CN115216460A - Preparation method and application of Mucor miehei glycoside hydrolase 12 family xyloglucanase - Google Patents

Abstract

The invention discloses a preparation method and application of a 12 family xyloglucanase of Rhizomucor militaris glycoside hydrolase. In the present invention, the FLD1 gene of Pichia pastoris and the Sso1 gene of Saccharomyces cerevisiae are co-expressed with the target protein RmXEG12A to construct a Pichia pastoris engineering bacterium producing xyloglucanase, and to improve the secretion and expression level of RmXEG12A. RmXEG12A is fermented at high density in a 5L fermenter, and the enzyme activity of the fermentation broth can reach 25700U/mL. The protein RmXEG12A provided by the invention has good hydrolysis characteristics, and can efficiently hydrolyze tamarind powder and tamarind gum rich in xyloglucan to generate xyloglucan oligosaccharides with different degrees of polymerization. Water holding capacity, improve the quality of yogurt. Therefore, the xyloglucanase RmXEG12A obtained by the present invention has important application potential and development prospect in the food industry.

Description

A kind of preparation method of Rhizomucor militaris glycoside hydrolase 12 family xyloglucanase and application

technical field

The invention relates to the field of biotechnology, in particular to a preparation method and application of a 12 family xyloglucanase of Rhizomucor miehei glycoside hydrolase.

Background technique

Xyloglucan is a type of main chain formed by D-glucopyranose residues connected by β-1,4-glycosidic bonds, and 60%-75% of the glucose residues O-6 on the main chain are α-D-pyridine residues. Long-chain polysaccharides formed by the substitution of xylanose. Usually, the xylose group of the xyloglucan side chain is further substituted by β-D-1,2-galactose and α-D-1,2-fucose, and sometimes a very small amount is also present in xyloglucan Arabic candy. Xyloglucan mainly contains glucose, xylose and galactose with a residue ratio of about 4:3:1 (Kiefer et al. Carbohydrate Research, 1990, 197: 139-158). Due to the complex side chain of xyloglucan, a systematic nomenclature is generally used to distinguish each unit of xyloglucan polymer (Fry et al. Physiologia Plantarum, 1993, 89: 1-3). Glucose residues not linked to xylose are represented by G, glucose residues linked to a xylose are represented by X, X to xylose to a galactose is represented to L, and to galactose to a fructose to L Represented by F. Xyloglucan is a hemicellulose that exists in the primary cell wall, intercellular layer and glial layer of higher plants, and is one of the main components of the primary cell wall of plants. Xyloglucan accounts for about 20-25% of the dry weight in the primary wall of dicots and about 2-5% in monocots and grasses (Benko et al. Enzyme and Microbial Technology, 2008, 43:109 -114).

Xyloglucanase (EC3.2.1.151) is a key enzyme in the hydrolysis of xyloglucan, which can randomly hydrolyze the β-1,4-glycosidic bonds in the main chain of xyloglucan to generate xyloglucan oligosaccharides (Menon et al. al. Journal of Biotechnology 2010, 148:233-239). Xyloglucanase is widely distributed in nature. Xyloglucanase is found in plants, animals and microorganisms, and microorganisms are the main source of xyloglucanase. Microbial production of xyloglucanase has many advantages, such as simple conditions for enzyme production, large amount of enzyme production, stable source, convenient extraction, and diverse enzymatic properties. Therefore, microbial-derived xyloglucanase is widely used in food, washing and other industries (Rashmi and Siddalingamurthy. Biocatalysis and Biotransformation, 2018, 36: 280-295).

Based on amino acid sequence homology, xyloglucanases can be divided into families of glycoside hydrolases 5, 12, 16, 44 and 74. Several xyloglucanases have been used to prepare xyloglucan oligosaccharides, such as those from Clostridium thermocellum (Martinez-Fleites et al. Journal of Biological Chemistry, 2006, 281:24922-24933) and Thermomonas Xyloglucanases of the genus Thermomonospora sp. can produce xyloglucan oligosaccharides (Pol et al. Extremophiles, 2012, 16:135-146). In order to meet the needs of industrial production, some xyloglucanase genes have been successfully expressed in yeast, but the expression level is still low, such as the enzyme activity of Myceliophthorathermophila VKPM F-244 xyloglucanase is 118U/ mL (Berezina et al. Applied Microbiology and Biotechnology, 2017, 101: 5653-5666) and Aspergillus cervinus xyloglucanase activity was 1000 U/mL (Rykov et al. Applied Microbiology and Biotechnology, 2019, 103: 7553-7566). Therefore, increasing the expression level of xyloglucanase is of great significance to its industrial production and application.

As a prebiotic, functional oligosaccharides can regulate intestinal microflora, improve immunity and antioxidant capacity, and have great application potential in food and feed industries (Zuniga et al. Current Issues in Molecular Biology, 2021 , 40:49-80; Rao Shiting et al. Feed Research, 2020, 9:129-131). At present, xyloglucan oligosaccharides are mainly prepared by chemical synthesis, acid hydrolysis and enzymatic hydrolysis. The enzymatic preparation of xyloglucose oligosaccharide 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 xyloglucose oligosaccharide. The raw materials for the preparation of xyloglucan oligosaccharides are mainly xyloglucan-rich plants. At present, most of the xyloglucan used in research and industrialization comes from tamarind. Among them, the raw materials commonly used to prepare xyloglucan are tamarind gum and tamarind powder, but the raw material concentration used during hydrolysis is low (≤10%) (Martinez-Fleites et al. Journal of Biological Chemistry, 2006, 281: 24922-24933; Satoh et al. Journal of the Japanese Society for Horticultural Science, 2013, 82: 270-276; Pol et al. Extremophiles, 2012, 16: 135-146; Matsuzawa et al. Applied Microbiology and Biotechnology, 2020, 104 :8761-8773). Tamarind gum is widely used in food, but xyloglucose oligosaccharide is mostly used to regulate plant growth and is less used in food (Busato et al. Phytochemistry, 2001, 58: 525-531; Mishra et al. Journal of Materials Chemistry, 2009, 19:8528-8536; Satoh et al. Journal of the Japanese Society for Horticultural Science, 2013, 82:270-276). Chinese Patent Application No. CN201811546164.9 discloses a method for making ready-to-eat composite natural nutritional jelly. Adding 0.5%-0.6% tamarind gum to the raw materials can improve the nutritional value of the jelly and increase the experience of consumers. Chinese Patent Application No. CN201810702200.X discloses a method for preparing instant black tea. Adding 35-45 parts of tamarind gum (the main ingredient is in parts by weight) into the raw materials can improve the aroma of the instant tea. Therefore, using xyloglucanase to hydrolyze raw materials rich in xyloglucan to produce xyloglucan oligosaccharides can enrich the functional oligosaccharide market in my country and has broad development prospects.

