WO2021218171A1 - 一种β-甘露聚糖酶的制备方法及在制备部分水解甘露聚糖中的应用 - Google Patents

一种β-甘露聚糖酶的制备方法及在制备部分水解甘露聚糖中的应用 Download PDF

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WO2021218171A1
WO2021218171A1 PCT/CN2020/135515 CN2020135515W WO2021218171A1 WO 2021218171 A1 WO2021218171 A1 WO 2021218171A1 CN 2020135515 W CN2020135515 W CN 2020135515W WO 2021218171 A1 WO2021218171 A1 WO 2021218171A1
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application
preparation
mannanase
gum
partially hydrolyzed
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French (fr)
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江正强
闫巧娟
王楠楠
李延啸
马俊文
温永平
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中国农业大学
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    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/127—Fermented milk preparations; Treatment using microorganisms or enzymes using microorganisms of the genus lactobacteriaceae and other microorganisms or enzymes, e.g. kefir, koumiss
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/123—Bulgaricus
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/21—Streptococcus, lactococcus
    • A23V2400/249—Thermophilus
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

  • the invention relates to a preparation method of ⁇ -mannanase and its application in the preparation of partially hydrolyzed mannans in the field of biotechnology.
  • Mannan is a class of linear polysaccharides connected by mannose with ⁇ -1,4-D-pyranoside bonds. Certain residues of the main chain can also be connected with ⁇ -1,4-glycosidic bonds. Glucose residues are substituted with galactose residues linked by ⁇ -1,6-glycosidic bonds. Mannan is the second major component of hemicellulose, and its content is second only to xylan. It is widely distributed in nature, in the endosperm of legume seeds, some plant gums (such as locust bean gum, couma gum, etc.), Coconut meat and konjac bulbs are rich in mannans (Hsu et al.
  • mannans can be divided into linear mannans, galactomannans, glucomannans and galacto-glucomannans (Petkowicz et al. Carbohydrate Polymers, 2001, 44:107-112).
  • ⁇ -Mannanase is a key enzyme for the hydrolysis of mannans. It can randomly hydrolyze ⁇ -1,4-glycosidic bonds in the main chain of mannans to generate mannoligosaccharides (Malgas et al. World Journal of Microbiology and Biotechnology, 2015,31:1167-1175).
  • ⁇ -mannanase is widely distributed in nature, and ⁇ -mannanase can be isolated and extracted from plants, lower animals and microorganisms, and microorganisms are the main source of ⁇ -mannanase.
  • Microbial ⁇ -mannanase has many advantages, such as simple enzyme production conditions, large enzyme production, stable source, convenient extraction, and diverse enzymatic properties. Therefore, ⁇ -mannanase derived from microorganisms is widely used in food, feed and other industries (Srivastava and Kapoor. Biotechnology Advances, 2017, 35:1-19).
  • ⁇ -mannanase can be divided into glycoside hydrolase 5, 26, 113 and 134 families (Srivastava and Kapoor. Biotechnology advances, 2017, 35:1-19).
  • the vast majority of ⁇ -mannanases belong to the 5 and 26 families of glycoside hydrolases (GH).
  • the GH5 family ⁇ -mannanase has been used to prepare partially hydrolyzed mannans, such as the GH5 family ⁇ -mannanase from Arabidopsis thaliana (Wang et al.Planta, 2014, 239:653-665) and Rhizomucor miehei CAU432 It can produce partially hydrolyzed guar gum (Li et al. International Journal of Biological Macromolecules, 2017, 105: 1171-1179).
  • the substrate is a galactomannan with more galactose substituents in the side chain
  • GH26 family ⁇ -mannanase has more advantages (von Freiesleben et al.
  • GH26 family ⁇ -mannanase has been successfully expressed in yeast, but the expression level is low, such as Thermophilic Bacillus subtilis (TBS2) ⁇ -mannanase (ReTMan26) ) Enzyme activity is 5435U/mL (Luo et al. Applied Biochemistry and Biotechnology, 2017, 182:1259-1275) and Aspergillus niger (Aspergillus niger) ⁇ -mannanase expressed in Pichia pastoris enzyme activity is 5069U/mL (Zhao et al. Bioresource Technology, 2011, 102: 7538-7547). Therefore, increasing the expression level of GH26 family ⁇ -mannanase is of great significance to its industrial application.
  • partially hydrolyzed mannan As a prebiotic, partially hydrolyzed mannan has the characteristics of low calorie, stable properties, promotes the proliferation of beneficial bacteria in the organism, and regulates metabolic circulation. It can be widely used in the food industry (Mudgil et al. International Journal of Biological Macromolecules, 2018) ,112:207-210; Kapoor et al. Journal of Function Foods,2016,24:207-220). At present, methods such as high temperature degradation, acid-base hydrolysis and enzymatic hydrolysis are mainly used to prepare partially hydrolyzed mannans.
  • Enzymatic preparation of partially hydrolyzed mannans has the advantages of easy process control, mild reaction conditions, and few side reactions, and has become the most commonly used method in the preparation of partially hydrolyzed mannans.
  • the substrates for preparing partially hydrolyzed mannans are mainly plant gums rich in mannans. Among them, the raw materials commonly used to prepare partially hydrolyzed mannans are konjac flour and guar gum, but the concentration of raw materials used during hydrolysis is low ( ⁇ 5%) (Mao et al.
  • Cipheral Patent No. ZL201610808817.0 discloses a method for preparing partially hydrolyzed guar gum by enzymatically hydrolyzing guar gum using mannanase derived from Rhizomucor miehei. The concentration of guar gum used is low, only 5%. .
  • cassia gum, couma gum and other plant gums rich in mannan are also good substrates for preparing partially hydrolyzed mannans, but there are few related reports and patents (Daas et al. Carbohydrate Polymers, 2017, 158: 102- 111).
  • Chinese Patent No. ZL201410024844.X discloses a method for preparing a thickener of couma gum. The endosperm of couma seeds is enzymatically treated with a biological enzyme solution, which improves the performance of couma gum powder;
  • Chinese patent application No. CN201710478917.6 Disclosed is a method for preparing a health food for preventing and treating hyperlipidemia.
  • ⁇ -mannanase water to solve plant gums such as gelatin and couma gum and increase the substrate concentration to produce partially hydrolyzed mannans can enrich the prebiotic market in my country and have broad development prospects.
  • Yogurt is a food made from fresh milk and fermented by Streptococcus thermophilus and Lactobacillus bulgaricus. Yogurt is delicate and smooth, thick in texture, sweet and sour, and has the advantages of easy digestion and absorption, promoting intestinal peristalsis, regulating the balance of intestinal flora, anti-oxidation and anti-bacterial, and lowering blood pressure (Farvin et al. Food Chemistry, 2010, 123: 1090-1097; Donkor et al. International Dairy Journal, 2007, 17: 1321-1331). Adding prebiotics to yogurt to prepare prebiotic yogurt can improve the physical and chemical properties of yogurt and increase the functional activity of yogurt, which has attracted wide attention (Cruz et al.
  • Chinese Patent Application No. CN201811327958.6 discloses a method for making brown oligosaccharide yogurt, adding 1-3g/L xylo-oligosaccharides, 15-22g/L fructo-oligosaccharides, 18-26g/L low The polyisomaltose is made into prebiotic yogurt, which is rich in nutrients, is beneficial to human health, and meets the consumption requirements of modern people for yogurt.
  • CN201910276659.2 discloses a method for preparing prebiotic yogurt, adding 0.02%-0.08% by mass of dietary fiber (citrus fiber) to raw milk, and then inoculating bacteria to ferment to obtain prebiotic yogurt. It can meet consumers' dual needs for taste and health, and is easily accepted by consumers. In order to enrich the types of prebiotic yogurt and meet market demand, partially hydrolyzed mannan can be added to yogurt to develop prebiotic yogurt.
  • Aspergillus niger is a heat-resistant filamentous fungus capable of secreting a variety of glycoside hydrolase enzymes.
  • the ⁇ -mannanase secreted by it has a variety of excellent enzymatic properties (Harnpicharnchai et al. Bioscience Biotechnology and Biochemistry, 2016, 80: 2298-2305).
  • the present invention provides a method for preparing ⁇ -mannanase and its application in preparing partially hydrolyzed mannan and yogurt.
  • the present invention first provides a method for preparing a protein with ⁇ -mannanase activity.
  • the method includes: introducing the rMan26 gene shown in SEQ ID No. 2 in the sequence list into biological cells to obtain recombinant cells, and culturing In the recombinant cell, the rMan26 gene is expressed to obtain a protein with ⁇ -mannanase activity.
  • the expression of the rMan26 gene is driven by a promoter named ATX1, and the ATX1 is (b1), (b2) or (b3):
  • (b2) A single-stranded DNA molecule with (b1) substitution and/or deletion and/or addition of one or several nucleotides and 75% or more identical to (b1);
  • nucleotide sequence of the rMan26 gene of the present invention can easily use known methods, such as directed evolution and point mutation methods, to mutate the nucleotide sequence of the rMan26 gene of the present invention.
  • Those artificially modified nucleotides that have 75% or more identity with the nucleotide sequence of the rMan26 gene of the present invention are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.
  • identity refers to sequence similarity to natural nucleic acid sequences. “Identity” includes 75% or higher, 80% or higher, or 85% or higher, or 90% or higher, or 95% or higher with the nucleotide sequence of the rMan26 gene of the present invention, Or a nucleotide sequence of 97% or higher, or 98% or higher, or 99% or higher identity. Identity can be evaluated with the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.
  • the stringent conditions can be as follows: 50°C, hybridization in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO 4 and 1 mM EDTA, at 50°C, 2 ⁇ SSC, 0.1 Rinse in %SDS; it can also be: 50°C, hybridize in a mixed solution of 7% SDS, 0.5M NaPO 4 and 1mM EDTA, and rinse in 50°C, 1 ⁇ SSC, 0.1% SDS; it can also be: 50°C , Hybridize in a mixed solution of 7% SDS, 0.5M NaPO 4 and 1 mM EDTA, and rinse at 50°C, 0.5 ⁇ SSC, 0.1% SDS; it can also be: 50°C, in 7% SDS, 0.5M NaPO 4 and Hybridization in a mixed solution of 1mM EDTA, rinsing at 50°C, 0.1 ⁇ SSC, 0.1% SDS; also: 50°C
  • the above-mentioned 75% or more than 75% identity may be 80%, 85%, 90% or more than 95% identity.
  • introducing the rMan26 gene shown in SEQ ID No. 2 in the sequence list into a biological cell may include introducing a recombinant vector containing the rMan26 gene and the ATX1 into the biological cell.
  • the recombinant vector may be pPAT19K-AnMan26, which is the DNA fragment between the BglII and BamHI recognition sequences of pPIC9K is replaced with a DNA molecule shown in positions 6-930 of SEQ ID No. 4 in the sequence table, And replace the DNA fragment between EcoRI and NotI recognition sequence with the recombinant vector obtained by rMan26 gene.
  • the protein can be the following A1), A2) or A3):
  • amino acid sequence is the protein of SEQ ID No. 3;
  • A2 A protein in which the amino acid sequence shown in SEQ ID No. 3 in the sequence list has been substituted and/or deleted and/or added by one or several amino acid residues and has ⁇ -mannanase function;
  • A3 A fusion protein obtained by attaching a tag to the N-terminus or/and C-terminus of A1) or A2).
  • amino terminal or carboxyl terminal of the protein consisting of the amino acid sequence shown in SEQ ID No. 3 in the sequence listing can be attached with the tag shown in the following table.
  • the protein in A2) above is a protein that is 75% or more identical to the amino acid sequence of the protein shown in SEQ ID No. 3 and has the same function.
  • the identity of 75% or more is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity .
  • the protein in A2) can be artificially synthesized, or its coding gene can be synthesized first, and then obtained by biological expression.
  • the protein coding gene in A2) above can be achieved by deleting one or several amino acid residue codons in the DNA sequence shown in SEQ ID No. 2, and/or performing one or several base pair missense mutations , And/or the coding sequence of the tag shown in the table above is attached to its 5'end and/or 3'end.
  • the DNA molecule shown in SEQ ID No. 2 encodes the protein shown in SEQ ID No. 3.
  • the protein may be the protein shown in SEQ ID No. 6 in the sequence listing.
  • the biological cell may be a microbial cell.
  • the microbial cell is a fungus.
  • the fungus may be yeast.
  • the yeast may be Pichia pastoris (such as Pichia pastoris GS115).
  • the recombinant cell is GS115-pPAT19K-AnMan26
  • the GS115-pPAT19K-AnMan26 is a recombinant bacteria obtained by introducing the pPAT19K-AnMan26 into Pichia pastoris GS115.
