用。
背景技术
β-1,3-葡聚糖(β-1,3-glucan)是一种在自然界中分布广泛的高分子多糖，其主链是由葡萄糖残基在C1和C3位上通过β-1,3-糖苷键连接而成。β-1,3-葡聚糖广泛分布于真菌、细菌和植物中，发挥着多种多样的生理功能。自然界中常见的β-1,3-葡聚糖包括昆布多糖、可得然多糖、酵母葡聚糖、香菇多糖、茯苓多糖以及裂褶多糖等。β-1,3-葡聚糖的降解产物β-1,3-葡寡糖(又称为昆布寡糖)是一种新型功能性低聚糖，具有促进肠道益生菌增值、抗肿瘤、降血糖等生物活性，在食品、农业、医学等领域具有广泛的应用前景(Ann Microbiol，2011，62:307–312；Appl Microbiol Biotechnol，2012，93:525–531)。
内切型β-1,3-葡聚糖酶(endo-β-1,3-glucanase，EC 3.2.1.39；简称β-1,3-葡聚糖酶)又称昆布多糖酶，是一种专一性水解β-1,3-葡聚糖中的β-1,3-糖苷键的酶类。β-1,3-葡聚糖酶从糖链的内侧水解聚糖，产物为一系列不同大小的寡糖。β-1,3-葡聚糖酶具有严格的底物特异性，对含有β-1,4-糖苷键的β-1,3-1,4-葡聚糖水解效率很低。β-1,3-葡聚糖酶分布广泛，主要来源于真菌、细菌、植物及一些低等动物和病毒，具有多种生物学功能(Acta Cryst，2013，D69，2027–2038)。根据碳水化合物代谢酶数据库(Carbohydrate-Active enZYmes Database；http://www.cazy.org)分类，已发现的β-1,3-葡聚糖酶根据序列进化关系可被归属于7个糖苷水解酶家族(Glycoside Hydrolase；GH)：GH16、17、55、64、81、128和132。微生物是β-1,3-葡聚糖酶的一个主要来源。细菌β-1,3-葡聚糖酶主要属于糖苷水解酶GH16家族。细菌分泌的胞外β-1,3-葡聚糖酶主要用于降解其生长环境中的真菌细胞壁或植物来源的β-1,3-葡聚糖，作为其生长繁殖的能源(J Bacteriol，1995，177:6937–6945)。此外，一些古生菌或线虫内也发现β-1,3-葡聚糖酶(Biochem J，2005，389:117–125)。真菌β-1,3-葡聚糖酶主要属于GH16家族与GH81家族，一般含有信号肽，定位于细胞膜附 近，也有一些真菌胞外分泌的β-1,3-葡聚糖酶(J Microbiol Methods，2010，81:6–10；Acta Cryst 2013，D69，2027–2038)。β-1,3-葡聚糖酶在真菌细胞壁部分消解及重塑的过程中发挥着重要作用。真菌β-1,3-葡聚糖酶与几丁质酶、转糖苷酶彼此协同，共同作用于细胞壁中的β-1,3-葡聚糖，完成真菌细胞的细胞壁的分裂及重铸过程(Front Microbiol，2013，4:81)。而植物来源的β-1,3-葡聚糖酶也有许多资料报道(Acta Cryst，2012，D68，713–723)，其通常涉及植物抗真菌机制。公开号CN102174547B的专利显示，将β-1,3-葡聚糖酶基因在植物内重组表达后能够显著提高植物抗真菌的能力。
β-1,3-葡聚糖酶制剂可广泛应用于啤酒工业、饲料工业、生物防治以及医药等领域(公开号[CN101565682B]；Appl Environ Microbiol，2011，77:983–990；J Appl Microbiol，2000，88:961–967)。此外，一些β-1,3-葡聚糖及一些小分子可溶β-1,3-葡寡糖是有效的免疫激活剂，对β-1,3-葡寡糖生物活性的研究近年来也多有报道。目前β-1,3-葡寡糖的制备方法主要包括酶法、化学法、物理法或多者方法耦合(Carbohyd Polym，2008，71:277–286；Food Sci Biotechnol，2014，23:799–806；Carbohyd Polym，2011，86:574–580)，其中酶法及化学法是较为常用的寡糖制备方法。迄今，酶法生产β-1,3-葡寡糖技术仍存在一些瓶颈，例如内切β-1,3-葡聚糖酶活性较低，水解时间较长，水解过程不易调控等。一些专利公开了β-1,3-葡聚寡糖的制备方法。如申请号[201210136867.0]公开一种β-1,3-葡寡糖的制备方法，利用酸解、酶解相结合制备β-1,3-葡寡糖，但方法中用到大量三氟乙酸、Ba(OH)2、氨水等试剂，污染较大，存在残留隐患，产品不宜用于食品、药品等领域应用。申请号[201510123040.X]公开一种利用热凝胶发酵液直接生产β-1,3-葡寡糖的方法，反应体系中含发酵液，且生产工艺需使用HCl、NaOH等化学试剂，不利于后续寡糖纯品的制备和应用。尽管目前已有一些β-1,3-葡聚糖酶及β-1,3-葡寡糖的制备相关研究，但基于食品、饲料等行业生产的实际需要，目前仍需开发具有良好稳定性及底物特异性的新型β-1,3-葡聚糖酶和环保、低能耗的β-1,3-葡寡糖的制备方法。另外，由于酶解产物聚合度不易控制，生产高聚合度β-1,3-葡寡糖仍然存在很大难度。