Critical Reviewsin Food Science and Nutrition,2020；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(Meybodi et al.International Dairy Journal,2020,109:104793；Cruz et al.Journal of Food Engineering,2013,114:323-330；Das etal.LWT-Food Science and Technology,2019,108:69-80；Fazilah et al.Journal ofFunctional 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，符合现代人对酸奶的消费要求。中国专利申请号CN201910267919.X中公开了一种益生元酸奶(石花菜酸奶)的制作方法，在复原乳中添加10-30份(w/v)石花菜琼胶寡糖，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。其中添加琼胶寡糖还具有抑菌和防腐作用，有利于酸奶的贮藏和运输。目前，尚无利用木葡寡糖制作益生元酸奶的报道。Yogurt is a dairy product made from raw cow (goat) milk, pasteurized and then inoculated with Streptococcus thermophilus and Lactobacillus bulgaricus. Yogurt has a thick texture, silky, delicate and smooth taste, and has the advantages of optimizing intestinal flora, adjusting intestinal function, improving immunity, regulating blood sugar and blood lipids (Baspinar and

Critical Reviews in Food Science and Nutrition, 2020; Donkor et al. International Dairy Journal, 2007, 17: 1321-1331). Adding prebiotics to yogurt to prepare prebiotic yogurt can improve the physicochemical properties of yogurt and improve the functional activity of yogurt, which has received extensive attention (Meybodi et al. International Dairy Journal, 2020, 109: 104793; 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). Chinese Patent Application No. CN201811327958.6 discloses a preparation method of oligosaccharide brown yogurt, adding 1-3g/L xylo-oligosaccharide, 15-22g/L fructooligosaccharide, 18-26g/L low The polyisomaltose is made into prebiotic yogurt, which is rich in nutrients, beneficial to human health, and meets the consumption requirements of modern people for yogurt. Chinese Patent Application No. CN201910267919.X discloses a preparation method of prebiotic yogurt (agaricus yogurt), adding 10-30 parts (w/v) of agaric oligosaccharide to the reconstituted milk, and then fermenting the inoculated bacteria to obtain Prebiotic yogurt, which can meet the dual needs of consumers for taste and health, is easily accepted by consumers. The addition of agar oligosaccharide also has bacteriostatic and antiseptic effects, which is beneficial to the storage and transportation of yogurt. At present, there is no report on the use of xyloglucose oligosaccharide to make prebiotic yogurt.

Rhizomucor miehei is a thermophilic filamentous fungus that can secrete a variety of glycoside hydrolases, and the xyloglucanase it secretes has a variety of excellent enzymatic properties (Song et al. Applied Microbiology and Biotechnology, 2013, 97 : 10013-10024).

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a preparation method and application of a rhizomucor niger glycoside hydrolase 12 family xyloglucanase.

In a first aspect, the present invention claims a method of producing a xyloglucanase.

The method for producing xyloglucanase as claimed in the present invention may include the following steps:

(A) The gene encoding the xyloglucanase XEG12A derived from Rhizomucor miehei CAU432, the gene encoding the FLD1 protein derived from Pichia pastoris, and the gene encoding the Sso1 protein derived from Saccharomyces cerevisiae were introduced into the recipient Pichia pastoris , to obtain recombinant Pichia pastoris.

Among them, Rhizomucor miehei CAU432 has been preserved in the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC for short, address: No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, zip code 100101) . Deposit date: June 21, 2011. The deposit number is CGMCC No.4967.

(B) fermenting and culturing the recombinant Pichia pastoris according to the following steps to obtain xyloglucanase XEG12A from the fermentation product:

B1) basal fermentation culture: the recombinant Pichia pastoris is inoculated in the BSM medium and cultivated, and the glycerol-supplemented fermentation stage is carried out completely when the glycerol is consumed;

B2) glycerol feeding fermentation stage: on the basis of step B1), glycerol is added to the fermentation system at a speed of 15-30mL/h/L (such as 30mL/h/L) starting fermentation broth, and the cells are treated When the wet weight reaches 180-220g/L (such as 200g/L), stop the flow of glycerol, starve it for 0.5h-1h (such as 0.5h), and then carry out the methanol feeding induction stage;

B3) Methanol feeding induction stage: on the basis of step B2), methanol flow was added to the fermentation system, and the flow rate was increased from 3.6mL/h/L initial fermentation broth to 10.9mL/h/L within 4h The fermentation broth was started, and then methanol was added to the fermentation broth at a rate of 10.9 mL/h/L until the fermentation was completed.

In step (A), the xyloglucanase XEG12A derived from Rhizomucor miehei CAU432 is any of the following proteins:

a1) The protein of SEQ ID No.1 consisting of amino acid residues from 21 to 242 of the N-terminal (the 1-20 positions of SEQ ID No. 1 are signal peptides);

a2) a protein consisting of the amino acid sequence shown in SEQ ID No.1;

a3) a protein with xyloglucanase activity obtained by subjecting the protein described in a1) or a2) to substitution and/or deletion and/or addition of one or several amino acid residues;

a4) A protein with more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the amino acid sequence defined in any of a1)-a3) and having xyloglucanase activity;

a5) A fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the proteins as defined in any of a1)-a4).

In step (A), the FLD1 protein derived from Pichia pastoris is any of the following proteins:

b1) a protein consisting of the amino acid sequence shown in SEQ ID No.2;

b2) a protein with the same activity obtained by substituting and/or deleting and/or adding one or more amino acid residues to the protein described in b1);

b3) A protein with more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology and the same activity as the amino acid sequence defined in any of b1)-b2);

b4) A fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of any of the proteins defined in b1)-b3).

In step (A), the Sso1 protein derived from Saccharomyces cerevisiae is any of the following proteins:

c1) a protein consisting of the amino acid sequence shown in SEQ ID No.3;

c2) a protein with the same activity obtained by substituting and/or deleting and/or adding one or more amino acid residues to the protein described in c1);

c3) A protein with more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology and the same activity as the amino acid sequence defined in any of c1)-c2);

c4) A fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of any of the proteins defined in c1)-c3).

In the above proteins, the tag refers to a polypeptide or protein that is fused and expressed with the target protein by using DNA recombination technology in vitro, so as to facilitate the expression, detection, tracking and/or purification of the target protein. The protein tag can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag and/or SUMO tag and the like.

In the above proteins, identity refers to the identity of amino acid sequences. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as the program, set the Expect value to 10, set all Filters to OFF, use BLOSUM62 as the Matrix, and set the Gap existence cost, Per residue gap cost and Lambda ratio to be respectively 11, 1 and 0.85 (default value) and search for the identity of a pair of amino acid sequences to calculate the identity value (%).

In the above proteins, the homology of more than 95% may be at least 96%, 97%, 98% identical. The above 90% homology may be at least 91%, 92%, 93%, 94% identical. The above 85% homology may be at least 86%, 87%, 88%, 89% identical. The above 80% homology may be at least 81%, 82%, 83%, 84% identical.

In step (A), the gene encoding the xyloglucanase XEG12A derived from Rhizomucor miehei CAU432 is introduced into the recipient Pichia in the form of recombinant vector 1 .

Further, the recombinant vector 1 is a recombinant plasmid obtained by inserting the encoding gene of the xyloglucanase XEG12A derived from Rhizomucor miehei CAU432 into the multiple cloning sites (eg EcoRI and NotI) of the pPIC9K vector.

In step (A), the gene encoding the FLD1 protein derived from Pichia pastoris is introduced into the recipient Pichia pastoris in the form of recombinant vector 2 .