  • the invention also provides a protein with ⁇ -mannanase activity prepared by the method for preparing a protein with ⁇ -mannanase activity.
  • the invention also provides any of the following products:
  • a set of reagents consisting of the rMan26 gene and the ATX1;
  • X6 A recombinant vector containing the rMan26 gene and/or the ATX1.
  • the set of reagents described in X4 can be used to prepare ⁇ -mannanase.
  • the expression cassette of X5 refers to DNA capable of expressing the protein encoded by the rMan26 gene in host cells.
  • the DNA may not only include a promoter for initiating the transcription of the rMan26 gene, but also a terminator for terminating the transcription of the rMan26 gene. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present invention include, but are not limited to: constitutive promoters, tissue, organ, and development-specific promoters, and inducible promoters. Specifically, the promoter in the expression cassette of X5 may be the ATX1.
  • the recombinant vector of X6 may be the pPAT19K-AnMan26.
  • the present invention also provides any of the following applications of the protein:
  • the vegetable gum can be b1) or b2) or b3) or b4) or b5): b1) locust bean gum; b2) cassia gum; b3) couma gum; b4) konjac flour; b5) rich Vegetable gum containing mannan.
  • the mannose oligosaccharides may be mannose oligosaccharides formed by the polymerization of 2-10 mannoses.
  • the "production of mannose oligosaccharides” and “production of partially hydrolyzed mannans” can be mannans or vegetable gums as substrates.
  • the vegetable gum is coumadin, and the ratio of the coumadin to the protein with ⁇ -mannanase activity is: 100-200 U of protein with ⁇ -mannanase activity is added per gram of coumadin
  • the hydrolysis temperature is 30°C-45°C; the solvent of the coumadin is citric acid buffer or water.
  • the vegetable gum may be cassia gum, and the ratio of the cassia gum to the protein with ⁇ -mannase activity is: 100-800 U of the protein with ⁇ -mannanase activity per gram of cassia gum
  • the hydrolysis temperature is 30°C-45°C; the solvent of the cassia gum is citric acid buffer or water.
  • the invention also provides any of the following applications of the product:
  • the vegetable gum can be b1) or b2) or b3) or b4) or b5): b1) locust bean gum; b2) cassia gum; b3) couma gum; b4) konjac flour; b5) rich in mannose Glycan of vegetable gum.
  • the partially hydrolyzed mannan prepared by using the protein or the product also belongs to the protection scope of the present invention.
  • the vegetable gum for producing the partially hydrolyzed mannan can be c1) or c2) or c3) or c4) or c5): c1) locust bean gum; c2) cassia gum; c3) couma gum; c4 ) Konjac flour; c5) Vegetable gum rich in mannan.
  • the preparation of the prebiotic yogurt also includes adding Streptococcus thermophilus and Lactobacillus bulgaricus to the system.
  • the present invention also provides a method for making prebiotic yogurt, the method comprising: using the protein or the product to make prebiotic yogurt.
  • the system in the above method contains vegetable gum.
  • the vegetable gum can be c1) or c2) or c3) or c4) or c5): c1) locust bean gum; c2) cassia gum; c3) couma gum; c4) konjac flour; c5) rich in mannan Of vegetable gum.
  • the system of the above method also contains Streptococcus thermophilus and Lactobacillus bulgaricus.
  • Figure 1 shows the enzyme production process diagram (A) and SDS-PAGE electrophoresis diagram (B) of recombinant ⁇ -mannanase in high-density fermentation in a 5L fermentor. Among them ( ⁇ ) enzyme activity, ( ⁇ ) protein concentration, ( ⁇ ) wet weight of bacteria. Lane M in B is the protein molecular weight standard, lanes 1-8 are the fermentation supernatants induced 0, 24, 48, 72, 96, 120, 144, 168h, respectively.
  • Figure 2 is the purification diagram of recombinant ⁇ -mannanase, where lane 1 is the crude enzyme solution, lane 2 is the pure enzyme purified by Sephacryl S-100HR gel column chromatography, and lane 3 is N-deglycosylation The result of enzyme deglycosylation.
  • Figure 3 is a graph showing the optimal pH determination curve of recombinant ⁇ -mannanase. Among them ( ⁇ ) citrate buffer (pH 3.0-6.0), ( ⁇ ) phosphate buffer (pH 6.0-8.0).
  • Figure 4 is a graph showing the determination of pH stability of recombinant ⁇ -mannanase. Among them ( ⁇ ) glycine-hydrochloric acid buffer (pH 2.0-3.0), ( ⁇ ) citrate buffer (pH 3.0-6.0), ( ⁇ ) phosphate buffer (pH 6.0-8.0).
  • Figure 5 is a graph showing the optimal temperature determination curve of recombinant ⁇ -mannanase.
  • Figure 6 is a graph showing the temperature stability determination of recombinant ⁇ -mannanase.
  • Fig. 7 is a thin layer chromatography chromatographic analysis diagram of AnMan26 as a product of ⁇ -mannanase hydrolyzed couma gum.
  • M, M2, M3, M4, M5, and M6 are mannose, mannobiose, mannotriose, mannotetraose, mannopentaose, and mannohexaose, respectively, and 1 is a partially hydrolyzed coumarin crude sugar liquid.
  • Fig. 8 is a gel permeation chromatographic analysis diagram of AnMan26 as a product of ⁇ -mannanase hydrolyzed couma gum. 0min represents unhydrolyzed couma gum.
  • Figure 9 is a thin layer chromatography chromatographic analysis diagram of AnMan26 as a product of ⁇ -mannanase water to resolve gelatin.
  • M, M2, M3, M4, M5, M6 are respectively mannose, mannobiose, mannotriose, mannotetraose, mannopentaose, mannohexaose, 1 is a partially water-resolved gelatin (cassia oligosaccharide) crude sugar solution.
  • Figure 10 is a gel permeation chromatographic analysis diagram of AnMan26 as a product of ⁇ -mannanase water to resolve gelatin. 0min represents unhydrolyzed cassia gum.
  • Figure 11 is a diagram showing the influence of the amount of partially hydrolyzed couma gum on the water holding capacity of prebiotic yogurt.
  • Streptococcus thermophilus in the following examples is a product of Chr-Hansen, Denmark.
  • Lactobacillus bulgaricus is a product of Chr-Hansen, Denmark.
  • the expression vector pPIC9K in the following examples is a product of Invitrogen from the United States, and the product number is V17520.
  • Pichia pastoris GS115 in the following examples is a product of Invitrogen from the United States, and the product number is C18100.
  • the ⁇ -mannanase activity is determined by the 3,5-dinitrosalicylic acid (DNS) method. Unless otherwise specified, the determination steps are as follows: (1) Take 0.9mL 0.5g/100mL locust bean gum solution (using locust Soybean gum and pH 5.0, 50mmol/L citric acid buffer solution), add 0.1mL of appropriately diluted enzyme solution to be tested, and place it in a constant temperature water bath at 45°C for 10 minutes; (2) After completing step (1), use DNS reagent terminates the reaction and reacts with the released reducing sugar, and the amount of reducing sugar (using mannose as the standard) is measured. Draw a standard curve with mannose standard solution.
  • the activity unit of ⁇ -mannanase is defined as: under the above reaction conditions, the amount of enzyme required to generate 1 ⁇ mol mannose per minute is one enzyme activity unit (1U).
  • Specific enzyme activity is defined as the unit of enzyme activity (U/mg) possessed by 1 mg of protein.
  • Locust bean gum Galactomannan, a product of American Sigma-Aldrich company, the product number is G0753.
  • Mannose It is a product of American Sigma-Aldrich Company, the article number is M2069.
  • ⁇ -mannanase gene XM_001397260.1
  • MAN26 gene The coding region of ⁇ -mannanase gene (MAN26 gene) XM_001397260.1 is 954bp, and its sequence is the ⁇ -mannanase shown in SEQ ID No. 1 in the sequence table and SEQ ID No. 3 in the coding sequence table MAN26.
  • the MAN26 gene is codon optimized, and the optimized gene is obtained, which is recorded as the rMan26 gene, and its sequence is SEQ ID No. 2 in the sequence table.
  • the rMan26 gene was artificially synthesized and used as a template to perform PCR amplification with specific primers rMan26AF and rMan26AR to obtain a PCR product.
  • the primer sequence is as follows:
  • rMan26AF 5'-CCG GAATTC GCTTCTAACCAAACTTTGTCTTACG-3' (underlined is the restriction enzyme EcoRI restriction site) (SEQ ID No. 7);
  • rMan26AR 5'-GAAT GCGGCCGC TTAAGCACCTTCCCAATTCAAAG-3' (underlined is the restriction endonuclease NotI restriction site) (SEQ ID No. 8).
  • pPAT19K-AnMan26 is to replace the DNA fragment between the recognition sequence of BglII and BamHI of pPIC9K with the DNA molecule shown at positions 6-930 of SEQ ID No. 4 in the sequence list, and replace the DNA fragment between the recognition sequence of EcoRI and NotI It is a recombinant vector derived from rMan26 gene.
  • pPAT19K-AnMan26 contains the DNA molecule shown in SEQ ID No. 5 in the sequence table (that is, the fusion gene formed by the rMan26 gene and a partial sequence on the vector, recorded as AnMan26 gene), and can express the DNA molecule shown in SEQ ID No. 6 in the sequence table
  • the fusion protein of is denoted as recombinant ⁇ -mannanase (AnMan26), and the expression of AnMan26 gene is driven by ATX1.
  • the 280-1233 of SEQ ID No. 5 in the sequence list is the sequence of the rMan26 gene, and the 1-279 is the sequence on the vector; the 94-410 of SEQ ID No. 6 is the amino acid sequence of MAN26, and the Position 1-93 is the polypeptide encoded by the sequence on the vector.
  • step 4 Collect the recombinant bacterial cells from step 3 with sterile water, and spread the appropriately diluted bacterial cells on YPD plates containing G418 at the concentrations of 1 mg/mL, 2 mg/mL, 3 mg/mL, and 4 mg/mL ( 1g/100mL yeast extract, 2g/100mL tryptone, 2g/100mL glucose), select single colonies that grow well at different G418 concentrations, and inoculate them in 5mL BMGY medium (the medium is composed of solvent and solute.
  • the solute and its concentration in the culture medium are 1g/100mL yeast extract, 2g/100mL peptone, 1.34g/100mL YNB (American Biomol product), 4 ⁇ 10 -5 g /100mL biotin and 1g/100mL glycerol), placed in 30°C, 200rpm shaker, shake culture, when the OD 600 is 2-6, centrifuge, discard the supernatant, collect the bacteria and transfer to 10mL BMMY culture Base (this medium is a medium obtained by replacing glycerol in BMGY medium with methanol, and the concentration of methanol in this medium is 0.5% (volume percentage)), placed in a shaker at 30°C and 200 rpm to induce expression .
  • this medium is a medium obtained by replacing glycerol in BMGY medium with methanol, and the concentration of methanol in this medium is 0.5% (volume percentage)
  • the fermentation tank is a 5L fermentation tank.
  • Seed medium BMGY, minimal medium BSM, glycerol fed-batch medium and 100% methanol induction medium are all configured according to the method in the literature.
  • the whole fermentation process is divided into four stages: seed liquid culture, basic culture, glycerol fed-batch culture and 100% methanol induction culture. Specific steps are as follows:
  • Seed culture inoculate GS115-pPAT19K-AnMan26 into a 500 mL Erlenmeyer flask containing 150 mL of BMGY medium, and cultivate it in a shaker at 30° C. and 200 rpm for more than 24 hours to obtain a seed solution with an OD 600 of 2-6.
  • Basic culture Inoculate the seed solution in 1) into a 5L sterilized fermentor (containing 1.5L of basic fermentation medium BSM), adjust the pH of the medium to 4.0 with ammonia, and add PTM1 4.35mL/L to start Fermentation broth, seed broth inoculum amount is 10%, the temperature during the culture process is 30°C, the rotation speed is 600rpm, when the dissolved oxygen DO value rises rapidly, and the glycerol is exhausted, it enters the glycerol fed culture stage.
  • BSM basic fermentation medium
  • Glycerin fed-batch culture the glycerin aqueous solution (glycerol concentration is 500g/L) flow acceleration is 30mL/h/L starting fermentation broth. Dissolved oxygen is always monitored at this stage, and the dissolved oxygen is maintained at DO>20% by adjusting the acceleration of the glycerin flow.
  • the temperature is controlled at 30°C and the pH of the fermentation broth is 4.0. When the wet weight of the bacteria reaches 180-220g/L, the feeding is stopped. The fermentation time of the whole process is 4-6h.