可得然多糖是由产碱杆菌产生的一种微生物胞外多糖，它是由400-500个D-葡萄糖以β-1,3-糖苷键线性连接而成，是自然界唯一的直链β-1,3-葡聚糖。可得然多糖由于其独特的凝胶特性，通常被用于食品增稠剂。可得然多糖可以溶于稀碱溶液，但是不溶于水，限制了它在医药和食品工业中的应用。降解可得然多糖成为一定分子量的β-1,3-葡寡糖，是解决其溶解性差的一种方法。此外，可得然多糖来源广泛，价格较低，也是是制备具有生物活性的β-1,3-葡寡糖的可靠来源。
发明公开
本发明的一个目的是提供一种蛋白质。
本发明提供的蛋白质是如下a)或b)或c)的蛋白质，将其命名为PbBgl64A，
a)氨基酸序列是序列表中序列2第29-449位氨基酸残基编码的蛋白质；
b)在序列表中序列2第29-449位所示的蛋白质的N端和/或C端连接标签得到的融合蛋白质；
c)将序列表中序列2第29-449位所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的且具有相同功能的蛋白质。
为了使a)中的蛋白质便于纯化，可在序列表中序列2第29-449位氨基酸残基编码的蛋白质的氨基末端或羧基末端连接上如表1所示的标签。
表1、标签的序列
标签 残基 序列
Poly-Arg 5-6(通常为5个) RRRRR
Poly-His 2-10(通常为6个) HHHHHH
FLAG 8 DYKDDDDK
Strep-tag II 8 WSHPQFEK
c-myc 10 EQKLISEEDL
上述c)中的蛋白质，所述一个或几个氨基酸残基的取代和/或缺失和/或添加为不超过10个氨基酸残基的取代和/或缺失和/或添加。
上述c)中的蛋白质可人工合成，也可先合成其编码基因，再进行生物表达得到。
上述c)中的蛋白质的编码基因可通过将序列1第85-1350位所示的DNA序列中缺失一个或几个氨基酸残基的密码子，和/或进行一个或几个碱基对的错义突变，和/或在其5′端和/或3′端连上表1所示的标签的编码序列得到。
本发明的另一个目的是提供与上述蛋白质相关的生物材料。
本发明提供的生物材料为下述B1)-B5)中的任一种：
B1)编码上述蛋白质的核酸分子；
B2)含有B1)所述核酸分子的表达盒；
B3)含有B1)所述核酸分子的重组载体或含有B2)所述表达盒的重组载体；
B4)含有B1)所述核酸分子的重组菌或含有B2)所述表达盒的重组菌或含有B3)所述重组载体的重组菌；
B5)含有B1)所述核酸分子的细胞系或含有B2)所述表达盒的细胞系或含有B3)所述重组载体的细胞系。
上述生物材料中，B1)所述核酸分子的为如下1)或2)或3)的DNA分子：
1)核苷酸序列是序列表中序列1第85-1350位所示的DNA分子；
2)与1)限定的DNA序列至少具有70％、至少具有75％、至少具有80％、至少具有85％、至少具有90％、至少具有95％、至少具有96％、至少具有97％、至少具有98％或至少具有99％同源性且编码所述蛋白质的DNA分子；
3)在严格条件下与1)或2)限定的DNA序列杂交且编码所述蛋白质的DNA分子。
其中，所述核酸分子可以是DNA，如cDNA、基因组DNA或重组DNA；所述核酸分子也可以是RNA，如mRNA或hnRNA等。
本领域普通技术人员可以很容易地采用已知的方法，例如定向进化和点突变的方法，对本发明的编码PbBgl64A的核苷酸序列进行突变。那些经过人工修饰的，具有编码PbBgl64A的核苷酸序列75％或者更高同一性的核苷酸，只要编码PbBgl64A且具有相同功能，均是衍生于本发明的核 苷酸序列并且等同于本发明的序列。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括与本发明的编码序列2第29-449位所示的氨基酸序列组成的蛋白质的核苷酸序列具有75％或更高，或85％或更高，或90％或更高，或95％或更高同一性的核苷酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。
上述75％或75％以上同一性，可为80％、85％、90％或95％以上的同一性。