Further, the recombinant vector 2 is a recombinant plasmid obtained by inserting the gene encoding the FLD1 protein derived from Pichia pastoris into the multiple cloning sites (eg, EcoRI and NotI) of the pPICZA vector.

In step (A), the gene encoding the Sso1 protein derived from Saccharomyces cerevisiae is introduced into the recipient Pichia in the form of recombinant vector 3 .

Further, the recombinant vector 3 is a recombinant plasmid obtained by inserting the encoding gene of the Sso1 protein derived from Saccharomyces cerevisiae into the multiple cloning sites (eg EcoRI and NotI) of the pGAPZB vector.

In the present invention, the Pichia pastoris is specifically Pichia pastoris GS115.

In step (A), the encoding gene of the xyloglucanase XEG12A derived from Rhizomucor miehei CAU432 can be a DNA molecule as shown in any of the following:

d1) the DNA molecule shown at positions 61-729 from the 5' end of SEQ ID No. 4 (positions 1-60 of SEQ ID No. 4 are the signal peptide coding sequence);

d2) the DNA molecule shown in SEQ ID No.4;

d3) a DNA molecule that hybridizes under stringent conditions to a DNA molecule defined in d1) or d2) and encodes the xyloglucanase XEG12A derived from Rhizomucor miehei CAU432;

d4) has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the DNA sequence defined in any of d1)-d3) and encodes the DNA sequence derived from Rhizomucor miehei CAU432. DNA molecule of xyloglucanase XEG12A.

In step (A), the coding gene of the FLD1 protein derived from Pichia pastoris can be any of the following DNA molecules:

e1) DNA molecule shown in SEQ ID No.5;

e2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in e1) and encodes the Pichia-derived FLD1 protein;

e3) has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the DNA sequence defined in any of e1)-e2) and encodes the Pichia-derived FLD1 protein DNA molecule.

In step (A), the coding gene of the Sso1 protein derived from Saccharomyces cerevisiae can be any of the following DNA molecules:

f1) DNA molecule shown in SEQ ID No.6;

f2) a DNA molecule that hybridizes to the DNA molecule defined in f1) under stringent conditions and encodes the Sso1 protein derived from Saccharomyces cerevisiae;

f3) has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the DNA sequence defined in any of f1)-f2) and encodes the Sso1 protein derived from Saccharomyces cerevisiae DNA molecule.

In the above DNA molecule, the stringent conditions may be as follows: 50°C, hybridization in a mixed solution of 7% sodium dodecyl _sulfate (SDS), 0.5M Na3PO4 and ₁ mM EDTA, at 50°C, 2 ×SSC, rinsed in 0.1% SDS; also: 50°C, hybridized in a mixed solution of ₇ % SDS, 0.5M Na3PO4 and 1mM EDTA, rinsed at 50°C, ₁ ×SSC, 0.1% SDS; Also: 50°C, hybridize in a mixed solution of 7% SDS, 0.5M Na ₃ PO ₄ and 1 mM EDTA, rinse at 50° C, 0.5×SSC, 0.1% SDS; also: 50° C, 7% Hybridize in a mixed solution of % SDS, 0.5M Na ₃ PO ₄ and 1 mM EDTA, rinse in 0.1×SSC, 0.1% SDS at 50°C; also: 50° C, in 7% SDS, 0.5M Na ₃ PO ₄ Hybridize with a mixed solution of 1mM EDTA, rinse in 0.1×SSC, 0.1% SDS at 65°C; alternatively: hybridize in a solution of 6×SSC, 0.5% SDS at 65°C, then use 2×SSC , 0.1% SDS and 1×SSC, 0.1% SDS to wash the membrane once.

In the above DNA molecules, identity refers to the identity of nucleotide sequences. Nucleotide sequence identity can be determined using homology search sites on the Internet, such as the BLAST page of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as the program, set the Expect value to 10, set all Filters to OFF, use BLOSUM62 as the Matrix, and set the Gap existence cost, Per residue gap cost and Lambda ratio to be respectively 11, 1 and 0.85 (default value) and search for the identity of a pair of nucleotide sequences to calculate the identity value (%).

In the above DNA molecules, the 95% or more homology may be at least 96%, 97%, 98% identical. The above 90% homology may be at least 91%, 92%, 93%, 94% identical. The above 85% homology may be at least 86%, 87%, 88%, 89% identical. The above 80% homology may be at least 81%, 82%, 83%, 84% identical.

In step B1), the recombinant Pichia pastoris inoculated in the BSM medium is the seed liquid of the recombinant Pichia pastoris.

Further, the seed liquid is prepared according to a method comprising the following steps: inoculating the recombinant Pichia pastoris obtained in step (A) in BMGY medium, and culturing with shaking at 30° C. and 200 rpm until the absorbance value of the fermentation broth at 600 nm reaches 2. -6 (eg, the absorbance value at 600nm is 4).

Wherein, the composition of the BMGY medium is as follows: the mass percentage concentration is 1% yeast extract, the mass percentage concentration is 2% peptone, the mass percentage concentration is 1.34% amino-free yeast nitrogen source YNB, and the mass percentage concentration is 4 x ^10-5 % biotin, 1% glycerol by volume, balance 100 mM pH 6.0 phosphate buffer.

In step B1), inoculating the seed liquid in the BSM medium is to inoculate in the fermentor containing the BSM medium, and the liquid filling amount of the BSM medium in the fermentor is the 1.5/5 of the volume of the fermenter.

In step B1), inoculating the seed liquid in the BSM medium is to inoculate in the fermenter equipped with the BSM medium (the inoculation amount is that the seed liquid is 1/10 volume of the BSM medium. ), the liquid filling amount of the BSM medium in the fermenter is 1.5/5 of the volume of the fermenter (eg, a 5-L fermenter is filled with 1.5L of the BSM medium).

In step B1), the composition of the BSM is as follows: each 1 L of the BSM medium contains 26.7 mL of phosphoric acid with a concentration of 85 g/100 mL, 40 g of glycerol, 0.93 g of CaSO ₄ , 18.2 of K ₂ SO ₄ , MgSO ₄ ·7H ₂ O 14.9g, KOH 4.13g, balance water.

In step B1), the step of adding PTM1 solution (Pichia pastoris trace metal salt solution) is also included.

Further, the amount of the PTM1 solution added is 4.35 mL of the PTM1 solution per 1 L of the initial fermentation broth.

The solvent of the PTM1 solution is water, and the solute and concentration are as follows: CuSO ₄ ·7H ₂ O 6.0g/L, NaI 0.08g/L, MnSO ₄ ·H ₂ O 3.0g/L, Na ₂ MoO ₄ ·2H ₂ O 0.2 g/L, boric acid 0.02g/L, CoCl ₂ 0.5g/L, ZnCl ₂ 20.0g/L, FeSO ₄ ·7H ₂ O 65.0g/L, biotin 0.2g/L, concentrated sulfuric acid 5.0mL/L.

In step B1), the temperature of the culture is 30° C., the pH is 4.0, and the rotation speed is 600 rpm.

In step B2), the glycerol is an aqueous glycerol solution of 500 g/L formed by being dissolved in water; the culture temperature of the glycerol-supplemented fermentation stage is 30° C., the pH is 4.0, and the dissolved oxygen is controlled to be greater than 20% (fermentation time 4). -6h).