  • 100% methanol feeding stage After stopping feeding glycerol, starve for about 30 minutes to exhaust the glycerin in the tank, adjust the pH of the fermentation broth to 6.0, rotate the speed to 800rpm, start feeding 100% methanol to induce enzyme production, and methanol is fed Increase from 3.6mL/h/L initial fermentation broth to 10.9mL/h/L initial fermentation broth, monitor the dissolved oxygen DO in the tank> 20% (if the dissolved oxygen DO is less than 20%, the flow acceleration can be appropriately reduced), Control the temperature at 30°C and ferment for 6-7 days.
  • wet weight of the bacteria m 2 -m 1 .
  • Protein content Using bovine serum albumin as the standard protein, the Lowry method (Lowry et al. The Journal of Biological Chemistry, 1951, 193(1): 265-275) is used to determine the protein content (mg/mL). The protein content is the total protein content.
  • the wet weight, protein content and ⁇ -mannanase enzyme activity changes are shown in Figure 1 A, and the SDS-PAGE electrophoresis of the protein during the fermentation process is shown in Figure 1 B.
  • the enzyme activity reached the highest in the high-density fermentation for 168h.
  • the ⁇ -mannanase enzyme activity in the supernatant of the fermentation broth was 22100U/mL, the protein content was 12.0mg/mL, and the wet weight of the bacteria was 355.8g/L.
  • the fermentation supernatant ie crude enzyme solution
  • 20mmol/L citrate buffer pH 5.0
  • the enzyme solution was concentrated to 0.6mL with a 10kDa ultrafiltration membrane, the concentrated sample was centrifuged at 11510 ⁇ g for 5min, and the supernatant was collected.
  • N-deglycosylation enzyme deglycosylation The steps of N-deglycosylation enzyme deglycosylation are as follows:
  • glycoprotein denaturation buffer (10 ⁇ ) (product of NEB, USA, product number P0702S).
  • GlycoBuffer3 (10 ⁇ ) (product of NEB, USA, item number P0702S).
  • the recovery rate refers to the percentage of the total enzyme activity of the pure enzyme in the total enzyme activity of the crude enzyme solution.
  • the purification factor refers to the ratio of the specific enzyme activity of the pure enzyme to the specific enzyme activity of the crude enzyme.
  • Example 2 Enzymatic properties of recombinant protein AnMan26 as ⁇ -mannanase
  • the buffers used are as follows: glycine-hydrochloric acid buffer (pH 2.0, 2.5 and 3.0), citrate buffer (pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 and 6.0), phosphate buffer (pH 6.0, 6.5) , 7.0, 7.5 and 8.0).
  • concentrations of the above buffers are all 50mmol/L.
  • the purified recombinant ⁇ -mannanase solution prepared in Example 1 was used as the sample solution to be tested, and the enzyme activity was determined as follows: Take 0.9mL 0.5g/100mL locust bean gum solution (using locust bean gum and pH 3.0 -8.0 range of different buffer systems to be tested), add 0.1mL of appropriately diluted enzyme solution to be tested, and place the resulting reaction system at 30°C for 10 minutes to determine the enzyme activity. Record the highest enzyme activity as 100%. The relative activity of ⁇ -mannanase under various pH conditions was calculated. As shown in Figure 3, the optimal pH value of recombinant ⁇ -mannanase was 5.0.
  • the recombinant ⁇ -mannanase prepared in Example 1 has a citric acid buffer solution at pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, and 6.0, and a phosphate buffer solution at pH 6.0, 6.5, 7.0, 7.5, and 8.0, respectively
  • the relative enzyme activities in the liquid were 1.4% ⁇ 0.4%, 20.5% ⁇ 0.5%, 44.7% ⁇ 0.5%, 77.9% ⁇ 1.3%, 100% ⁇ 0.4%, 87.6% ⁇ 0.7%, 54.7% ⁇ 0.3%, 36.3% ⁇ 0.3%, 17.9% ⁇ 0.9%, 3.7% ⁇ 0.1%, 0.7% ⁇ 0.03% and 0.2% ⁇ 0.02%.
  • the purified recombinant ⁇ -mannanase solution prepared in Example 1 was diluted with the above-mentioned buffer by an appropriate multiple, so that the recombinant ⁇ -mannanase was placed in the above-mentioned buffer at each pH, and then the diluted The enzyme solution was placed in a water bath at 30°C for 30 minutes, then quickly taken out and immediately ice-bathed for 30 minutes to determine the residual ⁇ -mannanase activity.
  • the control is the dilution of the recombinant ⁇ -mannanase solution (ie the recombinant ⁇ -mannanase obtained in Example 1) without the above treatment (the above treatment refers to the first 30°C water bath for 30 minutes, and then the rapid ice bath for 30 minutes). Dilution of pure enzyme solution). Taking the enzyme activity of the control as 100%, the relative activity of the enzyme after treatment with different pH buffers was calculated. The result is shown in Figure 4. The enzyme remained stable in the pH range of 2.5-6.0, and the residual enzyme activity remained stable after 30 minutes of treatment. Keep it above 80%.
  • Example 1 The recombinant ⁇ -mannanase prepared in Example 1 was subjected to a glycine-hydrochloric acid buffer with pH of 2.0, 2.5, and 3.0, a citric acid buffer with a pH of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, and 6.0, respectively.
  • the relative enzyme activities after treatment with phosphate buffer with pH 6.0, 6.5, 7.0, 7.5 and 8.0 were 9.4% ⁇ 0.2%, 78.9% ⁇ 2.3%, 82.3% ⁇ 5.1%, 96.2% ⁇ 3.7%, 91.0%, respectively ⁇ 2.4%, 92.7% ⁇ 3.7%, 92.2% ⁇ 5.1%, 93.5% ⁇ 2.8%, 92.5 ⁇ 1.6%, 95.7% ⁇ 4.3%, 69.4% ⁇ 3.0%, 66.6% ⁇ 2.1%, 65.4% ⁇ 2.3% , 59.0% ⁇ 2.1% and 26.0% ⁇ 1.0%.
  • the recombinant ⁇ -mannanase solution prepared in Example 1 was used as the enzyme solution to be tested, and the pH 5.0, 50mmol/L citric acid buffer system was used to react at different temperatures in the range of 30°C to 80°C to determine the difference For the enzyme activity at temperature, take the highest enzyme activity as 100%, and calculate the relative activity at each reaction temperature. The results are shown in Figure 5, the optimal temperature of recombinant ⁇ -mannanase is 45°C.
  • the recombinant ⁇ -mannanase prepared in Example 1 operates at 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C and 80°C
  • the relative enzyme activities at °C were 42.0% ⁇ 3.6%, 42.7% ⁇ 3.7%, 66.6% ⁇ 5.0%, 72.5% ⁇ 5.4%, 80.6% ⁇ 2.7%, 100% ⁇ 2.0%, 79.2% ⁇ 4.6%, 40.2% ⁇ 2.1%, 18.6% ⁇ 0.6%, 9.8% ⁇ 1.7%, 7.2% ⁇ 0.5%, 6.1% ⁇ 0.2%, and 4.6% ⁇ 0.3%.
  • the recombinant ⁇ -mannanase solution prepared in Example 1 was diluted with pH 5.0, 50mmol/L citric acid buffer to an appropriate multiple, and placed in a water bath at different temperatures for 30 minutes (water bath temperature was 20°C, 25°C, 30 °C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C or 80°C), quickly take out and ice bath for 30min, and determine the residual ⁇ -mannanase activity .
  • the control is a recombinant ⁇ -mannanase solution dilution that has not been processed by the above steps (the above treatment refers to a water bath for 30 minutes and a rapid ice bath for 30 minutes). Take the control enzyme activity as 100%.
  • the relative activity results of the test enzyme solution after the above-mentioned various temperature treatments are shown in Fig. 6.
  • the recombinant ⁇ -mannanase retains more than 80% of the activity below 40°C.
  • the recombinant ⁇ -mannanase prepared in Example 1 was subjected to 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C and 80°C
  • the relative enzyme activities after °C treatment were 100% ⁇ 3.7%, 100% ⁇ 3.8%, 100% ⁇ 4.0%, 100% ⁇ 3.9%, 93.8% ⁇ 5.5%, 73.8% ⁇ 4.0%, 19.4% ⁇ 0.3% , 12.6% ⁇ 1.9%, 3.6% ⁇ 0.3%, 1.7% ⁇ 0.1%, 1.4% ⁇ 0.1%, 1.2% ⁇ 0.1% and 1.1% ⁇ 0.1%.
  • the recombinant ⁇ -mannanase solution prepared in Example 1 was used as the test enzyme solution to determine the ⁇ -mannanase activity of different substrates.
  • the substrates were locust bean gum, konjac flour, cassia gum, coumadin and The concentration of carboxymethyl cellulose and the substrate in the reaction system are both 0.5g/100mL.
  • konjac flour is a product of Hubei Qiangsen Konjac Technology Co., Ltd., the item number is KJ-30; cassia gum is a product of Beijing Guarrun Technology Co., Ltd.; coumadin is a product of Beijing Guarrun Technology Co., Ltd.; carboxymethyl
  • the cellulose is a product of Sigma-Aldrich, USA, and the product number is C4888-500G.
  • Recombinant ⁇ -mannanase has the highest specific enzyme activity for locust bean gum, reaching 2869.0 U/mg; followed by konjac flour, with a specific enzyme activity of 1905.0 U/mg; the specific enzyme activity for couma gum is the lowest, at 341.1 U /mg, no hydrolysis ability to carboxymethyl cellulose.
  • Example 3 Using recombinant ⁇ -mannanase (AnMan26) to hydrolyze couma gum to produce partially hydrolyzed couma gum
  • the reaction system was inactivated in a boiling water bath for 20 minutes, centrifuged at 11510 ⁇ g for 10 minutes, and the precipitate and the supernatant were collected.
  • the precipitate was the hydrolysate residue, and the supernatant was the partially hydrolyzed coumarin crude sugar solution.
  • the 3,5-dinitrosalicylic acid method was used to determine the reducing sugar content in the crude sugar solution, and the reducing sugar yield, hydrolysis rate and product yield were calculated.
  • the composition of the crude sugar liquid was qualitatively analyzed by thin-layer chromatography.
  • the weight-average molecular weight of partially hydrolyzed couma gum was analyzed by gel permeation chromatography.
  • Reducing sugar yield (%) total reducing sugar mass in the system after hydrolysis / dry weight of raw materials added before hydrolysis ⁇ 100%;
  • Hydrolysis rate (%) (dry weight of raw materials added before hydrolysis-dry weight of hydrolysate residue)/dry weight of raw materials added before hydrolysis ⁇ 100%. Remeasured
  • Product yield (%) product dry weight/dry weight of raw materials added before hydrolysis ⁇ 100%, product dry weight is measured by placing the partially hydrolyzed couma gum crude sugar liquid in an oven at 105°C to a constant weight.
  • the standard control is a mixture of standard products of mannose, mannobiose, mannotriose, mannotetraose, mannopentaose and mannohexaose. Place an appropriate amount of the sample to be tested on the thin-layer chromatography chromatographic analysis plate, put the layering agent in the layer twice, completely soak and blow dry with the developer, and bake at 130°C for color development.
  • mannose is produced by Sigma-Aldrich in the United States, the product number is M2069; mannobiose, mannotriose, mannotetraose, mannopentaose and mannohexaose are all Irish Megazyme products, and the product numbers are O-MBI and O-MTR respectively. , O-MTE, O-MPE, O-MHE.
  • Gel permeation chromatography detection conditions TSKgel GMPW XL aqueous gel chromatography column (7.8 ⁇ 300mm); column temperature: 60°C; RID detector; injection volume: 20 ⁇ L; mobile phase: 100 mM NaNO 3 ; flow rate: 0.6 mL/ min; Use polyoxyethylene standard product (molecular weight 300-500000Da, product of Japan Tosoh Corporatio) to make the standard curve.
  • ⁇ -mannanase can hydrolyze couma gum to produce partially hydrolyzed couma gum with a yield of 73.5%.
  • the composition is mannose oligosaccharides with a degree of polymerization of 2-6, and a large amount of polymerization degree is greater than 6. Mannan.
  • the average degree of polymerization of partially hydrolyzed couma gum is less than or equal to 10, and the weight average molecular weight is 1.8 ⁇ 10 3 Da.
  • Example 4 Using recombinant ⁇ -mannanase (AnMan26) water to solve gelatin production and partial water solution to gelatin (cassia oligosaccharide)
  • the reaction system is heated in a boiling water bath to inactivate the enzyme for 20 minutes to obtain an enzymatic hydrolysate. After centrifuging the enzymatic hydrolysate at 11510 ⁇ g for 10 min, the supernatant is collected as the crude sugar solution.
  • the 3,5-dinitrosalicylic acid method was used to determine the reducing sugar content in the crude sugar solution, and the reducing sugar yield, hydrolysis rate and product yield were calculated.