上述生物材料中，A2)所述的含有编码PbBgl64A的核酸分子的表达盒(PbBgl64A基因表达盒)，是指能够在宿主细胞中表达PbBgl64A的DNA，该DNA不但可包括启动PbBgl64A转录的启动子，还可包括终止PbBgl64A转录的终止子。进一步，所述表达盒还可包括增强子序列。可用于本发明的启动子包括但不限于：组成型启动子；组织、器官和发育特异的启动子及诱导型启动子。启动子的例子包括但不限于：花椰菜花叶病毒的组成型启动子35S：来自西红柿的创伤诱导型启动子，亮氨酸氨基肽酶("LAP",Chao等人(1999)Plant Physiol 120：979-992)；来自烟草的化学诱导型启动子，发病机理相关1(PR1)(由水杨酸和BTH(苯并噻二唑-7-硫代羟酸S-甲酯)诱导)；西红柿蛋白酶抑制剂II启动子(PIN2)或LAP启动子(均可用茉莉酮酸甲酯诱导)；热休克启动子(美国专利5,187,267)；四环素诱导型启动子(美国专利5,057,422)；种子特异性启动子，如谷子种子特异性启动子pF128(CN101063139B(中国专利200710099169.7))，种子贮存蛋白质特异的启动子(例如，菜豆球蛋白、napin,oleosin和大豆beta conglycin的启动子(Beachy等人(1985)EMBO J.4：3047-3053))。它们可单独使用或与其它的植物启动子结合使用。此处引用的所有参考文献均全文引用。合适的转录终止子包括但不限于：农杆菌胭脂碱合成酶终止子(NOS终止子)、花椰菜花叶病毒CaMV35S终止子、tml终止子、豌豆rbcS E9终止子和胭脂氨酸和章鱼氨酸合酶终止子(参见，例如：Odell等人(I985)Nature 313:810；Rosenberg等人(1987)Gene,56:125；Guerineau等人(1991)Mol.Gen.Genet, 262:141；Proudfoot(1991)Cell,64:671；Sanfacon等人Genes Dev.,5:141；Mogen等人(1990)Plant Cell,2:1261；Munroe等人(1990)Gene,91:151；Ballad等人(1989)Nucleic Acids Res.17:7891；Joshi等人(1987)Nucleic Acid Res.,15:9627)。
上述生物材料中，所述载体可为质粒、黏粒、噬菌体或病毒载体。
上述生物材料中，所述微生物可为酵母、细菌、藻或真菌，如农杆菌。
本发明的另一个目的是提供上述蛋白质的新用途。
本发明提供了上述蛋白质在作为β-1,3-葡聚糖酶中的应用。
本发明还提供了上述蛋白质在水解β-1,3-葡聚糖中的应用。
上述应用中，所述β-1,3-葡聚糖为可得然多糖、昆布多糖或酵母葡聚糖。
本发明还提供了上述蛋白质在制备β-1,3-葡寡糖和/或高聚合度β-1,3-葡寡糖中的应用。
上述应用中，所述聚合度为2-21。
本发明的另一个目的是提供一种重组菌。
本发明提供的重组菌是将上述蛋白质的编码基因导入宿主菌中得到的菌。
上述重组菌中，所述蛋白质的编码基因是通过重组载体导入宿主菌；
所述重组载体为将上述蛋白质的编码基因插入表达载体的多克隆位点中得到的载体。所述重组载体具体为在载体pET-28a(+)的Nhe Ⅰ和Xho Ⅰ位点间插入了序列表序列1中第85-1350位所示的DNA片段，且保持载体pET-28a(+)的其他序列不变得到的载体。
上述重组菌中，所述蛋白质的编码基因是序列表中序列1第85-1350位所示的DNA分子。
上述重组菌中，所述宿主菌为大肠杆菌BL21(DE3)。
本发明还有一个目的是提供一种β-1,3-葡聚糖酶的制备方法。
本发明提供的β-1,3-葡聚糖酶的制备方法包括如下步骤：诱导培养上述重组菌，得到β-1,3-葡聚糖酶。
本发明的最后一个目的是提供一种生产高聚合度β-1,3-葡寡糖的方法。
本发明提供的生产高聚合度β-1,3-葡寡糖的方法包括如下步骤：用上述蛋白质水解β-1,3-葡聚糖，得到高聚合度β-1,3-葡寡糖。
上述方法中，所述蛋白质与所述β-1,3-葡聚糖的配比为1500U:1kg。
上述方法中，所述权利要求1所述的蛋白质的浓度为0.06U/mL。
上述方法中，所述β-1,3-葡聚糖的质量分数为4％。
上述方法中，所述水解的条件为：pH 5.0，50-60℃水解4-6h；所述水解的条件具体为：pH 5.0，55℃水解5h。
上述方法中，所述β-1,3-葡聚糖为可得然多糖。
本发明进一步通过下面的实施例进行阐述。
附图说明
图1显示了大肠杆菌重组PbBgl64A经Ni-IDA纯化的纯化图。其中，泳道M代表低分子量标准蛋白，泳道1代表破壁后的上清液，泳道2代表经Ni-IDA亲和层析后的蛋白。
图2显示了温度和pH变化对PbBgl64A酶活力和稳定性的影响。其中，(A)最适pH，(B)pH稳定性，(C)最适温度，(D)温度稳定性；所用缓冲液为：McIlvaine缓冲液(■)，醋酸-醋酸钠缓冲液(●)以及Tris–HCl缓冲液(▲)。