In step B3), the methanol is anhydrous methanol; the culture temperature of the methanol feeding induction expression stage is 30° C., the pH is 6.0, the dissolved oxygen is controlled to be greater than 20%, and the fermentation time is 6-7 days (such as 7 days) .

In a specific embodiment of the present invention, the time of the fermentation and cultivation in step (B) is specifically 168 hours (ie, 7 days).

In step (B), after fermenting and culturing the recombinant Pichia pastoris, it also includes the following steps: performing dialysis and desalting on the supernatant of the fermentation broth (the molecular weight cut-off of the used dialysis bag is 3.5kDa), and then performing an anion exchange layer. The purified xyloglucanase XEG12A was collected by linear elution with 20 mM pH 7.0 MOPS buffer containing 0-500 mM NaCl.

Further, the anion exchange chromatography is Q-Sepharose FF (Q-Sepharose FF) ion exchange chromatography.

The elution procedure for linear elution with 20 mM pH 7.0 MOPS buffer containing 0-500 mM NaCl is as follows: the concentration of NaCl in the eluate increases linearly from 0 to 500 mM within 50 min.

The anion exchange chromatography (Q-Sepharose FF, Q-Sepharose FF ion exchange chromatography) was performed with column equilibration using 20 mM MOPS buffer pH 7.0. When performing the anion exchange chromatography (Q-Sepharose FF, Q-Sepharose FF ion exchange chromatography), the sample loading flow rate was 0.5 mL/min. After loading, 5 column volumes were eluted with 20 mM MOPS buffer pH 7.0, followed by linear elution with 20 mM MOPS buffer pH 7.0 containing 0-500 mM NaCl. The elution flow rate was 1 mL/min.

After performing the anion exchange chromatography (Q-Sepharose FF, Q-Sepharose FF ion exchange chromatography), it also includes the step of collecting the eluate with xyloglucanase activity, and collecting the eluate with enzyme activity. The step of dialysis again against 20 mM MOPS buffer pH 7.0 after dehydration and pooling.

In the second aspect, the present invention claims the xyloglucanase XEG12A prepared by the method described in the first aspect above.

In a third aspect, the present invention claims recombinant Pichia pastoris.

The recombinant Pichia pastoris claimed in the present invention is the recombinant Pichia pastoris described in step (A) in the first aspect above.

In the fourth aspect, the present invention claims to be applied as shown in any of the following:

P1, the application of the xyloglucanase XEG12A described in the second aspect above in the preparation of xyloglucan oligosaccharides;

P2, the application of the xyloglucanase XEG12A described in the second aspect above in the enzymatic hydrolysis of xyloglucan-rich plants;

P3, the application of the xyloglucanase XEG12A described in the second aspect above in the preparation of a product for enzymatic hydrolysis of a plant rich in xyloglucan;

P4, the application of the xyloglucanase XEG12A described in the second aspect above in the preparation of prebiotic yogurt;

P5, the application of the xyloglucanase XEG12A of claim 7 in food industry;

P6. Use of the recombinant Pichia pastoris described in the third aspect above in the production of the xyloglucanase XEG12A described in the second aspect above.

Further, in P1, the xyloglucose oligosaccharide is a xyloglucose oligosaccharide obtained by polymerizing 2-10 pieces of glucose, xylose and/or galactose.

Further, in P1, the preparation of xyloglucan oligosaccharide uses xyloglucan or a plant rich in xyloglucan (including plant tissue or organ) as a substrate.

Further, the plant is tamarind, apple and/or blueberry (including tissues or organs of these plants).

In the fifth aspect, the present invention claims any of the following methods or utilizes the method to prepare the resulting product:

Q1, a method for hydrolyzing tamarind powder, comprising the steps of: hydrolyzing tamarind powder with the xyloglucanase XEG12A described in the second aspect above, and the ratio of the tamarind powder and the xyloglucanase XEG12A is 1g of tamarind powder: 200-1000U of the xyloglucanase XEG12A (eg 1g of tamarind powder: 500U of the xyloglucanase XEG12A), hydrolyzed at 30-60°C (eg 55°C) for 8h, after the hydrolysis is complete , take the reaction system in a boiling water bath (eg, 20min), centrifuge (eg, 11510g for 10min) to take the supernatant to obtain the following three high molecular weight xyloglucan oligosaccharides: XXXG, XXLG (XLXG) and XLLG.

Further, the tamarind powder is dissolved in MOPS buffer or water.

Q2. A method for hydrolyzing tamarind gum, comprising the steps of: hydrolyzing tamarind gum with the xyloglucanase XEG12A described in the second aspect above, and the ratio of the tamarind gum and the xyloglucanase XEG12A is: 1g tamarind gum: 200-1000U xyloglucanase XEG12A (eg 1g tamarind gum: 500U xyloglucanase XEG12A), hydrolyze at 30-60°C (eg 55°C) for 8h, after the hydrolysis is completed , take the reaction system in a boiling water bath (eg, 20min), centrifuge (eg, 11510g for 10min) to take the supernatant to obtain the following three high molecular weight xyloglucan oligosaccharides: XXXG, XXLG (XLXG) and XLLG.

Further, the tamarind gum is dissolved in MOPS buffer or water.

Q3, a Lactobacillus coryneformis NRRL B-4391, Lactobacillus brevis NRRL B-4527, Lactobacillus casei AS1.62, Lactobacillus delbrueckii NRRL B-548, Lactobacillus rhamnosus AS 1.2466 and/or Bifidobacterium bifidum A method for proliferating Fidobacteria NRRL B-41410, comprising the steps of: producing xyloglucan oligosaccharide with the xyloglucanase XEG12A described in the second aspect, and then using the xyloglucan oligosaccharide to promote Lactobacillus coryneformis NRRL B-4391 , Lactobacillus brevis NRRL B-4527, Lactobacillus casei AS1.62, Lactobacillus delbrueckii NRRL B-548, Lactobacillus rhamnosus AS 1.2466 and/or Bifidobacterium bifidum NRRL B-41410 were propagated.

Q4. A method for preparing prebiotic yogurt, comprising the steps of: producing xyloglucan oligosaccharide with the xyloglucanase XEG12A described in the second aspect, and then utilizing the xyloglucan oligosaccharide, fresh milk, sucrose and Prebiotic yogurt was prepared by Streptococcus thermophilus and Lactobacillus bulgaricus.

Further, the method includes the steps of: taking the fresh milk whose mass percentage is 93-91% (such as 92.5-91%, then 92-91%, and further such as 91.5-91%), and the mass percentage is 0-2% (excluding 0 points) (such as 0.5-2%, such as 1-2%, further such as 1.5-2%) of the xyloglucose oligosaccharide and 7% sucrose by mass (the sum of the three mass percentages is 100%) mixed, sterilized (such as pasteurization) and then inoculated with the Streptococcus thermophilus (Streptococcus thermophilus) and the Lactobacillus bulgaricus (Lactobacillus bulgaricus). It is 1×10 ⁶ CFU/mL, and then fermentation, demulsification and chilling, and refrigerating and post-cooking are carried out in sequence to obtain the prebiotic yogurt.