  • the composition of the crude sugar liquid was qualitatively analyzed by thin-layer chromatography. Gel permeation chromatography was used to analyze part of the water to solve the weight average molecular weight of gelatin (cassia oligosaccharide).
  • the calculation method of reducing sugar yield, hydrolysis rate and product yield, TLC chromatographic conditions and gel permeation chromatographic analysis conditions are the same as in Example 3.
  • the results of gel permeation chromatography analysis are shown in Figure 10.
  • the weight-average molecular weight of cassia gum is 1.7 ⁇ 10 6 Da.
  • part of the water resolves the rapid decline of the weight-average molecular weight of gelatin gum.
  • the weight-average molecular weight is 1188 Da.
  • ⁇ -mannanase can produce partial water-resolved gelatin (cassia oligosaccharides) with a yield of 74.7%.
  • the main components are mannose oligosaccharides with a degree of polymerization of between 1-6. Mannan with a degree of polymerization greater than 6.
  • the average degree of polymerization of partial water solution gelatin gum is less than 7, and the weight average molecular weight is 1188 Da.
  • Example 5 Using partially hydrolyzed mannan to produce prebiotic yogurt
  • the partially hydrolyzed mannan used in this example is the partially hydrolyzed couma gum obtained in Example 3.
  • Partially hydrolyzed couma gum powder is spray-dried to obtain partially hydrolyzed couma gum powder (spray drying conditions: air inlet temperature 180°C, air outlet temperature 80°C).
  • Partially hydrolyzed mannan to produce prebiotic yogurt is made from the following raw materials (1000 parts) by weight: 925-910 parts of fresh milk, 5-20 parts of partially hydrolyzed couma gum powder, and 70 parts of sucrose.
  • the first step mix fresh milk, partially hydrolyzed couma gum and sucrose according to the above weight parts, and then preheat at 65°C until the material is completely dissolved, homogenize at 40MPa, and pasteurize at 95°C for 5 minutes to obtain a mixture.
  • the second step After cooling the mixture to 40°C, add Streptococcus thermophilus and Lactobacillus bulgaricus for inoculation.
  • the contents of the two added bacteria in the system are both 1 ⁇ 10 6 cfu/ml.
  • the third step Put the inoculated mixture in a constant temperature fermentation room and ferment at 42°C for 4 hours (to pH 4.6).
  • the fourth step demulsify the prebiotic yogurt obtained by fermentation at 350 rpm for 1 min, chill it to 20°C, put it in the refrigerator and cook for 16 hours to obtain the yogurt.
  • Water holding capacity take the yogurt that has been ripened for 16 hours for determination, weigh the mass of the sterilized centrifuge tube and record it as m 1 , add 20 g of yogurt to each centrifuge tube and weigh the total mass, record it as m 2 , use a centrifuge Centrifuge at 3000 rpm for 10 minutes, discard the supernatant, and weigh the centrifuge tube and the lower layer as m 3 .
  • water holding capacity (%) (m 3 -m 1 )/(m 2 -m 1 ) ⁇ 100%.
  • Counting of Streptococcus thermophilus Choose 2-3 consecutive appropriate dilutions, and draw 1 mL of sample homogenate into a sterilized dish for each dilution. After transferring the diluent into the petri dish, pour the MC medium cooled to 48°C into the petri dish about 15 mL, and rotate the petri dish to make the mixture uniform. 36°C ⁇ 1°C aerobic culture for 72h ⁇ 2h, count after culture.
  • Lactobacillus count select 2-3 consecutive appropriate dilutions, and draw 1 mL of sample homogenate into a sterilized petri dish for each dilution. After transferring the diluent into the petri dish, pour the MRS agar medium cooled to 48°C into the petri dish for about 15 mL, and rotate the petri dish to make the mixture uniform. Anaerobic culture at 36°C ⁇ 1°C for 72h ⁇ 2h, count after culture.
  • Table 3 is the sensory evaluation data of yogurt with partially hydrolyzed couma The score is 6.6.
  • the main difference is that the flavor and viscosity score of yogurt with partially hydrolyzed couma gum is lower than that of yogurt without partially hydrolyzed couma gum.
  • the total score of yogurt with 1% partially hydrolyzed couma gum is 6.3, which is close to the total score of yogurt without partially hydrolyzed couma gum, 6.6, and can be used in the production of prebiotic yogurt.
  • the mixture in Table 1 is made of the following raw materials (1000 parts) by weight: 925-910 parts of fresh milk, 5-20 parts of partially hydrolyzed couma gum powder, and 70 parts of sucrose.
  • Figure 11 shows the water retention results of partially hydrolyzed couma gum yogurt. It can be seen from Figure 11 that the water retention capacity of yogurt after adding partially hydrolyzed couma gum is between 36.8% and 38.6%, which is higher than that of yogurt without partially hydrolyzed couma gum. The water holding capacity is 34.7%.
  • the amount of partially hydrolyzed couma gum is 0-2.0%
  • the water holding capacity of yogurt increases with the increase of the amount of partially hydrolyzed couma gum, indicating that the addition of partially hydrolyzed couma gum helps to increase the water holding capacity of yogurt
  • the addition of partially hydrolyzed couma gum can effectively enhance the gel structure of yogurt, make the yogurt more effectively retain water, prevent the precipitation of whey, and improve the tissue state of the yogurt.
  • Table 4 shows the effect of partially hydrolyzed coumadin yogurt on the viable counts of Lactobacillus and Streptococcus thermophilus. It can be seen from Table 4 that the number of viable lactobacilli and the viable number of Streptococcus thermophilus in the yogurt without adding partially hydrolyzed couma gum are 1.1 ⁇ 10 8 CFU/mL and 2.75 ⁇ 10 8 CFU/mL, respectively , And the number of viable lactobacilli in the yogurt after adding partially hydrolyzed couma gum is between 4.05 ⁇ 10 8 CFU/mL-1.075 ⁇ 10 9 CFU/mL, and the number of viable streptococcus thermophilus is 2.82 ⁇ 10 8 CFU /mL-6.9 ⁇ 10 8 CFU/mL, which is higher than the number of viable bacteria of Lactobacillus and the number of Streptococcus thermophilus in yogurt without partially hydrolyzed couma gum.
  • the number of viable lactobacilli in yogurt increased first and then decreased with the increase of the amount of partially hydrolyzed coumarin.