图3显示了PbBgl64A对不同底物的水解特性。其中，(A)为PbBgl64A水解β-1,3-葡聚糖(可得然多糖)的水解历程薄层层析分析结果。(B)为PbBgl64A水解β-1,3-葡寡糖(昆布二糖、昆布三糖、昆布四糖、昆布五糖及昆布六糖)产物薄层层析分析结果，(B)图中每组寡糖的分析结果中左侧为反应前，右侧为反应后。
图4显示了PbBgl64A水解可得然多糖水解产物的MALDI-TOF MS分析图谱。图中显示水解产物的聚合度分布在2-21，其中，五糖丰度最高。
图5显示了不同初始pH条件对PbBgl64A水解可得然多糖效率的影响。
图6显示了不同初始温度条件对PbBgl64A水解可得然多糖效率的影响。
图7显示了不同加酶量对PbBgl64A水解可得然多糖效率的影响。
图8显示了不同底物浓度对PbBgl64A水解可得然多糖最终水解液中 还原糖浓度的影响。
图9为PbBgl64A水解可得然多糖产物分析图。其中，(A)为水解历程薄层层析分析结果；(B)为最终水解产物组分离子色谱分析峰图。
实施发明的最佳方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中的巴伦葛兹类芽孢杆菌(Paenibacillus barengoltzii)CAU904已保存于中国微生物菌种保藏中心(简称CGMCC，地址：北京市朝阳区大屯路)，保藏编号为CGMCC No.9530，在文献“Biotechnol Biofuel 2014,7:174”中公开过。
实施例1、基因PbBgl64A及蛋白PbBgl64A的获得
依据Genbank数据库中已提交的巴伦葛兹类芽孢杆菌基因组序列信息，去除第1-28位氨基酸残基形成的信号肽片段，设计上游引物PbBgl64A-up(5’-TGACTGCTAGCGCTGATTTCACTCAAGGAGCGG-3’，下划线示Nhe Ⅰ酶切位点)和下游引物PbBgl64A-down(5’-TGACTCTCGAGTTACCAGCCCACTCTGACGATG-3’，下划线示Xho Ⅰ酶切位点)，并以巴伦葛兹类芽孢杆菌CAU904的基因组DNA为模板进行PCR扩增，得到PCR扩增产物。
PCR扩增条件为：94℃预变性5min；94℃变性30s，54℃退火30s，72℃延伸90s，循环35次；最后72℃后延伸5min。
PCR产物经过1％琼脂糖凝胶电泳回收，以Nhe Ⅰ和Xho Ⅰ双酶切。将该双酶切后的产物与经相同双酶切过的原核表达载体pET-28a(+)(美国Novagen公司，产品编号：69864-3)片段以T4DNA连接酶进行连接，得到重组质粒，并将其转化至宿主大肠杆菌DH5α。挑选菌落PCR(PCR所用的引物和扩增条件与本段前述PCR的相同)验证为阳性的转化子测序。
测序结果表明：重组质粒为在载体pET-28a(+)的Nhe Ⅰ和Xho Ⅰ位点间插入了序列表序列1中第85-1350位所示的DNA片段，阳性转化子即为含有上述重组质粒的大肠杆菌DH5α。
将序列表序列1中第85-1350位所示的基因命名为基因PbBgl64A，将该基因所编码的蛋白命名为蛋白PbBgl64A，其氨基酸序列如序列表序列2中第29-449位所示。
实施例2、重组β-1,3-葡聚糖酶(PbBgl64A)的表达和纯化及其性质检测
一、重组β-1,3-葡聚糖酶(PbBgl64A)的表达和纯化
将实施例1中的重组质粒转化表达至宿主大肠杆菌BL21(DE3)(北京博迈德基因技术有限公司，产品编号：BC201-01)，得到重组菌，并将其接种至1L LB液体培养基(含50μg mL-1卡那霉素)，在37℃，200rpm条件下培养至OD600在0.6-0.8之间，加入IPTG(异丙基-β-D-硫代半乳糖苷)至终浓度为1mM，30℃诱导过夜。离心收集菌体后，将菌体按照1:10(v/v)的比例，用缓冲液A(20mM Tris-Hcl冲液，0.5M NaCl，20mM咪唑,pH 7.9)重悬浮，然后于冰水浴中超声破碎(200W，超声3s，间歇4s，120次)，再离心收集上清液即为粗酶液，粗酶液中含有重组蛋白PbBgl64A，即重组β-1,3-葡聚糖酶(PbBgl64A)。
基于pET28-a(+)质粒中有编码His-Tag标签蛋白的序列，使用琼脂糖Ni-IDA亲和柱纯化重组蛋白PbBgl64A(即在序列表序列2所示氨基酸序列的N端连接了His-Tag标签序列(HHHHHH)的重组蛋白)。具体纯化步骤如下：