Further, the fermentation conditions are fermentation at 42° C. for 4 hours (pH is about 4.6). The demulsification and cooling are as follows: demulsification at 350 rpm for 1 min, and cooling to 20°C. The refrigerated post-cooking is: 4° C. post-cooking for 16 hours.

In Q3 and Q4, the method for producing xyloglucan oligosaccharide with the xyloglucanase XEG12A described in the second aspect above is Q1 or Q2.

In the present invention, the FLD1 gene of Pichia pastoris and the Sso1 gene of Saccharomyces cerevisiae are co-expressed with the target protein RmXEG12A to construct a Pichia pastoris engineering bacterium producing xyloglucanase, and to improve the secretion and expression level of RmXEG12A. RmXEG12A is fermented at high density in a 5L fermenter, and the enzyme activity of the fermentation broth can reach 25700U/mL. The optimum pH of xyloglucanase is 7.0, and the optimum temperature is 65° C. The protein RmXEG12A provided by the present invention has good hydrolysis characteristics, and can efficiently hydrolyze tamarind powder and tamarind gum rich in xyloglucan to generate different Xyloglucose oligosaccharide of degree of polymerization. Xyloglucoside can promote Lactobacillus coryneformis NRRL B-4391, Lactobacillus brevis NRRL B-4527, Lactobacillus casei AS1.62, Lactobacillus delbrueckii NRRL B-548, Lactobacillus rhamnosus AS 1.2466 and Bifidobacterium bifidum Proliferation of Bacillus NRRL B-41410. The prebiotic yogurt is prepared by adding xyloglucose oligosaccharide to the yogurt, which increases the water holding capacity of the yogurt, increases the viable counts of Lactobacillus and Streptococcus thermophilus, and improves the quality of the yogurt. Therefore, the xyloglucanase RmXEG12A has important application potential and development prospects in the food industry.

Description of drawings

Figure 1 is the vector map of the recombinant vector pPIC9k-RmXEG12A.

Figure 2 is a vector map of the recombinant vector pPICZA-FLD1.

Figure 3 is the vector map of the recombinant vector pGAPZB-Sso1.

FIG. 4 is the enzyme production process diagram (A) and the SDS-PAGE electrophoresis diagram (B) of xyloglucanase in high-density fermentation in a 5L fermenter. Among them (■) enzyme activity, (▲) protein concentration, (●) wet weight of bacteria. Lane M: low molecular weight standard protein; lanes 1-8: fermentation supernatants induced for 0, 24, 48, 72, 96, 120, 144, and 168 h, respectively.

Figure 5 is a diagram of the purification of xyloglucanase, wherein the lane marked 1 is the crude enzyme solution, and the lane marked 2 is the pure enzyme solution.

Figure 6 is a graph showing the optimal pH value determination curve of recombinant protein RmXEG12A as xyloglucanase. Among them (▲) citrate buffer (pH 3.0-5.5), (◆) MES (pH 5.0-6.5), (■) MOPS (pH 6.0-8.0), (△) Tris-HCl (pH 7.0-9.0), (□) Gly-NaOH (pH 8.5-11.0).

Figure 7 is a graph showing the pH stability assay of recombinant protein RmXEG12A as xyloglucanase. Among them (▲) citrate buffer (pH 3.0-5.5), (◆) MES (pH 5.0-6.5), (■) MOPS (pH 6.0-8.0), (△) Tris-HCl (pH 7.0-9.0), (□) Gly-NaOH (pH 8.5-11.0).

Figure 8 is a graph showing the optimal temperature determination curve of recombinant protein RmXEG12A as xyloglucanase.

Figure 9 is a graph showing the temperature stability assay of recombinant protein RmXEG12A as xyloglucanase.

Figure 10 is the thin-layer chromatography analysis chart of recombinant protein RmXEG12A as xyloglucanase hydrolyzed tamarind powder (G1: glucose; G2: cellobiose; G3: cellotriose; G4: cellotetraose; xyloglucose oligosaccharide Mixture of XXXG, XXLG/XLXG and XLLG standards), of which 1 is hydrolyzed tamarind powder.

Figure 11 is a thin-layer chromatography analysis chart of recombinant protein RmXEG12A as xyloglucanase hydrolysis of tamarind gum (G1: glucose; G2: cellobiose; G3: cellotriose; G4: cellotetraose; xyloglucose oligosaccharide XXXG, a mixture of XXLG/XLXG and XLLG standards), where 1 is hydrolyzed tamarind gum.

Detailed ways

The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product specification. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

The experiments in the following examples have been set up to repeat the experiments three times, and the results are the average of the results of the three experiments.

The enzyme activity of xyloglucanase was determined by 3,5-dinitrosalicylic acid (DNS) method (Miller, Analytical Chemistry, 1959, 31:426-428), and the specific determination steps were as follows: (1) Take 100 μL of 0.5% ( mass volume fraction, ie 0.5 g/100 mL) xyloglucan (tamarind seed) solution (prepared with pH 7.0, 50 mM MOPS buffer), then 50 μL of 50 mM pH 7.0 MOPS buffer was added and mixed. Preheat the reaction at 65 °C for 3 min, add 50 μL of the enzyme solution to be tested appropriately diluted, and place it in a constant temperature water bath at 65 °C for 10 min; (2) After completing the reaction in step (1), use 200 μL of DNS reagent (1% 3,5-dinitrogen) Base salicylic acid, 1% NaOH and 0.2% phenol, both are mass volume fractions, that is, % means 1g/100mL), terminate the reaction and react with the released reducing sugar, add 200 μL saturated potassium sodium tartrate solution after boiling for 15min, and after cooling Absorbance was measured at a wavelength of 540 nm with glucose as the standard. Taking glucose as the mark, the activity unit of xyloglucanase is defined as: under the above reaction conditions, the amount of enzyme required to generate 1 μmol of glucose per minute is one unit of enzyme activity (1U).

Specific enzyme activity is defined as the unit of enzyme activity per mg protein (U/mg).

Example 1. Preparation of recombinant xyloglucanase

1. PCR amplification of the xyloglucanase gene of Rhizomucor miehei

The coding region of the xyloglucanase gene JQ901459 is 729 bp, encoding 242 amino acids. The upstream primer RmXEG12AF and the downstream primer RmXEG12AR were designed respectively. The sequence is as follows:

RmXEG12AF: 5'-CTCAG GAATTC ATTCCACTTGAAAAGCGCGC-3' (the restriction endonuclease EcoRI cleavage site is underlined)

RmXEG12AR: 5'-TGACT GCGGCCGC TTAGTTGACAGAGACCGAGTAAGC-3' (the restriction endonuclease NotI cleavage site is underlined)

Using the cDNA of Rhizomucor miehei CAU432 (CGMCC No. 4967) as a template, PCR amplification of the coding gene sequence of the protein (the 61st-729th position of SEQ ID No.4, the 21st-729th position of coding SEQ ID No.1) The protein shown at position 242. Positions 1-20 of SEQ ID No. 1 are signal peptides, encoded by positions 1-60 of SEQ ID No. 4). The PCR reaction program was: pre-denaturation at 94 °C for 10 min; denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 50 s, 30 cycles; and extension at 72 °C for 5 min. PCR products were detected by 1% agarose gel electrophoresis. Recovery of PCR products.