  • the number of viable streptococcus thermophilus in yogurt With the increase of the amount of partially hydrolyzed couma gum, the number of Lactobacilli in the yogurt is the highest 1.075 ⁇ 10 9 CFU/mL when 1.5% partially hydrolyzed couma gum is added, and in the yogurt when 2.0% partially hydrolyzed couma gum is added
  • the highest number of viable bacteria of Streptococcus thermophilus is 6.9 ⁇ 10 8 CFU/mL.
  • the number of viable bacteria of Lactobacillus and Streptococcus thermophilus in the partially hydrolyzed couma gum yogurt were higher than those of Lactobacillus and Streptococcus thermophilus without the addition of partially hydrolyzed couma gum yogurt. It shows that the addition of partially hydrolyzed couma gum can effectively increase the number of viable bacteria of Lactobacillus and Streptococcus thermophilus in yogurt, and improve the quality of yogurt.
  • the present invention optimizes the codon of Aspergillus niger ⁇ -mannanase, and transforms the pAOX1 promoter to obtain the pATX1 promoter and construct the expression vector pATX19K to transform into Pichia pastoris GS115 for high-density expression, thereby obtaining high-enzyme activity ⁇ -mannanase, its enzyme activity can reach 22100U/mL.
  • the optimal pH of the ⁇ -mannanase obtained in the present invention is 5.0, and the optimal temperature of the enzyme is 45°C. It can solve the mannan-rich vegetable gums such as gelatin and couma gum, and obtain different polymerization degrees.
  • the partially hydrolyzed mannan of mannose oligosaccharides is significantly different from the existing ⁇ -mannanase water solution gelatin and coumadin, which can enrich the partially hydrolyzed mannan market.
  • Partially hydrolyzed mannan is added to yogurt to prepare prebiotic yogurt, which can increase the water holding capacity of yogurt, the number of viable bacteria of Lactobacillus and Streptococcus thermophilus, and improve the quality of yogurt.
  • the ⁇ -mannanase of the present invention and the preparation method thereof have great application value in the food industry.

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Abstract

一种β-甘露聚糖酶的制备方法及在制备部分水解甘露聚糖中的应用。β-甘露聚糖酶的制备方法包括：将SEQ ID NO.2所示的rMan26基因导入生物细胞中，培养所得重组细胞使rMan26基因得到表达，得到具有β-甘露聚糖酶活性的蛋白质；rMan26基因的表达由SEQ ID NO.4的第6-930位所示ATX1启动子驱动。将得到的重组细胞进行高密度表达，得到了高酶活的β-甘露聚糖酶，其酶活可达22100U/ml，可以水解富含甘露聚糖的植物胶，进而得到包含不同聚合度的并可用于制备酸奶的甘露聚糖的部分水解甘露聚糖。β-甘露聚糖酶及其制备方法在食品行业中有很大的应用价值。

Description

一种β-甘露聚糖酶的制备方法及在制备部分水解甘露聚糖中的应用 技术领域
本发明涉及生物技术领域中，一种β-甘露聚糖酶的制备方法及在制备部分水解甘露聚糖中的应用。
背景技术
甘露聚糖是一类由甘露糖以β-1,4-D-吡喃糖苷键连接而成的线性多糖，主链的某些残基还可以被以β-1,4-糖苷键连接的葡萄糖残基和以α-1,6-糖苷键连接的半乳糖残基取代。甘露聚糖是半纤维素的第二大组成成分，含量仅次于木聚糖，广泛分布在于自然界中，豆科植物种子的胚乳、一些植物胶(如槐豆胶、香豆胶等)、椰肉、魔芋球茎等都含有丰富的甘露聚糖(Hsu et al.Applied Microbiology and Biotechnology,2018,102:1737-1747)。根据甘露聚糖的结构和理化特性的差异，甘露聚糖可分为线性甘露聚糖、半乳甘露聚糖、葡甘露聚糖和半乳葡甘露聚糖(Petkowicz et al.Carbohydrate Polymers,2001,44:107-112)。β-甘露聚糖酶是水解甘露聚糖的关键酶，能随机水解甘露聚糖主链中的β-1,4-糖苷键，生成甘露寡糖(Malgas et al.World Journal of Microbiology and Biotechnology,2015,31:1167-1175)。β-甘露聚糖酶在自然界分布广泛，从植物、低等动物和微生物中均可以分离提取出β-甘露聚糖酶，而微生物是β-甘露聚糖酶的主要来源。微生物β-甘露聚糖酶具有很多优点，如产酶条件简单、产酶量大、来源稳定、提取方便、酶学性质多样等。因此，微生物来源的β-甘露聚糖酶广泛用于食品、饲料等行业中(Srivastava and Kapoor.Biotechnology Advances,2017,35:1-19)。
根据催化域氨基酸序列的同源性，β-甘露聚糖酶可划分为糖苷水解酶5、26、113和134家族(Srivastava and Kapoor.Biotechnology advances,2017,35:1-19)。绝大多数β-甘露聚糖酶属于糖苷水解酶(GH)5家族和26家族。GH5家族β-甘露聚糖酶已用于制备部分水解甘露聚糖，如来自Arabidopsis thaliana(Wang et al.Planta,2014,239:653-665)和Rhizomucor miehei CAU432的GH5家族β-甘露聚糖酶可生产部分水解瓜尔豆胶(Li et al.International Journal of Biological Macromolecules,2017,105:1171-1179)。当底物为侧链含有较多半乳糖取代基的半乳甘露聚糖时，GH26家族β-甘露聚糖酶有更多优势(von Freiesleben et al.Enzyme and Microbial Technology,2016,83:68-77)。为满足工业生产需求，一些GH26家族β-甘露聚糖酶已成功在酵母中表达，但表达水平较低，如嗜热枯草芽孢杆菌(Thermophilic Bacillus subtilis)(TBS2)β-甘露聚糖酶(ReTMan26)酶活为5435U/mL(Luo et al.Applied Biochemistry and Biotechnology,2017,182:1259-1275)和黑曲霉(Aspergillus niger)β-甘露聚糖酶在毕赤酵母中表达酶活为5069U/mL(Zhao et al.Bioresource Technology,2011,102: 7538-7547)。因此，提高GH26家族β-甘露聚糖酶的表达水平对其工业应用具有重要意义。
部分水解甘露聚糖作为一种益生元具有低热量、性质稳定、促进生物体内有益菌群的增殖、调节代谢循环等特点，可广泛应用于食品工业(Mudgil et al.International Journal of Biological Macromolecules,2018,112:207-210；Kapoor et al.Journal of Function Foods,2016,24:207-220)。目前主要利用高温降解、酸碱水解及酶法水解等方法制备部分水解甘露聚糖。酶法制备部分水解甘露聚糖具有过程容易控制、反应条件温和、副反应少等优点，已经成为部分水解甘露聚糖制备中最常用的方法。制备部分水解甘露聚糖的底物主要为富含甘露聚糖的植物胶类。其中，常用来制备部分水解甘露聚糖的原料是魔芋粉和瓜尔豆胶，但是水解时所用的原料浓度较低(≤5％)(Mao et al.Carbohydrate Polymers,2018,181:368-375；Li et al.International Journal of Biological Macromolecules,2017,105:1171-1179；Liu et al.Bioresource Technology,2020,295:122257)。中国专利号ZL201610808817.0中公开了使用米黑根毛霉来源的甘露聚糖酶酶解瓜尔豆胶制备部分水解瓜尔豆胶的方法，其所用瓜尔豆胶浓度较低，仅为5％。此外，决明子胶、香豆胶等富含甘露聚糖的植物胶也是制备部分水解甘露聚糖的良好底物，但是相关报道和专利很少(Daas et al.Carbohydrate Polymers,2017,158:102-111)。中国专利号ZL201410024844.X中公开了制备香豆胶增稠剂的方法，通过生物酶液对香豆子种子胚乳进行酶处理，改善了香豆胶胶粉的性能；中国专利申请号CN201710478917.6中公开了一种防治高血脂症保健食品的制作方法，在原料中加入5％-15％香豆提取物可减少胆固醇，得到降血脂的保健食品。因此，利用β-甘露聚糖酶水解决明子胶、香豆胶等植物胶，并提高底物浓度生产部分水解甘露聚糖，可以丰富我国的益生元市场，有广阔的发展前景。
酸奶是一种以鲜奶为原料，经过嗜热链球菌和保加利亚乳杆菌等发酵制得的食品。酸奶细腻爽滑、质地稠厚、酸甜可口，有易于消化吸收、促进肠道蠕动、调节肠道内菌群平衡、抗氧化抗菌、降血压等优点(Farvin et al.Food Chemistry,2010,123:1090-1097；Donkor et al.International Dairy Journal,2007,17:1321-1331)。将益生元添加到酸奶中制备益生元酸奶能改善酸奶的理化性质，提高酸奶的功能活性，受到广泛关注(Cruz et al.Journal of Food Engineering,2013,114:323-330；Das et al.LWT-Food Science and Technology,2019,108:69-80；Fazilah et al.Journal of Functional Foods,2018,48:387-399)。中国专利申请号CN201811327958.6中公开了一种低聚糖褐色酸奶的制作方法，在酸奶中添加1-3g/L低聚木糖，15-22g/L低聚果糖，18-26g/L低聚异麦芽糖制成益生元酸奶，该酸奶营养丰富，有益于人体健康，符合现代人的对酸奶的消费要求。中国专利申请号CN201910276659.2中公开了一种益生元酸奶的制作方法，在生牛乳中添加0.02％-0.08％质量百分比 的膳食纤维(柑橘纤维)，然后接种菌发酵得到益生元酸奶，该酸奶可以满足消费者对口感和健康的双重需求，易被消费者接受。为了丰富益生元酸奶的种类，满足市场需求，可将部分水解甘露聚糖添加至酸奶中研制益生元酸奶。
黑曲霉是一株能够分泌多种糖苷水解酶的耐热丝状真菌，其分泌的β-甘露聚糖酶具有多种优良的酶学特性(Harnpicharnchai et al.Bioscience Biotechnology and Biochemistry,2016,80:2298-2305)。
发明公开
本发明提供一种β-甘露聚糖酶的制备方法及在制备部分水解甘露聚糖与酸奶中的应用。
本发明首先提供了一种具有β-甘露聚糖酶活性的蛋白质的制备方法，所述方法包括：将序列表中SEQ ID No.2所示的rMan26基因导入生物细胞中，得到重组细胞，培养所述重组细胞，使所述rMan26基因得到表达，得到具有β-甘露聚糖酶活性的蛋白质。
上述方法中，所述rMan26基因的表达由名称为ATX1的启动子驱动，所述ATX1为(b1)、(b2)或(b3)：
(b1)序列表中SEQ ID No.4的第6-930位所示的DNA分子；
(b2)将(b1)经过一个或几个核苷酸的取代和/或缺失和/或添加且与(b1)具有75％或75％以上同一性的单链DNA分子；
(b3)在严格条件下与(b1)或(b2)限定的核苷酸序列杂交的DNA分子。