将粗酶液上样于Ni-IDA柱进行纯化。纯化的具体步骤为(流速为1mL min-1)：先用缓冲液A(20mM Tris-Hcl冲液，0.5M NaCl，20mM咪唑，pH 7.9)洗脱至OD280小于0.05，然后用缓冲液B(20mM Tris-Hcl冲液，0.5M NaCl，50mM咪唑，pH 7.9)洗脱至OD280小于0.05，最后用缓冲液C(20mM Tris-Hcl冲液，0.5M NaCl，200mM咪唑，pH 7.9)洗脱。收集缓冲液C洗脱部分，得到纯化的重组β-1,3-葡聚糖酶(PbBgl64A)的溶液。
经SDS-PAGE(Laemmli，U.K.Nature，1970，227(5259):680-685)检测蛋白纯度(图1)。结果显示重组蛋白PbBgl64A经Ni-IDA亲和柱一步纯化可得电泳纯蛋白，分子量大小约为45kDa。
二、重组β-1,3-葡聚糖酶(PbBgl64A)的性质检测
1、PbBgl64A的酶活力
步骤一制备得到的重组β-1,3-葡聚糖酶(PbBgl64A)的酶活力为207.2U mg-1。β-1,3-葡聚糖酶的酶活力的测定方法具体如下：在1.5mL离心管中先后加入350μL胶体可得然多糖底物及50μL适当稀释的酶液，55℃反应20min，随后采用DNS法测定反应液中的还原糖含量。酶活力单位(1U)定义为：在上述反应条件下，每分钟生成1μmol的葡萄糖所需要的酶量。其中，胶体可得然多糖制备方法：将1.2g可得然多糖用30mL缓冲液(50mM醋酸-醋酸钠缓冲液pH 5.0，100mL NaCl)重悬，形成4％(w/v)可得然多糖悬液。将悬液置于磁力搅拌水浴锅中55℃搅拌加热4h。经过这一处理，可得然多糖悬液即形成均一的胶体。
2、PbBgl64A的最适pH测定
分别以下述不同pH值的缓冲体系测定该酶最适pH值：McIlvaine缓冲液(pH 3.0-7.0)、醋酸-醋酸钠缓冲液(pH 4.0-5.5)以及Tris-HCl缓冲液(7.0-9.0)。然后在55℃按照步骤1中的方法分别测定各个pH下步骤一制备得到的纯化的重组β-1,3-葡聚糖酶的酶活力，以酶活力最高值作为100％，结果如图2A所示。图2A显示重组β-1,3-葡聚糖酶(PbBgl64A)的最适pH值为pH 5.5(醋酸-醋酸钠缓冲液)。
3、PbBgl64A的pH稳定性测定
分别用如下3种不同pH的缓冲液稀释纯化的重组β-1,3-葡聚糖酶(PbBgl64A)的溶液：McIlvaine缓冲液(pH 3.0-7.0)、醋酸-醋酸钠缓冲液(pH 4.0-5.5)以及Tris-HCl缓冲液(7.0-9.0)，将稀释好的酶液在30℃恒温水浴锅中保温30min，然后在冰水浴中冷却30min，最后在55℃和pH 5.5条件下按照步骤1中的方法测定步骤一制备得到的纯化的重组β-1,3-葡聚糖酶的酶活力，以未经处理的酶液的酶活力作为对照，分别计算经过不同酸碱条件处理后酶液的残余酶活力。以残余酶活力占对照酶活力的百分比计算相对酶活力。结果如图2B所示。图2B显示重组β-1,3-葡聚糖酶(PbBgl64A)具有较宽的pH稳定范围，当pH为pH 4.5-8.5时，残余酶活力大多在80％以上。
4、PbBgl64A的最适反应温度测定
将步骤一制备得到的纯化的重组β-1,3-葡聚糖酶的溶液在50mM的醋酸-醋酸钠缓冲液(pH 5.5)中适当稀释，然后分别在不同温度下：30℃、40℃、50℃、60℃、70℃和80℃按照步骤1中的方法测定β-1,3-葡聚糖酶的酶活力，以酶活力的最高值作为100％，结果如图2C所示。图2C显示重组β-1,3-葡聚糖酶(PbBgl64A)的最适温度为70℃。
5、PbBgl64A的温度稳定性测定
将步骤一制备得到的纯化的重组β-1,3-葡聚糖酶的溶液在50mM的醋酸-醋酸钠缓冲液(pH 5.5)中适当稀释，然后分别在不同温度下：30℃、40℃、50℃、60℃、70℃和80℃，保温30min，随后在冰水浴中冷却30min，最后在70℃和pH 5.5条件下按照步骤1中的方法测定β-1,3-葡聚糖酶的酶活力，以未经热处理的酶液作为对照，分别计算不同热处理后酶液的残余酶活力。以残余酶活力占对照酶活力的百分比计算相对酶活力。结果如图2D所示。图2D显示重组β-1,3-葡聚糖酶(PbBgl64A)在65℃以下较为稳定，酶活力能保持80％以上。
6、PbBgl64A水解特性
(1)薄层层析法(thin layer chromatography，TLC)监测水解产物
为了分析重组β-1,3-葡聚糖酶(PbBgl64A)的水解特性，分别以β-1,3-葡聚糖(可得然多糖)以及β-1,3-葡寡糖(昆布二糖、昆布三糖、昆布四糖、昆布五糖和昆布六糖)作为底物，分析其水解后生成的产物。反应条件：50mM醋酸-醋酸钠缓冲液pH 5.0，1％(w/v)底物浓度，加酶量5U/mL；55℃保温2h。为了监测反应进程，分别在不同的时间点取样，沸水浴5min终止反应。采用Kieselgel 60硅胶板(Merck)分析水解产物，展层液为正丁醇：乙酸：水(2:1:1，v/v/v)。样品经10000×g离心1min后点样2μL于硅胶板点样点，将硅胶板用展层剂展开两次，吹干后在其表面均匀用显色剂硫酸：甲醇(5:95,v/v)溶液浸湿，然后在130℃烘箱中烘烤5min显色。