2. Expression of recombinant xyloglucanase (RmXEG12A)

1. The PCR product and plasmid pPIC9k (product of Novagen) obtained by double digestion with EcoRI and NotI were carried out with T4 DNA ligase to obtain a recombinant vector, which was transferred into DH5α and coated on LB containing ampicillin (100 μg/mL). Plate (coated on LB Amp ⁺ plate), pick a single colony for colony PCR, sequence the colony with the correct band, and obtain a positive clone after comparison. The recombinant vector obtained after being verified to be correct by sequencing was named pPIC9k-RmXEG12A (Fig. 1).

2. Artificially synthesize the FLD1 gene of Pichia pastoris (SEQ ID No. 5, encoding the protein shown in SEQ ID No. 2), and by seamless cloning, the FLD1 gene and the vector pPICZA that has been double digested by EcoRI and NotI (Novagen Company) was connected, and the recombinant expression vector pPICZA-FLD1 was obtained after the sequence was verified to be correct (Fig. 2). In addition, the Sso1 gene of Saccharomyces cerevisiae (SEQ ID No. 6, encoding the protein shown in SEQ ID No. 3) was chemically synthesized and inserted into the pGAPZB vector (Novagen) between the EcoRI and NotI restriction sites, and the sequence was verified to be correct. Then the recombinant expression vector pGAPZB-Sso1 was obtained (Fig. 3).

3. The SalI linearized recombinant vector pPIC9k-RmXEG12A, the SacI linearized recombinant vector pPICZA-FLD1 and the AvrII linearized recombinant vector pGAPZB-Sso1 were recovered by alcohol precipitation, and then co-transformed by electric shock to Pichia GS115, and spread on MD/Zeocin ^TM plates , in a 30 ℃ incubator upside-down culture for 2-3d, until the colony grows so far, to obtain the recombinant bacteria.

4. Collect the recombinant bacterial cells with sterile water, and spread the appropriately diluted bacterial cells on the G418 screening plates with concentrations of 1 mg/mL, 2 mg/mL, 3 mg/mL, and 4 mg/mL, respectively, and select different G418 cells. A single colony that grows well under the concentration is inoculated in 5 mL of BMGY medium (recipe: yeast extract with a concentration of 1% by mass, peptone with a concentration of 2% by mass, and amino-free yeast with a concentration of 1.34% by mass). Nitrogen source YNB, 4× ^10-5 % biotin by mass, 1% glycerol by volume, and balance 100mM pH 6.0 phosphate buffer), placed in a shaker at 30°C, 200rpm, and shaken for culture , when the OD ₆₀₀ is 2-6, centrifuge, discard the supernatant, collect the cells and transfer them to 10 mL of BMMY medium (replace glycerol with 0.5% methanol by volume), and place them at 30°C, 200rpm in a shaker to induce expression . Methanol was added every 24h to a final concentration of 0.5% by volume, induced for 3d, and the enzyme activity of the fermentation broth was determined according to the aforementioned method. Results: The recombinant Pichia pastoris (named as recombinant bacteria A) with an enzyme production of up to 359 U/mL was obtained for subsequent high-density fermentation.

4. High-density fermentation

High density fermentation was carried out with reference to the method of the document "Picha Fermentation Process Guidelines (Version B, 053002, Invitrogen)". The fermenter is a 5L fermenter. Seed medium BMGY, basic medium BSM (85% phosphoric acid (85% phosphoric acid is analytical grade, % indicates g/100mL) 40 mL, CaSO ₄ 1.4 g, K ₂ SO ₄ 27.3 g, MgSO ₄ ·7H ₂ O 22.4 g, KOH 6.19g, glycerol 60g, add distilled water to 1.5L), glycerol fed-batch medium and 100% methanol induction medium were prepared according to the methods in the literature. The whole fermentation process is divided into four stages: seed liquid culture, basal culture, glycerol fed fermentation stage and 100% methanol fed induced expression stage. Specific steps are as follows:

1) Seed cultivation: Inoculate the recombinant bacterial A in step 3 into a 500 mL Erlenmeyer flask containing 150 mL of BMGY medium, and cultivate at 30° C. and 200 rpm shaker for more than 24 hours to obtain a seed solution with an OD ₆₀₀ of 4.

2) basal culture: inoculate the seed liquid in 1) into a 5L sterilized fermenter (1.5L fermentation basic medium BSM is housed), adjust the pH of the medium to 4.0 with ammonia, add PTM1 4.35mL/L to start Fermentation liquid, seed liquid inoculation amount is 10% (volume percentage), the temperature is 30 ° C and the rotation speed is 600 rpm in the cultivation process. Among them, PTM1 formula: CuSO ₄ ·7H ₂ O 6.0g, NaI 0.08g, MnSO ₄ ·H ₂ O 3.0g, Na ₂ MoO ₄ ·2H ₂ O 0.2g, boric acid 0.02g, CoCl ₂ 0.5g, ZnCl ₂ 20.0 g, FeSO ₄ ·7H ₂ O 65.0 g, biotin 0.2 g, concentrated sulfuric acid 5.0 mL, add water to 1 L).

3) Glycerol fed-feed fermentation stage: the flow rate of glycerol aqueous solution (glycerol concentration is 500g/L) is 30mL/h/L initial fermentation broth. Dissolved oxygen is always monitored at this stage, and dissolved oxygen is maintained at 20%-70%. The temperature was controlled at 30°C, and the pH of the fermentation broth was 4.0. When the wet weight of the cells reached 200 g/L, the feeding was stopped. The fermentation time of the whole process was 6h.

4) 100% methanol feeding induction expression stage: after stopping the feeding of glycerol, starvation for 0.5 h to exhaust the glycerol in the tank, adjust the pH of the fermentation broth to 6.0, the speed to 800 rpm, and start the feeding of 100% methanol to induce enzyme production, within 4 h The methanol flow rate was increased from 3.6mL/h/L initial fermentation broth to 10.9mL/h/L initial fermentation broth, and then methanol was added at a rate of 10.9mL/h/L initial fermentation broth to the end of fermentation. Monitor the dissolved oxygen in the tank to keep the dissolved oxygen at 20%-70%, control the temperature to 30°C, and ferment for 7 days. During the fermentation process, samples were taken to determine the wet weight, protein content and xyloglucanase activity of bacteria. The measurement conditions were: 50 mM MOPS buffer (pH 7.0), and the reaction temperature was 65° C. (see above for the specific method).

The measurement steps are as follows:

1) Bacterial wet weight: Weigh the mass of the centrifuge tube, record it as m1, add 1 mL of fermentation broth to the centrifuge tube, use a centrifuge at 11510 × g for 3 minutes, discard the supernatant, and weigh the centrifuge tube and the lower layer material and record it. is m2.

The formula for calculating the wet weight of the bacteria is: wet weight of the bacteria = m2-m1.

2) 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) was used to determine the protein content (mg/mL). The protein content is the total protein content.

The changes in the wet weight of the bacteria, the protein content and the activity of xyloglucanase during the fermentation process are shown in Figure 4A, and the SDS-PAGE electrophoresis of the protein during the fermentation process is shown in Figure 4B. The enzyme activity reached the highest at high density fermentation for 168h. The xyloglucanase activity in the supernatant of the fermentation broth was 25700U/mL, the protein concentration was 5.1mg/mL, and the wet weight of the bacteria was 426g/L.