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rMan26基因的核苷酸序列进行突变。那些经过人工修饰的，具有与本发明的rMan26基因的核苷酸序列75％或者更高同一性的核苷酸，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rMan26基因的核苷酸序列具有75％或更高，80％或更高，或85％或更高，或90％或更高，或95％或更高，或97％或更高，或98％或更高，或99％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。
上述方法中，所述严格条件可为如下：50℃，在7％十二烷基硫酸钠(SDS)、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在50℃，2×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在50℃，1×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在50℃，0.5×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在50℃，0.1×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在65℃，0.1×SSC，0.1％SDS中漂洗；也可为：在6×SSC， 0.5％SDS的溶液中，在65℃下杂交,然后用2×SSC,0.1％SDS和1×SSC,0.1％SDS各洗膜一次；也可为：2×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次5min，又于0.5×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次15min；也可为：0.1×SSPE(或0.1×SSC)、0.1％SDS的溶液中，65℃条件下杂交并洗膜。
上述75％或75％以上同一性，可为80％、85％、90％或95％以上的同一性。
上述方法中，将序列表中SEQ ID No.2所示的rMan26基因导入生物细胞中可包括将含有所述rMan26基因和所述ATX1的重组载体导入所述生物细胞中。
所述重组载体可为pPAT19K-AnMan26，所述pPAT19K-AnMan26为将pPIC9K的BglⅡ和BamHⅠ识别序列间的DNA片段替换为序列表中SEQ ID No.4的第6-930位所示的DNA分子，并将EcoRI和NotI识别序列间的DNA片段替换为rMan26基因得到的重组载体。
上述方法中，所述蛋白质可为如下A1)、A2)或A3)：
A1)氨基酸序列是SEQ ID No.3的蛋白质；
A2)将序列表中SEQ ID No.3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/或添加且具有β-甘露聚糖酶功能的蛋白质；
A3)在A1)或A2)的N端或/和C端连接标签得到的融合蛋白质。
为了使A1)中的蛋白质便于纯化，可在由序列表中SEQ ID No.3所示的氨基酸序列组成的蛋白质的氨基末端或羧基末端连接上如下表所示的标签。
表：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A2)中的蛋白质，为与SEQ ID No.3所示蛋白质的氨基酸序列具有75％或75％以上同一性且具有相同功能的蛋白质。所述具有75％或75％以上同一性为具有75％、具有80％、具有85％、具有90％、具有95％、具有96％、具有97％、具有98％或具有99％的同一性。
上述A2)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A2)中的蛋白质的编码基因可通过将SEQ ID No.2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′端连上上表所示的标签的编码序列得到。其中，SEQ ID No.2所示的DNA分子编码SEQ ID No.3所示的蛋白质。
具体的，A3)所述蛋白质可为序列表中SEQ ID No.6所示的蛋白质。
上述方法中，所述生物细胞可为微生物细胞。进一步，所述微生物细胞为真菌。更进一步，所述真菌可为酵母。所述酵母可为毕赤酵母(如毕赤酵母GS115)。
在本发明的一个实施例中，所述重组细胞为GS115-pPAT19K-AnMan26，所述GS115-pPAT19K-AnMan26为将所述pPAT19K-AnMan26导入毕赤酵母GS115中得到的重组菌。
本发明还提供了利用所述具有β-甘露聚糖酶活性的蛋白质的制备方法制备的具有β-甘露聚糖酶活性的蛋白质。
本发明还提供了下述任一产品：
X1、所述重组细胞；
X2、所述rMan26基因；
X3、所述ATX1；
X4、由所述rMan26基因和所述ATX1组成的成套试剂；
X5、含有所述rMan26基因和/或所述ATX1的表达盒；
X6、含有所述rMan26基因和/或所述ATX1的重组载体。
X4所述成套试剂可用于制备β-甘露聚糖酶。
X5所述表达盒是指能够在宿主细胞中表达rMan26基因编码蛋白质的DNA，该DNA不但可包括启动rMan26基因转录的启动子，还可包括终止rMan26基因转录的终止子。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，和诱导型启动子。具体的，X5所述表达盒中的启动子可为所述ATX1。
X6所述重组载体可为所述pPAT19K-AnMan26。
本发明还提供了所述蛋白质的下述任一应用：
a1)作为β-甘露聚糖酶的应用；
a2)在制备有β-甘露聚糖酶活性产品中的应用；
a3)在制备甘露寡糖中的应用；
a4)在制备用于制备甘露寡糖产品中的应用；
a5)在降解甘露聚糖中的应用；
a6)在制备用于降解甘露聚糖产品中的应用；
a7)在制备部分水解甘露聚糖中的应用；
a8)在制备用于制备部分水解甘露聚糖产品中的应用；
a9)在降解植物胶中的应用；
a10)在制备用于降解植物胶产品中的应用。
上述应用中，所述植物胶可为b1)或b2)或b3)或b4)或b5)：b1)槐豆胶；b2)决明子胶；b3)香豆胶；b4)魔芋粉；b5)富含甘露聚糖的植物胶。
所述甘露寡糖可以为2-10个甘露糖聚合而成的甘露寡糖。
所述“生产甘露寡糖”和“生产部分水解甘露聚糖”可以甘露聚糖或植物胶作为底物。
所述植物胶为香豆胶，所述香豆胶与所述具有β-甘露聚糖酶活性的蛋白质的比例为：每克香豆胶加入具有β-甘露聚糖酶活性的蛋白质100-200U；水解温度为30℃-45℃；所述香豆胶的溶剂为柠檬酸缓冲液或水。
所述植物胶可为决明子胶，所述决明子胶与所述具有β-甘露聚糖酶活性的蛋白质的比例为：每克决明子胶加入所述具有β-甘露聚糖酶活性的蛋白质100-800U；水解温度为30℃-45℃；所述决明子胶的溶剂为柠檬酸缓冲液或水。
本发明还提供了所述产品的下述任一应用：
a2)在制备有β-甘露聚糖酶活性产品中的应用；
a3)在制备甘露寡糖中的应用；
a4)在制备用于制备甘露寡糖产品中的应用；
a5)在降解甘露聚糖中的应用；
a6)在制备用于降解甘露聚糖产品中的应用；
a7)在制备部分水解甘露聚糖中的应用；
a8)在制备用于制备部分水解甘露聚糖产品中的应用；
a9)在降解植物胶中的应用；
a10)在制备用于降解植物胶产品中的应用。
其中，所述植物胶可为b1)或b2)或b3)或b4)或b5)：b1)槐豆胶；b2)决明子胶；b3)香豆胶；b4)魔芋粉；b5)富含甘露聚糖的植物胶。
利用所述蛋白质或所述产品制备的部分水解甘露聚糖，也属于本发明的保护范围。
利用所述蛋白质制备部分水解甘露聚糖在制作益生元酸奶中的应用，也属于本发明的保护范围。
上述应用中，生产所述部分水解甘露聚糖的植物胶可为c1)或c2)或c3)或c4)或c5)：c1)槐豆胶；c2)决明子胶；c3)香豆胶；c4)魔芋粉；c5)富含甘露聚糖的植物胶。
所述益生元酸奶制备中还包括向体系中添加嗜热链球菌(Streptococcus thermophilus)和保加利亚乳杆菌(Lactobacillus bulgaricus)。
本发明还提供了制作益生元酸奶的方法，所述方法包括：利用所述蛋白质或所述产品制作益生元酸奶。
上述方法中的体系中含有植物胶。所述植物胶可为c1)或c2)或c3)或c4)或c5)：c1)槐豆胶；c2)决明子胶；c3)香豆胶；c4)魔芋粉；c5)富含甘露聚糖的植物胶。
上述方法的体系中还含有嗜热链球菌(Streptococcus thermophilus)和保加利亚乳杆菌(Lactobacillus bulgaricus)。
附图说明
图1为重组β-甘露聚糖酶在5L发酵罐中高密度发酵的产酶历程图(A)和SDS-PAGE电泳图(B)。其中(■)酶活、(▲)蛋白浓度、(●)菌体湿重。B中泳道M为蛋白质分子量标准，泳道1-8分别为诱导0、24、48、72、96、120、144、168h的发酵上清液。
图2为重组β-甘露聚糖酶纯化图，其中泳道1为粗酶液，泳道2为经Sephacryl S-100HR凝胶柱层析纯化后的纯酶，泳道3为用N-去糖基化酶去糖基后的结果。
图3为重组β-甘露聚糖酶的最适pH值测定曲线图。其中(◆)柠檬酸缓冲液(pH 3.0-6.0)、(▲)磷酸缓冲液(pH 6.0-8.0)。
图4为重组β-甘露聚糖酶的pH稳定性测定曲线图。其中(●)甘氨酸-盐酸缓冲液(pH 2.0-3.0)、(◆)柠檬酸缓冲液(pH 3.0-6.0)、(▲)磷酸缓冲液(pH 6.0-8.0)。
图5为重组β-甘露聚糖酶的最适温度测定曲线图。
图6为重组β-甘露聚糖酶温度稳定性测定曲线图。
图7为AnMan26作为β-甘露聚糖酶水解香豆胶的产物的薄层层析色谱分析图。M、M2、M3、M4、M5、M6分别为甘露糖、甘露二糖、甘露三糖、甘露四糖、甘露五糖、甘露六糖，1为部分水解香豆胶粗糖液。
图8为AnMan26作为β-甘露聚糖酶水解香豆胶的产物的凝胶渗透色谱分析图。0min表示未经水解的香豆胶。
图9为AnMan26作为β-甘露聚糖酶水解决明子胶的产物的薄层层析色谱分析图。M、M2、M3、M4、M5、M6分别为甘露糖、甘露二糖、甘露三糖、甘露四糖、甘露五糖、甘露六糖，1为部分水解决明子胶(决明子寡糖)粗糖液。
图10为AnMan26作为β-甘露聚糖酶水解决明子胶的产物的凝胶渗透色谱分析图。0min表示未经水解的决明子胶。
图11为部分水解香豆胶添加量对益生元酸奶持水力影响图。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。下述实施例中的实验方法，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、仪器等，如无特殊说明，均可从商业途径得到。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。下述实施例中，如无特殊说明，序列表中各核苷酸序列的第1位均为相应DNA/RNA的5′末端核苷酸，末位均为相应DNA/RNA的3′末端核苷酸。
下述实施例中的嗜热链球菌(Streptococcus thermophilus)为丹麦Chr-Hansen公司产品。
下述实施例中的保加利亚乳杆菌(Lactobacillus bulgaricus)为丹麦Chr-Hansen公司产品。
下述实施例中的表达载体pPIC9K为美国Invitrogen产品，货号V17520。
下述实施例中的毕赤酵母GS115为美国Invitrogen产品，货号C18100。
β-甘露聚糖酶活测定采用3,5-二硝基水杨酸(DNS)法，如无特殊说明，测定步骤如下：(1)取0.9mL 0.5g/100mL槐豆胶溶液(利用槐豆胶和pH 5.0、50mmol/L的柠檬酸缓冲液配制)，加入0.1mL适当稀释的待测酶液，置于45℃恒温水浴中反应10min；(2)完成步骤(1)反应后，采用DNS试剂终止反应并与释放的还原糖反应，测定还原糖(以甘露糖作为标准)量。以甘露糖标准液绘制标准曲线，β-甘露聚糖酶的活力单位定义为：在上述反应条件下，每分钟反应生成1μmol甘露糖所需要的酶量为一个酶活力单位(1U)。
比酶活力定义为1mg蛋白所具有的酶活力单位(U/mg)。
槐豆胶：半乳甘露聚糖，为美国Sigma-Aldrich公司产品，货号为G0753。
甘露糖：为美国Sigma-Aldrich公司产品，货号为M2069。
实施例1、重组β-甘露聚糖酶的制备
一、黑曲霉的β-甘露聚糖酶基因的PCR扩增
β-甘露聚糖酶基因(MAN26基因)XM_001397260.1的编码区为954bp，其序列为序列表中SEQ ID No.1，编码序列表中SEQ ID No.3所示的β-甘露聚糖酶MAN26。将MAN26基因进行密码子优化，得到优化后的基因，记为rMan26基因，其序列为序列表中SEQ ID No.2。
人工合成rMan26基因，以其为模板，利用特异性引物rMan26AF和rMan26AR进行PCR扩增，得到PCR产物。引物序列如下：
rMan26AF：5′-CCG GAATTCGCTTCTAACCAAACTTTGTCTTACG-3′(下划线为限制性内切酶EcoRI的酶切位点)(SEQ ID No.7)；
rMan26AR：5′-GAAT GCGGCCGCTTAAGCACCTTCCCAATTCAAAG-3′(下划线为限制性内切酶NotI的酶切位点)(SEQ ID No.8)。
二、重组β-甘露聚糖酶(AnMan26)的表达
1、去除毕赤酵母的醇氧化酶启动子AOX1基因序列中的TATA box序列，改造后的新启动子命名为ATX1，其序列为序列表中SEQ ID No.4的第6-930位。人工合成SEQ ID No.4所示的DNA片段，利用BglⅡ和BamHⅠ双酶切回收酶切大片段，将回收产物与表达载体pPIC9K经BglⅡ和BamHⅠ双酶切得到的载体骨架相连，将得到的序列正确的重组载体记为pATX19K。
2、利用EcoRI和NotI分别双酶切步骤一得到的PCR产物及pPAT19K，回收PCR产物的酶切大片段以及载体骨架并连接，将得到的序列正确的重组载体记为pPAT19K-AnMan26。
pPAT19K-AnMan26为将pPIC9K的BglⅡ和BamHⅠ识别序列间的DNA片段替换为序列表中SEQ ID No.4的第6-930位所示的DNA分子，并将EcoRI和NotI 识别序列间的DNA片段替换为rMan26基因得到的重组载体。pPAT19K-AnMan26含有序列表中SEQ ID No.5所示的DNA分子(即rMan26基因与载体上部分序列所形成的融合基因，记为AnMan26基因)，能表达序列表中SEQ ID No.6所示的融合蛋白质，记为重组β-甘露聚糖酶(AnMan26)，AnMan26基因的表达由ATX1驱动。