(2)基质辅助激光解析电离飞行时间质谱法(Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry，Maldi-tof-MS)分析水解产物
为了鉴定PbBgl64A的最终水解中寡糖组成，采用MALDI-TOFMS法 分析PbBgl64A水解可得然多糖水解产物。样品制备条件：50mM醋酸-醋酸钠缓冲液pH 5.0，2％(w/v)底物浓度，加酶量5U/mL；55℃保温2h，沸水浴5min终止反应。将反应液10000g离心1min，取上清稀释100倍，与等体积10mg/mL 2,5-羟基苯甲酸水溶液混合均匀。样品(1μL)上样于Maldi载片使用AB SCIEX TOF/TOFTM 5800系统采用阳离子模式分析样品。
实验结果显示，PbBgl64A能够水解可得然多糖生成一系列的低聚糖，包含昆布二糖、昆布三糖、昆布四糖、昆布五糖及一系列高聚合度(DP>5)的昆布寡糖(图3A)。经过两小时的水解，底物中的可得然多糖几乎被完全水解，其中昆布五糖为该酶的主要水解产物。PbBgl64A的这一特点与已有报道的GH64家族β-1,3-葡聚糖酶类似，是一个昆布五糖型β-1,3-葡聚糖酶(laminaripentaose-producingβ-1,3-glucanase)(J Ferment Bioeng1998，85:459–464)。PbBgl64A对不同β-1,3-葡寡糖的水解情况如图3B所示，PbBgl64A对于实验所测的昆布二糖、昆布三糖、昆布四糖、昆布五糖和昆布六糖均无水解能力，也无转糖苷产物生成，这与已报到的其他家族β-1,3-葡聚糖酶的水解特性均不相同。PbBgl64A水解可得然多糖的水解产物经MALDI-TOF MS鉴定，发现其中低聚糖含量极为丰富，能够检测到的低聚糖聚合度从2连续分布到21，其中五糖含量最高，与TLC检测结果相同(图4)。
实施例3、重组β-1,3-葡聚糖酶(PbBgl64A)在酶法制备β-1,3-葡寡糖中的应用
一、PbBgl64A水解可得然多糖水解条件优化
可得然多糖是一种线性无支链的水不溶性β-1,3-葡聚糖，作为一种细菌胞外多糖，通常由产碱杆菌(Agrobacterium sp.)分泌。由于其良好的成胶特性，可得然多糖作为一种增稠剂被广泛应用于食品工业中。因此，可得然多糖也是一种用于制备β-1,3-葡寡糖的安全可靠的原料。
PbBgl64A水解可得然多糖反应初始条件参照酶的最适反应条件：pH5.0，60℃，底物浓度2％，加酶量0.05U/mL，水解时间8h。水解液体积200mL，在恒温摇床中80rpm/min震荡水解，反应结束后在沸水浴中保 温10min终止反应。随后进行还原糖浓度测定、TLC分析。反应液还原糖浓度采用DNS法测定，以葡萄糖为标准品测定标准曲线。TLC分析过程同实施例2中的步骤。酶解条件的优化主要包括初始pH、加酶量、酶解温度和酶解时间及底物浓度。具体实验结果如下：
1、不同初始pH条件对PbBgl64A水解可得然多糖效率的影响
pH对β-1,3-葡聚糖酶的水解效率有一定影响，不仅影响β-1,3-葡聚糖酶的蛋白构像，还有可能影响到β-1,3-葡聚糖酶和底物结合的能力，因此，需要考察酶解时反应液的pH值。
由图5可以看出，酶解时的pH对反应液还原糖浓度有一定的影响，随着pH的增加，酶解相同时间还原糖浓度先升高后降低，在pH 5.0的时候酶解效果最好，水解8h后还原糖浓度(以葡萄糖计)达到4.68mg/mL，与该酶的最适pH相同。
2、不同温度对PbBgl64A水解可得然多糖效率的影响
由图6可以看出，反应温度是决定最终反应液还原糖浓度的重要条件。随着温度的增加，酶解效率显著提高，还原糖得率也大幅增加，当酶解温度为55℃时达到顶点，此时还原糖浓度为3.97mg/mL。反应温度对水解反应具有极大影响，在30-45℃反应条件下，水解反应进行缓慢，但当反应温度上升到50℃时，反应速率急剧提升。这主要是由于可得然多糖形成的凝胶按其性质可分为低位凝胶和高位凝胶(低位凝胶：把可得然胶的水分散液加热到约55-65℃，形成的热可逆性的凝胶；高位凝胶：而当低位凝胶被加热到80℃以上，形成的热不可逆性凝胶)。当水解反应温度达到50℃，可得然胶溶胀形成低位凝胶，类似于淀粉颗粒糊化过程，当可得然胶形成低位凝胶后，可得然胶颗粒吸水溶胀，酶分子可以更加充分的与β-1,3-葡聚糖糖链结合，因此水解反应速率有效提高。继续增加温度反应液还原糖得率小幅降低，可能是酶稳定性下降失活，造成酶解效率降低。