3. Purification of recombinant xyloglucanase

Q-Sepharose FF (Q-Sepharose FF) ion exchange chromatography: put 5mL of fermentation supernatant into a dialysis bag (molecular weight cut-off of 3.50kDa) and put it into 20mM MOPS (pH 7.0) for overnight dialysis. The good enzyme solution was centrifuged at 11510 × g for 5 min. The supernatant was loaded on a Q-Sepharose FF column (10×50mm) equilibrated with MOPS buffer (20mM, pH 7.0) for purification. The flow rate of the loading process was 0.5mL/min. After loading, 5 column volumes were eluted with the same buffer, followed by linear elution with 0-500 mM NaCl solution (20 mM MOPS buffer, pH 7.0) (linear NaCl from 0 to 500 mM in 50 min), elution flow rate 1 mL/min, collect the fraction with enzymatic activity, combine the obtained pure enzymes and dialyze with 20 mM MOPS (pH 7.0) for 4 h for use. The entire purification process is shown in Table 1, and the SDS-PAGE results are shown in Figure 5, where lane 1 is the crude enzyme solution, and lane 2 is the pure enzyme purified by the Q-Sepharose chromatography column. The results in Figure 5 show that the recombinant xyloglucanase showed a single molecular weight band, corresponding to a molecular weight of 21.9 kDa, which was consistent with the expected results.

Table 1. Purification of recombinant xyloglucanase

Purification step Total enzyme activity (U) Total protein (mg) Specific enzyme activity (U/mg) Recovery rate(%) Purification fold Crude enzyme solution 128500 25.5 5039.2 100 1.0 pure enzyme 117150 14.8 7915.5 91.2 1.6

In Table 1, the recovery rate is the percentage of the total enzyme activity of the pure enzyme in the total enzyme activity of the crude enzyme solution. Purification fold is the ratio of the specific enzyme activity of pure enzyme to the specific enzyme activity of crude enzyme.

Example 2. Enzymatic properties of recombinant protein RmXEG12A as xyloglucanase

The details of each buffer are as follows: (▲) citrate buffer (pH 3.0-5.5), (◆) MES (2-(N-morpholino)ethanesulfonic acid) buffer (pH 5.0-6.5), (■) MOPS (3-morpholinopropanesulfonic acid) buffer (pH 6.0-8.0), (△) Tris-HCl (tris(hydroxymethyl)aminomethane-hydrochloric acid) buffer (pH 7.0-9.0), (□) Gly -NaOH (glycine-sodium hydroxide) buffer (pH 8.5-11.0). The concentrations of the above buffers were all 50 mM.

1. The optimum pH value of recombinant xyloglucanase

Take the purified recombinant xyloglucanase solution prepared in Example 1 as the sample solution to be tested, dilute it by appropriate times in the above buffer (different buffer systems in the range of pH 3.0-11.0), and measure the enzyme according to the above method at 45°C live. When diluting, use different buffer systems in the range of pH 3.0-11.0 to dilute the pure enzyme solution, and use the corresponding dilution buffer when measuring. The purified recombinant xyloglucanase solution prepared in Example 1 was used as the sample solution to be tested, and its enzyme activity was determined as follows: (1) Take 100 μL of 0.5% (mass volume fraction, that is, 0.5 g/100 mL) wood Dextran (tamarind seed) solution (prepared with xyloglucan and 50 mM pH 3.0-11.0 different buffers to be tested), then add 50 μL of 50 mM corresponding buffer, and mix well. Preheat the reaction at 45°C for 3 min, add 50 μL of appropriately diluted enzyme solution to be tested, and place it in a constant temperature water bath at 45°C for 10 min; (2) After completing the reaction in step (1), use 200 μL of DNS reagent (1% 3,5-dichloromethane) Nitrosalicylic acid, 1% NaOH and 0.2% phenol, all are mass and volume fractions, i.e. 1% 3,5-dinitrosalicylic acid is 1g/100mL 3,5-dinitrosalicylic acid), terminated React and react with the released reducing sugar, add 200 μL of saturated potassium sodium tartrate solution after boiling for 15 min, and measure the absorbance value at a wavelength of 540 nm after cooling, using glucose as the standard. Taking glucose as the mark, the activity unit of xyloglucanase is defined as: under the above reaction conditions, the amount of enzyme required to generate 1 μmol of glucose per minute is one unit of enzyme activity (1U). Take the maximum value as 100%. The relative enzyme activity at each pH was calculated separately, and the highest enzyme activity was 100% to calculate the relative activity of xyloglucanase at each pH value. The results are shown in Figure 6. The optimum pH of the recombinant xyloglucanase The value is 7.0.

2. pH stability of recombinant xyloglucanase

The purified recombinant xyloglucanase solution prepared in Example 1 was diluted with the above buffer to an appropriate number of times, placed in a 40°C water bath for 30 minutes, and then quickly taken out and immediately ice-bathed for 30 minutes. The method measures residual xyloglucanase activity. The control was the dilution of the recombinant xyloglucanase solution without the above-mentioned treatment (the above-mentioned treatment refers to a water bath at 40° C. for 30 minutes, followed by a rapid ice bath for 30 minutes). Taking the enzyme activity of the control as 100%, the relative activity of the enzyme after treatment with different pH buffers was calculated. The results are shown in Figure 7. The enzyme remained stable in the pH range of 3.5-9.0, and the residual enzyme activity remained after 30min of treatment. above 80%.

3. The optimum temperature of recombinant xyloglucanase

The purified recombinant xyloglucanase solution prepared in Example 1 was used as the enzyme solution to be tested, and the MOPS of pH 7.0 and 50 mM was diluted to an appropriate multiple, and reacted at different temperatures in the range of 30 ° C to 80 ° C, and then according to the specific implementation The xyloglucanase activity was determined by the method described in Example 1 in the following manner, and the relative activity at each reaction temperature was calculated by taking the highest enzyme activity as 100%. The results are shown in Fig. 8, and the optimum temperature of the recombinant xyloglucanase was 65°C.

4. Temperature stability of recombinant xyloglucanase

The purified recombinant xyloglucanase solution prepared in Example 1 was diluted to an appropriate multiple with MOPS of pH 7.0 and 50 mM, and placed in a water bath for 30 min at different temperatures (the water bath temperatures were 30 °C, 35 °C, 40 °C, and 45 °C). , 50°C, 55°C, 60°C, 65°C, 70°C, 75°C or 80°C), quickly take it out and take an ice bath for 30min, and measure the residual xyloglucanase according to the method described in Example 1 under the specific embodiments. vitality. The control was the dilution of the recombinant xyloglucanase solution without the above-mentioned steps (the above-mentioned treatment refers to a water bath for 30 minutes and a rapid ice bath for 30 minutes). The enzyme activity of the control was taken as 100%. The relative activity results of the tested enzyme solution after each temperature treatment are shown in Figure 9, and the recombinant xyloglucanase is stable below 60°C.