其中，序列表中SEQ ID No.5的第280-1233位为rMan26基因的序列，第1-279位为载体上序列；SEQ ID No.6的第94-410位为MAN26的氨基酸序列，第1-93位为载体上序列编码的多肽。
3、SalI线性化重组载体pPAT19K-AnMan26，醇沉回收后电击转毕赤酵母GS115，涂MD平板，于30℃培养箱中倒置培养2-3d，直至长出菌落为止，得到重组菌，将该重组菌记为GS115-pPAT19K-AnMan26。
4、用无菌水收集步骤3的重组菌菌体，并将适当稀释的菌体涂布于含有G418的浓度分别为1mg/mL、2mg/mL、3mg/mL、4mg/mL的YPD平板(1g/100mL酵母提取物，2g/100mL胰蛋白胨，2g/100mL葡萄糖)上，挑选出不同G418浓度下生长良好的单菌落，接种于5mL BMGY培养基(该培养基的由溶剂和溶质组成，溶剂为100mmol/L pH 6.0磷酸缓冲液，溶质及其在培养基中的浓度分别为1g/100mL酵母提取物，2g/100mL蛋白胨，1.34g/100mL YNB(美国Biomol产品)，4×10 -5g/100mL生物素和1g/100mL的甘油)中，置于30℃，200rpm摇床中，振荡培养，待OD 600为2-6时，离心，弃上清，收集菌体转接至10mL BMMY培养基(该培养基为将BMGY培养基中的甘油替换为甲醇得到的培养基，甲醇在该培养基中的浓度为0.5％(体积百分比))中，置于30℃，200rpm摇床中诱导表达。每隔24h加入甲醇至终浓度为0.5％，诱导3d，收集发酵液，11510×g离心5min,收集上清液，将上清液装入透析袋(截流分子量为3.50kDa)并放入20mmol/L柠檬酸缓冲液(pH 5.0)中透析过夜，测定透析后上清液β-甘露聚糖酶活，GS115-pPAT19K-AnMan26产酶可高达467U/mL。
5、高密度发酵
参照文献“Picha Fermentation Process Guidelines(Version B,053002,Invitrogen)”的方法利用步骤4得到的重组菌进行高密度发酵。发酵罐为5L发酵罐。种子培养基BMGY、基本培养基BSM、甘油分批补料培养基和100％甲醇诱导培养基均参照文献中的方法配置。整个发酵过程分为种子液培养、基础培养、甘油流加培养和100％甲醇诱导培养四个阶段。具体步骤如下：
1)种子培养：将GS115-pPAT19K-AnMan26接种到装有150mL BMGY培养基的500mL的三角瓶中，在30℃，200rpm摇床中培养24h以上，得到OD 600为2-6的种子液。
2)基础培养：将1)中的种子液接种到5L灭菌后的发酵罐中(装有1.5L发酵基本培养基BSM)，用氨水调节培养基pH 4.0，加入PTM1 4.35mL/L起始发酵液，种子液接种量10％，培养过程中温度为30℃，转速600rpm，待溶氧 DO值迅速上升，甘油耗尽时，进入甘油流加培养阶段。其中，PTM1(CuSO 4·7H 2O 6.0g，NaI 0.08g，MnSO 4·H 2O 3.0g，Na 2MoO 4·2H 2O 0.2g，硼酸0.02g，CoCl 2 0.5g，ZnCl 2 20.0g，FeSO 4·7H 2O 65.0g，生物素0.2g，浓硫酸5.0mL，加水至1L。)。
3)甘油流加培养：甘油水溶液(甘油浓度为500g/L)流加速度为30mL/h/L起始发酵液。该阶段始终监测溶氧，通过调节甘油流加速度，使溶氧保持DO＞20％。控制温度30℃、发酵液pH 4.0，待菌体湿重达180-220g/L，停止流加，整个过程发酵时间为4-6h。
4)100％甲醇流加阶段：停止流加甘油后，饥饿30min左右，使罐内甘油耗尽，调整发酵液pH至6.0、转速至800rpm，开始流加100％甲醇诱导产酶，甲醇流加从3.6mL/h/L起始发酵液增加至10.9mL/h/L起始发酵液，监控罐内溶氧DO＞20％(如溶氧DO低于20％，可适当减少流加速度)，控制温度30℃，发酵6-7天。
发酵过程中取样测定菌体湿重、蛋白质含量和β-甘露聚糖酶活。测定条件为：50mmol/L柠檬酸缓冲液(pH 5.0)，反应温度为45℃。测定步骤如下：
1)菌体湿重：称取离心管质量，记为m 1，在离心管中加入1mL发酵液，使用离心机11510×g离心3min，弃去上清液，将离心管及下层物质称重记为m 2。
菌体湿重计算公式为：菌体湿重＝m 2-m 1。
2)蛋白质含量：以牛血清蛋白为标准蛋白，采用Lowry法(Lowry et al The Journal of Biological Chemistry,1951,193(1):265-275)测定蛋白含量(mg/mL)。蛋白质含量为蛋白质总含量。
发酵过程中菌体湿重、蛋白质含量和β-甘露聚糖酶酶活力变化如图1中A所示，发酵过程中蛋白的SDS-PAGE电泳图如图1中B所示。高密度发酵168h时酶活力达到最高，发酵液的上清液中β-甘露聚糖酶酶活力为22100U/mL，蛋白含量为12.0mg/mL，菌体湿重为355.8g/L。
三、重组β-甘露聚糖酶的纯化
将10mL高密度发酵168h时的发酵上清液(即粗酶液)装入透析袋(截流分子量为3.50kDa)并放入20mmol/L柠檬酸缓冲液(pH 5.0)中透析过夜，将透析好的酶液用10kDa超滤膜浓缩至0.6mL，将浓缩后的样品11510×g离心5min，收集上清液。
将所得上清液上样于用缓冲液(该缓冲液的溶剂为20mmol/L柠檬酸缓冲液(pH 5.0，溶质及其在缓冲液中的浓度为150mmol/L NaCl)平衡好的Sephacryl S-100HR(1×100cm)柱中，凝胶柱分离过程的流速为0.3mL/min。收集有酶活的部分，将得到的纯酶合并后用20mmol/L柠檬酸缓冲液(pH 5.0)透析，即得到纯化后的重组β-甘露聚糖酶酶液(纯酶液)。整个纯化过程见表1，SDS-PAGE结果见图2，其中泳道1为粗酶液，泳道2为经Sephacryl S-100HR 凝胶柱层析纯化后的纯酶，泳道3为用N-去糖基化酶去糖基后的结果。
结果表明，粗酶液和纯化后的酶均为单一分子量条带，对应分子量为65.3kDa，为重组β-甘露聚糖酶，比酶活为2869.0U/mg。为检测重组β-甘露聚糖酶是否发生了糖基化，将所得纯化后的酶经N-去糖基化酶处理，去糖基后重组β-甘露聚糖酶溶液显示单一分子量条带，对应分子量为38.7kDa，与预期大小一致。经去糖基化酶去糖基后重组β-甘露聚糖酶的比酶活为191.2U/mg。
N-去糖基化酶去糖基的步骤如下：
1)在离心管中加入4μg纯酶，加入1μL糖蛋白变性缓冲液(10×)，加水至总反应体积至10μL，沸水浴10min使纯酶变性。
2)在1)体系中加入2μL GlycoBuffer3(10×)，2μL去糖基化酶(美国NEB公司产品，货号P0702S)，用水补至反应体积至20μL，置于37℃水浴60min后，用SDS-PAGE电泳检测蛋白。
其中，糖蛋白变性缓冲液(10×)(美国NEB公司产品，货号P0702S)。GlycoBuffer3(10×)(美国NEB公司产品，货号P0702S)。
表1、重组β-甘露聚糖酶的纯化
Figure PCTCN2020135515-appb-000001
表1中，回收率指的是纯酶的总酶活力占粗酶液总酶活力的百分含量。纯化倍数指的是纯酶的比酶活与粗酶的比酶活的比值。
实施例2、重组蛋白AnMan26作为β-甘露聚糖酶的酶学性质
所用各缓冲液具体如下：甘氨酸-盐酸缓冲液(pH 2.0、2.5和3.0)、柠檬酸缓冲液(pH 3.0、3.5、4.0、4.5、5.0、5.5和6.0)、磷酸缓冲液(pH 6.0、6.5、7.0、7.5和8.0)。以上缓冲液的浓度均为50mmol/L。
一、重组β-甘露聚糖酶的最适pH值
将实施例1制备的纯化后的重组β-甘露聚糖酶溶液作为待测样品溶液，测定其酶活力，方法如下：取0.9mL 0.5g/100mL槐豆胶溶液(利用槐豆胶和pH 3.0-8.0范围的不同待测缓冲体系中配制)，加入0.1mL适当稀释的待测酶液，将所得反应体系分别置于30℃下反应10分钟测定酶活力，将最高酶活力记为100％，计算各个pH值条件下β-甘露聚糖酶的相对活力，结果如图3所示，重组β-甘露聚糖酶的最适pH值为5.0。
实施例1制备的重组β-甘露聚糖酶在pH分别为3.0、3.5、4.0、4.5、5.0、5.5和6.0的柠檬酸缓冲液、pH分别为6.0、6.5、7.0、7.5和8.0的磷酸缓冲液下的相对酶活力分别为1.4％±0.4％、20.5％±0.5％、44.7％±0.5％、 77.9％±1.3％、100％±0.4％、87.6％±0.7％、54.7％±0.3％、36.3％±0.3％、17.9％±0.9％、3.7％±0.1％、0.7％±0.03％和0.2％±0.02％。
二、重组β-甘露聚糖酶的pH稳定性
将实施例1制备的纯化后的重组β-甘露聚糖酶溶液分别用上述缓冲液稀释适当倍数，使重组β-甘露聚糖酶分别处于上述各pH下的缓冲液中，然后将稀释后的酶液置于30℃水浴中保温30min，再迅速取出并立即冰浴30min，测定残余的β-甘露聚糖酶活力。对照为未经上述处理(上述处理指的是先30℃水浴30min，再迅速冰浴30min)的重组β-甘露聚糖酶溶液的稀释液(即实施例1得到的重组β-甘露聚糖酶纯酶液的稀释液)。以对照的酶活力为100％，计算用不同pH缓冲液处理后的酶的相对活力，结果如图4所示，该酶在pH2.5-6.0范围内保持稳定，处理30min后残余酶活力仍然保持在80％以上。
实施例1制备的重组β-甘露聚糖酶经pH分别为2.0、2.5和3.0的甘氨酸-盐酸缓冲液、pH分别为3.0、3.5、4.0、4.5、5.0、5.5和6.0的柠檬酸缓冲液、pH分别为6.0、6.5、7.0、7.5和8.0的磷酸缓冲液处理后的相对酶活力分别为9.4％±0.2％、78.9％±2.3％、82.3％±5.1％、96.2％±3.7％、91.0％±2.4％、92.7％±3.7％、92.2％±5.1％、93.5％±2.8％、92.5±1.6％、95.7％±4.3％、69.4％±3.0％、66.6％±2.1％、65.4％±2.3％、59.0％±2.1％和26.0％±1.0％。
三、重组β-甘露聚糖酶的最适温度
将实施例1中制备的重组β-甘露聚糖酶溶液作为待测酶液，利用pH 5.0、50mmol/L的柠檬酸缓冲体系，在30℃-80℃范围内的不同温度下反应，测定不同温度下的酶活力，以最高酶活力为100％，计算在各个反应温度下的相对活力。结果如图5所示，重组β-甘露聚糖酶的最适温度为45℃。
实施例1制备的重组β-甘露聚糖酶在20℃、25℃、30℃、35℃、40℃、45℃、50℃、55℃、60℃、65℃、70℃、75℃和80℃下的相对酶活力分别为42.0％±3.6％、42.7％±3.7％、66.6％±5.0％、72.5％±5.4％、80.6％±2.7％、100％±2.0％、79.2％±4.6％、40.2％±2.1％、18.6％±0.6％、9.8％±1.7％、7.2％±0.5％、6.1％±0.2％和4.6％±0.3％。
四、重组β-甘露聚糖酶的温度稳定性
将实施例1制备的重组β-甘露聚糖酶溶液用pH 5.0、50mmol/L的柠檬酸缓冲液稀释至适当倍数，置于不同温度下分别水浴30min(水浴温度为20℃、25℃、30℃、35℃、40℃、45℃、50℃、55℃、60℃、65℃、70℃、75℃或80℃)，迅速取出并冰浴30min，测定残余的β-甘露聚糖酶活力。对照为未经上述步骤处理(上述处理指的是水浴30min，迅速冰浴30min)的重组β-甘露聚糖酶溶液稀释液。以对照的酶活力作为100％。上述各个温度处理后的待测酶液相对活力结果如图6所示，重组β-甘露聚糖酶在40℃以下保留80％以上的活力。
实施例1制备的重组β-甘露聚糖酶经20℃、25℃、30℃、35℃、40℃、45℃、50℃、55℃、60℃、65℃、70℃、75℃和80℃处理后的相对酶活力分别 为100％±3.7％、100％±3.8％、100％±4.0％、100％±3.9％、93.8％±5.5％、73.8％±4.0％、19.4％±0.3％、12.6％±1.9％、3.6％±0.3％、1.7％±0.1％、1.4％±0.1％、1.2％±0.1％和1.1％±0.1％。
五、底物特异性的测定
将实施例1制备的重组β-甘露聚糖酶溶液作为待测酶液，测定不同底物的β-甘露聚糖酶活力，底物为槐豆胶、魔芋粉、决明子胶、香豆胶和羧甲基纤维素，底物在反应体系中的浓度均为0.5g/100mL。
其中，魔芋粉为湖北强森魔芋科技有限公司产品，货号为KJ-30；决明子胶为北京瓜尔润科技股份有限公司产品；香豆胶为北京瓜尔润科技股份有限公司产品；羧甲基纤维素为美国Sigma-Aldrich公司产品，货号为C4888-500G。
以重组β-甘露聚糖酶对槐豆胶的酶活力为100％，计算该酶对槐豆胶、魔芋粉、决明子胶、香豆胶和羧甲基纤维素的相对活力和比酶活力。
结果见表2。重组β-甘露聚糖酶对槐豆胶的比酶活力最高，达2869.0U/mg；其次为魔芋粉，比酶活力达1905.0U/mg；对香豆胶的比酶活力最低，为341.1U/mg，对羧甲基纤维素没有水解能力。
表2 重组β-甘露聚糖酶的底物特异性
Figure PCTCN2020135515-appb-000002
“-”表示未检测到活性
实施例3、利用重组β-甘露聚糖酶(AnMan26)水解香豆胶生产部分水解香豆胶
称取10g香豆胶在200rpm搅拌下溶于100mL pH 5.0、50mmol/L的柠檬酸缓冲液中，配制成10g/100mL的香豆胶溶液，向香豆胶溶液中添加实施例1的重组β-甘露聚糖酶得到反应体系，加酶量为每克香豆胶加入200U重组β-甘露聚糖酶，将该反应体系置于40℃下水解8h，得到水解液。水解完成后，将反应体系沸水浴灭酶20min，11510×g离心10min，收集沉淀和上清液，沉淀即为水解液残渣，上清液即为部分水解香豆胶粗糖液。
用3,5-二硝基水杨酸法测定粗糖液中的还原糖含量，计算还原糖得率、水解率和产物得率。采用薄层层析色谱定性分析粗糖液的组成。采用凝胶渗透色谱分析部分水解香豆胶的重均分子量。
还原糖得率、水解率、产物得率的计算公式分别为：
还原糖得率(％)＝水解后体系中总还原糖质量/水解前加入原料干重×100％；
水解率(％)＝(水解前加入原料干重-水解液残渣干重)/水解前加入原料干重×100％，水解液残渣干重为将水解液残渣置于105℃烘箱中烘至恒重测量所得；
产物得率(％)＝产物干重/水解前加入原料干重×100％，产物干重为将部分水解香豆胶粗糖液置于105℃烘箱中烘至恒重测量所得。