3、不同加酶量对PbBgl64A水解可得然多糖效率的影响
由图7可以看出，随着加酶量的增加，酶解之后产生的还原糖含量小幅增加，当加酶量为0.06U/mL时，继续增加酶量，酶解所产生的还原糖含量增幅较小。加酶量为0.06U/mL时，酶解最终还原糖浓度达到4.29 mg/mL。结果显示，在加酶量为0.02-0.12U/mL时，水解产物还原糖浓度总体提升不大，表明在该水解条件下加酶量对最终产物浓度影响较小。
4、底物浓度对PbBgl64A水解可得然多糖效率的影响
底物浓度是影响酶解效率和最终寡糖得率的关键因素之一。较高的底物浓度能够提高酶解效率，降低酶解及寡糖后续处理成本，但底物浓度过高可能形成产物抑制作用，降低酶解效率，和最终产物得率，同时延长酶解时间。实验分别在2-8％的底物浓度下测定PbBgl64A水解可得然多糖最终水解液中还原糖浓度与可溶性寡糖产物得率(图8、表1)。可溶性寡糖产物得率为水解液离心所得的可溶性寡糖经冷冻干燥称重，减去缓冲液无机盐质量后计算所得。实验结果显示随着底物浓度的增加，水解液中的还原糖浓度逐渐增加，水解液还原糖浓度基本在4h左右即达到顶点。在2％、4％、6％和8％的不同底物浓度下，可溶性寡糖得率分别为80.9％、75.4％、68.0％与69.7％。综合考虑产物得率及后续寡糖纯化工序，选取4％的底物浓度、水解4-6h为最优条件。
经条件优化后最终的PbBgl64A水解可得然多糖条件为：pH 5.0，55℃，底物浓度4％，加酶量0.06U/mL，水解时间4-6h。在该水解条件下，最终可溶性寡糖得率为75.4％以上。
表1、不同底物浓度时PbBgl64A水解可得然多糖产物最终寡糖得率的变化统计情况
Figure PCTCN2016105187-appb-000001
二、高聚合度β-1,3-葡寡糖制备
1、高聚合度β-1,3-葡寡糖制备方法
将0.8kg可得然多糖加入20L去离子水中(w/v,4％)，搅拌均匀，用冰醋酸调节pH至5.0左右，使其混合物成为胶状悬液，随后按0.06U/mL(以β-1,3-葡聚糖酶活力计)比例加入PbBgl64A酶液，混匀后将混合液置于55℃、80-150rpm/min水解5h，所得反应液即为高聚合度β-1,3-葡寡糖溶液。
2、水解产物的TLC检测
将水解产物进行TLC检测。具体步骤如下：于1、2、3、4和6h分别从水解液中取样，将样品在沸水浴中保持10min使酶失活，然后离心取上清液，通过TLC分析比较水解产物。TLC分析操作：取1μL不同水解液分别点样于TLC层析板，将TLC层析板置于展层剂(正丁醇：乙酸：水＝2:1:1)中。待展层结束后用显色剂(硫酸：甲醇＝5:95)130℃烘烤显色。
在该水解条件下，最终可溶性寡糖得率为80％以上。TLC结果显示(图9A)，PbBgl64A在4％底物浓度下该水解条件所得寡糖组成与初始条件无差异，均为昆布五糖为主的一系列昆布寡糖。
3、水解产物的离子色谱(HPAEC)定量
采用离子色谱定量最终反应液寡糖组成。具体步骤如下：将反应液10000×g离心1min，将上清用100mM NaOH溶液稀释100倍，采用HPAEC-PAD(ICS-5000+,Thermo,USA)结合CarboaPacTM PA1(4×250mm，Thermo,USA)阴离子交换柱检测样品。上样量25μL，洗脱条件：100mM NaOH溶液包含0–350mM醋酸钠线性洗脱，洗脱时间20min，流速1mL/min，柱温30℃。
利用离子色谱对寡糖定量可得最终水解液中昆布五糖含量为1.81mg/mL，占所有寡糖含量的49.70％，其次含量较高的寡糖依次分别为昆布四糖、昆布六糖、昆布七糖(图9B及表2)。
表2、PbBgl64A水解可得然多糖产物中不同寡糖组分的离子色谱定量数据
  G L2 L3 L4 L5 L6 L7 L8 L9 L10
浓度(mg/mL) 0 0.0056 0.139 0.692 1.81 0.288 0.258 0.211 0.144 0.094
质量百分比(％) 0 0.15 3.83 19.00 49.70 7.91 7.08 5.79 3.95 2.58
三、β-1,3-葡寡糖产品精制
1、工艺一
将步骤二制备的β-1,3-葡寡糖水解液经板框过滤(200-400目，压力0.3MPa)或离心(3000-5000rpm)除去不容物。收集可溶寡糖水解液，随后将料液泵入离子交换柱，采用阴阳离子交换树脂(大孔弱碱性苯乙烯系阴离子交换树脂(D301型)；强酸性苯乙烯系阳离子交换树脂(001×7型))脱去料液中的离子。将脱离子后的料液旋转蒸发(70-80℃)浓缩至 糖液固形物含量30-60％，喷雾干燥得到β-1,3-葡寡糖粉末。
2、工艺二
将步骤二制备的β-1,3-葡寡糖水解液经板框过滤(200-400目，压力0.3MPa)或离心(3000-5000rpm)除去不容物。收集可溶寡糖水解液，采用膜过滤系统(纳滤膜：截留量150；操作压力0.4-0.8mP)将料液浓缩至糖液固形物含量20-30％，期间脱去料液中的大部分离子，得到寡糖糖浆。最后将寡糖糖浆喷雾干燥得到β-1,3-葡寡糖粉末。
工业应用
本发明所提供的蛋白质(在实施例中具体为重组蛋白PbBgl64A)具有β-1,3-葡聚糖酶活性，具有207.2U mg-1的比酶活力，最适反应pH为5.5，并且在较宽的pH范围内(4.5-8.5)保持稳定；最适反应温度为70℃，并且在65℃以下保持较高酶活力，具有较好的耐热性；该蛋白质作为β-1,3葡聚糖酶可以高效水解可得然寡糖制备高聚合度β-1,3-葡寡糖，所得寡糖聚合度分布在2-21，水解所得寡糖得率达到80％以上。本发明提供的蛋白质具有良好的β-1,3-葡聚糖酶酶学性质，在低聚糖制备以及食品、饲料等行业中具有良好的应用价值。

Claims (18)

  1. 蛋白质，是如下a)或b)或c)的蛋白质：
    a)氨基酸序列是序列表中序列2第29-449位氨基酸残基编码的蛋白质；
    b)在序列表中序列2第29-449位所示的蛋白质的N端和/或C端连接标签得到的融合蛋白质；
    c)将序列表中序列2第29-449位所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的且具有相同功能的蛋白质。
  2. 与权利要求1所述蛋白质相关的生物材料，为下述B1)-B5)中的任一种：
    B1)编码权利要求1所述蛋白质的核酸分子；
    B2)含有B1)所述核酸分子的表达盒；
    B3)含有B1)所述核酸分子的重组载体或含有B2)所述表达盒的重组载体；
    B4)含有B1)所述核酸分子的重组菌或含有B2)所述表达盒的重组菌或含有B3)所述重组载体的重组菌；
    B5)含有B1)所述核酸分子的细胞系或含有B2)所述表达盒的细胞系或含有B3)所述重组载体的细胞系。
  3. 根据权利要求2所述的相关生物材料，其特征在于：B1)所述核酸分子的为如下1)或2)或3)的DNA分子：
    1)核苷酸序列是序列表中序列1第85-1350位所示的DNA分子；
    2)与1)限定的DNA序列至少具有70％、至少具有75％、至少具有80％、至少具有85％、至少具有90％、至少具有95％、至少具有96％、至少具有97％、至少具有98％或至少具有99％同源性且编码所述蛋白质的DNA分子；
    3)在严格条件下与1)或2)限定的DNA序列杂交且编码所述蛋白质的DNA分子。
  4. 权利要求1所述蛋白质在作为β-1,3-葡聚糖酶中的应用。
  5. 权利要求1所述蛋白质在水解β-1,3-葡聚糖中的应用。
  6. 根据权利要求5所述的应用，其特征在于：所述β-1,3-葡聚糖为可 得然多糖、昆布多糖或酵母葡聚糖。
  7. 权利要求1所述蛋白质在制备β-1,3-葡寡糖和/或高聚合度β-1,3-葡寡糖中的应用。
  8. 根据权利要求7所述的应用，其特征在于：所述聚合度为2-21。
  9. 一种重组菌，是将权利要求1所述蛋白质的编码基因导入宿主菌中得到的菌。
  10. 根据权利要求9所述的重组菌，其特征在于：所述宿主菌为大肠杆菌BL21(DE3)。
  11. 一种β-1,3-葡聚糖酶的制备方法，包括如下步骤：诱导培养权利要求9或10所述的重组菌，得到β-1,3-葡聚糖酶。
  12. 一种生产高聚合度β-1,3-葡寡糖的方法，包括如下步骤：用权利要求1所述的蛋白质水解β-1,3-葡聚糖，得到高聚合度β-1,3-葡寡糖。
  13. 根据权利要求12所述的方法，其特征在于：所述权利要求1所述的蛋白质与所述β-1,3-葡聚糖的配比为1500 U:1 kg。
  14. 根据权利要求13所述的方法，其特征在于：
    所述权利要求1所述的蛋白质的浓度为0.06 U/mL。
  15. 根据权利要求13所述的方法，其特征在于：
    所述β-1,3-葡聚糖的质量分数为4％。
  16. 根据权利要求12所述的方法，其特征在于：所述水解的条件为：pH5.0，50-60℃水解4-6h。
  17. 根据权利要求16所述的方法，其特征在于：所述水解的条件为：pH5.0，55℃水解5h。
  18. 根据权利要求12所述的方法，其特征在于：所述β-1,3-葡聚糖为可得然多糖。
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