薄层层析色谱检测条件：展层剂为正丁醇：乙醇：水＝2：1：1，显色剂为甲醇：硫酸＝95：5。标准对照为甘露糖、甘露二糖、甘露三糖、甘露四糖、甘露五糖和甘露六糖的标准品组成的混合物。在薄层层析色谱分析板上点适量待测样本，放入展层剂展层两次后用显色剂完全浸湿吹干，130℃烘烤显色。
其中，甘露糖为美国Sigma-Aldrich生产，货号为M2069；甘露二糖、甘露三糖、甘露四糖、甘露五糖和甘露六糖均为爱尔兰Megazyme产品，货号分别为O-MBI、O-MTR、O-MTE、O-MPE、O-MHE。
凝胶渗透色谱检测条件：TSKgel GMPW XL水相凝胶色谱柱(7.8×300mm)；柱温：60℃；RID检测器；进样量：20μL；流动相：100mM NaNO 3；流速：0.6mL/min；以聚氧乙烯标准品(分子量300-500000Da，为日本Tosoh Corporatio公司产品)做标准曲线。
结果显示，AnMan26水解香豆胶的水解率为82.2％，还原糖得率为43.8％，产物得率为73.5％。薄层层析色谱分析结果见图7(Mn为标准对照，从上至下依次为甘露糖、甘露二糖、甘露三糖、甘露四糖、甘露五糖、甘露六糖)。结果表明，重组β-甘露聚糖酶水解香豆胶生成聚合度在2-6之间的甘露寡糖，大量聚合度大于6的甘露聚糖。
凝胶渗透色谱分析结果见图8，香豆胶的重均分子量为4.9×10 6Da，加入AnMan26后，部分水解香豆胶的重均分子量迅速下降，水解结束后，重均分子量为1.8×10 3Da，部分水解香豆胶的平均聚合度≤10。
由此可知，采用重组β-甘露聚糖酶可以水解香豆胶生产73.5％得率的部分水解香豆胶，其成分为聚合度在2-6之间的甘露寡糖，大量聚合度大于6的甘露聚糖。部分水解香豆胶的平均聚合度≤10，重均分子量为1.8×10 3Da。
实施例4、利用重组β-甘露聚糖酶(AnMan26)水解决明子胶生产部分水解决明子胶(决明子寡糖)
称取10g决明子胶在200rpm搅拌下溶于100mL pH 5.0、50mmol/L的柠檬酸缓冲液，配制成10g/100mL的决明子胶溶液，将其置于40℃保温30min，使决明子胶均匀分散在缓冲液中，然后向该反应体系中加入重组β-甘露聚糖酶(AnMan26)，加酶量为800U/g决明子胶，在40℃下水解，水解过程以600rpm的搅拌速度不断搅拌水解体系。水解至8h，得到水解液。水解完成后，将反应体系沸水浴灭酶20min，得到酶解液。将酶解液于11510×g离心10min后，收集上清液即为粗糖液。
用3，5-二硝基水杨酸法测定粗糖液中的还原糖含量，计算还原糖得率、水解率和产物得率。采用薄层层析色谱定性分析粗糖液的组成。采用凝胶渗透色谱分析部分水解决明子胶(决明子寡糖)的重均分子量。还原糖得率、水解率和产物得率计算方法，薄层层析色谱条件和凝胶渗透色谱分析条件同实施例3。
结果显示，AnMan26水解决明子胶的水解率为83.3％，还原糖得率为43.9％，产物得率为74.7％。薄层层析色谱部分水解决明子胶的实验结果见图9(Mn为标准对照，从上至下依次为甘露糖、甘露二糖、甘露三糖、甘露四糖、甘露五糖、甘露六糖)。结果表明，重组β-甘露聚糖酶水解决明子胶主要生成甘露糖、甘露二糖、甘露三糖、甘露四糖、聚合度在4-6之间的甘露寡糖和少量聚合度大于6的甘露聚糖。
凝胶渗透色谱分析结果见图10，决明子胶的重均分子量为1.7×10 6Da，加入AnMan26后，部分水解决明子胶的重均分子量迅速下降，水解结束后，重均分子量为1188Da。
由此可知，采用重组β-甘露聚糖酶可以水解决明子胶生产74.7％得率的部分水解决明子胶(决明子寡糖)，其成分主要为聚合度在1-6之间的甘露寡糖，少量聚合度大于6的甘露聚糖。部分水解决明子胶的平均聚合度＜7，重均分子量为1188Da。
实施例5、利用部分水解甘露聚糖生产益生元酸奶
本实施例使用的部分水解甘露聚糖为实施例3得到的部分水解香豆胶。部分水解香豆胶糖液经喷雾干燥后得到部分水解香豆胶粉(喷雾干燥条件：进风口温度180℃，出风口温度80℃)。部分水解甘露聚糖生产益生元酸奶由如下重量份的原料(1000份)制成：鲜牛奶925份-910份，部分水解香豆胶粉5份-20份，蔗糖70份。
本实施例所述的益生元酸奶的制作方法包括如下步骤：
第一步：将鲜牛奶、部分水解香豆胶和蔗糖按照上述重量份混合均匀后于65℃预热至物料完全溶解，在40MPa下进行均质，95℃巴氏杀菌5min，得到混合料。
第二步：将所述混合料降温至40℃后，加入嗜热链球菌(Streptococcus thermophilus)和保加利亚乳杆菌(Lactobacillus bulgaricus)进行接种，所加入的两种菌在体系中的活菌含量均为1×10 6cfu/ml。
第三步：将接种后的混合料放于恒温发酵室中，在42℃条件下发酵4h(至pH 4.6左右)。
第四步：将发酵所得的益生元酸奶于350rpm破乳1min，打冷至20℃，放入冷藏库后熟16h，即得到酸奶。
效果评价：
1.感官评价：请20位有酸奶感官评价经验的人员对酸奶的口感、风味、色 泽、粘稠度和整体评价进行感官评分。评分范围为0-10分，其中口感顺滑为高分(7-10分)，颗粒感为低分(0-3分)；发酵风味淡为低分(0-3分)，发酵风味浓郁为高分(7-10分)；色泽白为0分，黄为10分；粘稠感越强分数越高；整体评价依据个人喜好进行评分。
2.持水力：取后熟16h的酸奶进行测定，称取灭菌离心管质量，记为m 1，在每个离心管中加入酸奶20g并称取总质量，记为m 2，使用离心机3000rpm离心10min，弃去上清液，将离心管及下层物质称重记为m 3。
持水力计算公式为：持水力(％)＝(m 3-m 1)/(m 2-m 1)×100％。
3.活菌计数
按照国标GB4789.35-2016《食品微生物学检验乳酸菌检验》中方法进行活菌数检测，具体如下：
嗜热链球菌计数：选择2-3个连续的适宜稀释度，每个稀释度吸取1mL样品匀液于灭菌平皿内。稀释液移入平皿后，将冷却至48℃的MC培养基倾注入平皿约15mL，转动平皿使混合均匀。36℃±1℃需氧培养72h±2h，培养后计数。
乳杆菌计数：选择2-3个连续的适宜稀释度，每个稀释度吸取1mL样品匀液于灭菌平皿内。稀释液移入平皿后，将冷却至48℃的MRS琼脂培养基倾注入平皿约15mL，转动平皿使混合均匀。36℃±1℃厌氧培养72h±2h，培养后计数。
4.结果
表3是部分水解香豆胶酸奶的感官评价数据，从表3重可以看出，添加部分水解香豆胶后酸奶总分5.3-6.3之间，低于未添加部分水解香豆胶的酸奶总分6.6，主要差别在添加部分水解香豆胶酸奶风味和粘稠度得分低于未添加部分水解香豆胶的酸奶风味和粘稠度得分。但添加1％部分水解香豆胶的酸奶总分6.3，与未添加部分水解香豆胶酸奶的总分6.6接近，可以应用于益生元酸奶的制作。
表3 部分水解香豆胶酸奶感官评价表
Figure PCTCN2020135515-appb-000003
注：表1中的混合料由如下重量份的原料(1000份)制成：鲜牛奶925份-910份，部分水解香豆胶粉5份-20份，蔗糖70份。
图11是部分水解香豆胶酸奶持水力结果，从图11可以看出，添加部分水解香豆胶后酸奶持水力在36.8％-38.6％之间，高于未添加部分水解香豆胶的酸奶的持水力34.7％。在添加部分水解香豆胶量为0-2.0％范围内，酸奶的持水力随部分水解香豆胶添加量的增加而增大，说明部分水解香豆胶的添加有助于酸奶持水力的增加，部分水解香豆胶的加入能有效地增强酸奶的凝胶结构，使酸奶更有效地截留水分，防止乳清析出，改善酸奶的组织状态。
表4是部分水解香豆胶酸奶对乳杆菌和嗜热链球菌的活菌数影响。从表4可以看出，未添加部分水解香豆胶的酸奶中的乳杆菌的活菌数和嗜热链球菌的活菌数分别为1.1×10 8CFU/mL和2.75×10 8CFU/mL，而添加部分水解香豆胶后酸奶中乳杆菌的活菌数在4.05×10 8CFU/mL-1.075×10 9CFU/mL之间，嗜热链球菌的活菌数在2.82×10 8CFU/mL-6.9×10 8CFU/mL之间，高于未添加部分水解香豆胶的酸奶中的乳杆菌的活菌数和嗜热链球菌数量的活菌数。在添加部分水解香豆胶量为0-2.0％范围内，酸奶中乳杆菌的活菌数随部分水解香豆胶添加量的增加先增加后而减少，酸奶中嗜热链球菌的活菌数随部分水解香豆胶添加量的增加而增加，在添加1.5％部分水解香豆胶时酸奶中乳杆菌的数量最高1.075×10 9CFU/mL，在添加2.0％部分水解香豆胶时酸奶中嗜热链球菌的活菌数最高6.9×10 8CFU/mL。部分水解香豆胶酸奶中乳杆菌的活菌数和嗜热链球菌的活菌数均高于不添加部分水解香豆胶酸奶的乳杆菌的活菌数和嗜热链球菌的活菌数，说明部分水解香豆胶的添加有效提高酸奶中乳杆菌和嗜热链球菌的活菌数，提高酸奶的品质。
表4 部分水解香豆胶酸奶对乳杆菌和嗜热链球菌的影响
Figure PCTCN2020135515-appb-000004
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。
工业应用
本发明将黑曲霉β-甘露聚糖酶进行密码子优化，并对pAOX1启动子进行改造，得到pATX1启动子并构建表达载体pATX19K转化至毕赤酵母GS115进行高密度表达，进而得到高酶活的β-甘露聚糖酶，其酶活可达22100U/mL。本发明所得β-甘露聚糖酶的最适pH为5.0，该酶的最适温度为45℃，可以水解决明子胶和香豆胶等富含甘露聚糖的植物胶，并得到包含不同聚合度的甘露寡糖的部分水解甘露聚糖，与现有β-甘露聚糖酶水解决明子胶和香豆胶相比有显著区别，可以丰富部分水解甘露聚糖市场。将部分水解甘露聚糖添加在酸奶中制得益生元酸奶，可以增加酸奶的持水力，乳杆菌和嗜热链球菌的活菌数，提高酸奶的品质。本发明的β-甘露聚糖酶及其制备方法在食品行业中有很大的应用价值。

Claims (20)

  1. 一种具有β-甘露聚糖酶活性的蛋白质的制备方法，包括：将序列表中SEQ ID No.2所示的rMan26基因导入生物细胞中，得到重组细胞，培养所述重组细胞，使所述rMan26基因得到表达，得到具有β-甘露聚糖酶活性的蛋白质。
  2. 根据权利要求1所述的方法，其特征在于：所述rMan26基因的表达由名称为ATX1的启动子驱动，所述ATX1为(b1)、(b2)或(b3)：
    (b1)序列表中SEQ ID No.4的第6-930位所示的DNA分子；
    (b2)将(b1)经过一个或几个核苷酸的取代和/或缺失和/或添加且与(b1)具有75％或75％以上同一性的单链DNA分子；
    (b3)在严格条件下与(b1)或(b2)限定的核苷酸序列杂交的DNA分子。
  3. 根据权利要求1或2所述的方法，其特征在于：所述生物细胞为微生物细胞。
  4. 根据权利要求3所述的方法，其特征在于：所述微生物细胞为真菌。
  5. 根据权利要求4所述的方法，其特征在于：所述真菌为酵母。
  6. 根据权利要求1或2所述的方法，其特征在于：所述蛋白质为如下A1)、A2)或A3)：
    A1)氨基酸序列是SEQ ID No.3的蛋白质；
    A2)将序列表中SEQ ID No.3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/或添加且具有β-甘露聚糖酶功能的蛋白质；
    A3)在A1)或A2)的N端或/和C端连接标签得到的融合蛋白质。
  7. 根据权利要求6所述的方法，其特征在于：所述生物细胞为微生物细胞。
  8. 根据权利要求7所述的方法，其特征在于：所述微生物细胞为真菌。
  9. 根据权利要求8所述的方法，其特征在于：所述真菌为酵母。
  10. 利用权利要求1-9中任一所述方法制备的具有β-甘露聚糖酶活性的蛋白质。
  11. 下述任一产品：
    X1、权利要求1-9中任一所述重组细胞；
    X2、权利要求1中所述rMan26基因；
    X3、权利要求2中所述ATX1；
    X4、由权利要求1中所述rMan26基因和权利要求2中所述ATX1组成的成套试剂；
    X5、含有权利要求1中所述rMan26基因和/或权利要求2中所述ATX1的表达盒；
    X6、含有权利要求1中所述rMan26基因和/或权利要求2中所述ATX1的重组载体。
  12. 权利要求1-9中任一所述蛋白质的下述任一应用：
    a1)作为β-甘露聚糖酶的应用；
    a2)在制备有β-甘露聚糖酶活性产品中的应用；
    a3)在制备甘露寡糖中的应用；
    a4)在制备用于制备甘露寡糖产品中的应用；
    a5)在降解甘露聚糖中的应用；
    a6)在制备用于降解甘露聚糖产品中的应用；
    a7)在制备部分水解甘露聚糖中的应用；
    a8)在制备用于制备部分水解甘露聚糖产品中的应用；
    a9)在降解植物胶中的应用；
    a10)在制备用于降解植物胶产品中的应用。
  13. 根据权利要求12所述的应用，其特征在于：所述植物胶为b1)或b2)或b3)或b4)或b5)：b1)槐豆胶；b2)决明子胶；b3)香豆胶；b4)魔芋粉；b5)富含甘露聚糖的植物胶。
  14. 权利要求11所述产品的下述任一应用：
    a2)在制备有β-甘露聚糖酶活性产品中的应用；
    a3)在制备甘露寡糖中的应用；
    a4)在制备用于制备甘露寡糖产品中的应用；
    a5)在降解甘露聚糖中的应用；
    a6)在制备用于降解甘露聚糖产品中的应用；
    a7)在制备部分水解甘露聚糖中的应用；
    a8)在制备用于制备部分水解甘露聚糖产品中的应用；
    a9)在降解植物胶中的应用；
    a10)在制备用于降解植物胶产品中的应用。
  15. 根据权利要求14所述的应用，其特征在于：所述植物胶为b1)或b2)或b3)或b4)或b5)：b1)槐豆胶；b2)决明子胶；b3)香豆胶；b4)魔芋粉；b5)富含甘露聚糖的植物胶。
  16. 利用权利要求1-9中任一所述蛋白质或权利要求11所述产品制备的部分水解甘露聚糖。
  17. 利用权利要求1-9中任一所述的蛋白质制备部分水解甘露聚糖在制作益生元酸奶中的应用。
  18. 根据权利要求17所述的应用，其特征在于：生产所述部分水解甘露聚糖的植物胶为c1)或c2)或c3)或c4)或c5)：c1)槐豆胶；c2)决明子胶；c3)香豆胶；c4)魔芋粉；c5)富含甘露聚糖的植物胶。
  19. 根据权利要求17或18所述的应用，其特征在于：所述益生元酸奶制备中还包括向体系中添加嗜热链球菌(Streptococcus thermophilus)和保加利亚乳杆菌(Lactobacillus bulgaricus)。
  20. 制作益生元酸奶的方法，包括：利用权利要求1-9中任一所述的蛋白质或权利要求11所述产品制作益生元酸奶。
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