5. Determination of substrate specificity

The purified recombinant xyloglucanase solution prepared in Example 1 was used as the enzyme solution to be tested, and the xyloglucanase activities of different substrates were determined, as follows: (1) Take 100 μL of 0.5% (mass volume fraction, that is, 0.5 g/100 mL) substrate solution (prepared with pH 7.0, 50 mM MOPS buffer), then 50 μL of 50 mM pH 7.0 MOPS buffer was added and mixed. Preheat the reaction at 65°C for 3min, add 50μL of appropriately diluted enzyme solution to be tested, and place it in a constant temperature water bath at 65°C to react for 10min; (2) After completing the reaction in step (1), use 200μL of DNS reagent (1% 3,5-dicarbonate) Nitrosalicylic acid, 1% NaOH and 0.2% phenol, all are mass volume fractions, that is, % means 1g/100mL), terminate the reaction and react with the released reducing sugar, add 200μL saturated potassium sodium tartrate solution after boiling for 15min, cool Then the absorbance value was measured at 540nm wavelength with glucose as the standard. Substrates include xyloglucan (tamarind seed), barley beta-glucan, lichenin, laminarin, keratin, microcrystalline cellulose, birch xylan, sodium carboxymethylcellulose (CMC), Locust bean gum (LBG), pullulan, soluble starch, etc., the substrate concentrations were all 0.5% (mass volume ratio, 0.5 g substrate/100 mL 50 mM MOPS pH7.0 buffer).

Taking the enzyme activity of recombinant xyloglucanase to xyloglucan (tamarind seed) as 100%, the enzyme activity of barley β-glucan, lichen polysaccharide, laminarin, keratin polysaccharide, microcrystalline cellulose, Relative and specific enzymatic activities of birch xylan, sodium carboxymethylcellulose (CMC), locust bean gum (LBG), pullulan and soluble starch.

The results are shown in Table 2. The specific enzyme activity of recombinant xyloglucanase to xyloglucan (tamarind seed) was the highest, which was 7915.5U/mg; the second was lichen polysaccharide, the specific enzyme activity was 110.8U/mg; The enzyme activity is the lowest at 87.1U/mg, which is effective against laminarin, keratin, microcrystalline cellulose, birch xylan, sodium carboxymethyl cellulose (CMC), locust bean gum (LBG), pullulan and soluble starch showed no activity.

Table 2. Substrate specificity of recombinant xyloglucanase

substrate Specific enzyme activity (U/mg) Relative Vitality (%) Xyloglucan (tamarind seed) 7915.5±132.3 100 Lichen Polysaccharide 110.8±4.0 Barley Beta-Glucan 87.1±4.1 1.1 Laminaria - - keratin polysaccharide - - microcrystalline cellulose - - birch xylan - - Sodium carboxymethyl cellulose - - Locust Bean Gum - - Pullulan - - soluble starch - -

Note: "-" indicates that no activity was detected.

Example 3. Using recombinant xyloglucanase (RmXEG12A) to hydrolyze tamarind powder to produce xyloglucan oligosaccharides

Weigh 15g tamarind powder, dissolve it in 100mL distilled water (distilled water can also be changed to pH 7.0, 50mM MOPS buffer), add 7500U recombinant xyloglucanase (RmXEG12A), stir well at 200rpm, and place it at 55°C for hydrolysis for 8h . After the hydrolysis was completed, the reaction system was bathed in boiling water for 20 min, centrifuged at 11510 × g for 10 min, and the supernatant was taken as the crude xyloglucose oligosaccharide solution. The xyloglucose oligosaccharide crude sugar solution is spray-dried to obtain xyloglucose oligosaccharide powder (spray drying conditions: air inlet temperature 180°C, air outlet temperature 80°C). The reducing sugar content in the crude sugar solution was determined by the 3,5-dinitrosalicylic acid method, and the reducing sugar yield, hydrolysis rate and product yield were calculated. The composition of the crude sugar solution was qualitatively analyzed by thin layer chromatography (TLC).

The calculation formulas of reducing sugar yield, hydrolysis rate and product yield are:

Reducing sugar yield (%) = total reducing sugar mass in the system after hydrolysis/mass of raw materials added before hydrolysis × 100;

Hydrolysis rate (%)=(1-Dry weight of precipitation after centrifugation of hydrolyzate/mass of raw materials added before hydrolysis)×100;

Product yield (%)=product dry weight/mass of raw materials added before hydrolysis×100.

Reducing sugar determination method: (1) Take 150 μL of distilled water (distilled water can also be replaced with MOPS buffer with pH 7.0 and 50 mM), add 50 μL of appropriately diluted xyloglucan oligosaccharide solution to be tested, and mix well. (2) After completing step (1), use 200 μL of DNS reagent (1% 3,5-dinitrosalicylic acid, 1% NaOH and 0.2% phenol, all by mass and volume fraction) to react with the released reducing sugar, After boiling for 15 min, add 200 μL of saturated potassium sodium tartrate solution, and after cooling, measure the absorbance at a wavelength of 540 nm, using glucose as the standard.

TLC detection conditions: the developing agent is n-butanol: ethanol: water = 2: 1: 1 (volume ratio v/v), and the developer is methanol: sulfuric acid = 95:5 (volume ratio v/v) ). Standard controls were a mixture of glucose, cellobiose, cellotriose, cellotetraose, xylo-oligosaccharide XXXG, XXLG/XLXG and XLLG standards. Spot an appropriate amount of the sample to be tested on the thin-layer chromatography analysis plate, put it in the developing agent to develop the layer twice, soak it completely with the color developer and blow dry, and bake it at 130°C to develop the color.

Among them, glucose, cellobiose, cellotriose and cellotetraose are all products of Shanghai Yuanye Technology Co., Ltd., and the product numbers are B21882, B25328, S46082 and S46724 respectively. The mixture of xyloglucose oligosaccharides XXXG, XXLG/XLXG and XLLG standard products was obtained from Megazyme, the product number is O-XGHON.

The results showed that the hydrolysis rate of RmXEC12A hydrolyzed tamarind powder was 56.6%, the yield of reducing sugar was 32.5%, and the yield of product was 50.8%. The results of thin layer chromatography analysis are shown in Figure 10 (M is the standard control, from top to bottom are glucose, cellobiose, cellotriose, cellotetraose, xyloglucosaccharide XXXG, XXLG/XLXG and XLLG standard composition mixture). The results showed that when tamarind powder xyloglucan was used as the substrate, three high molecular weight xyloglucan oligosaccharides were mainly produced, namely XXXG, XXLG (XLXG) and XLLG.

Example 4. Using recombinant xyloglucanase (RmXEG12A) to hydrolyze tamarind gum to produce xyloglucan oligosaccharides

Weigh 20g tamarind gum, dissolve it in 100mL distilled water (distilled water can also be changed to pH 7.0, 50mM MOPS buffer), add 10000U recombinant xyloglucanase (RmXEG12A), stir well at 200rpm, and place it at 55°C for hydrolysis for 8h . After the hydrolysis was completed, the reaction system was bathed in boiling water for 20 min, centrifuged at 11510 × g for 10 min, and the supernatant was taken as the crude xyloglucose oligosaccharide solution. The xyloglucose oligosaccharide crude sugar solution is spray-dried to obtain xyloglucose oligosaccharide powder (spray drying conditions: air inlet temperature 180°C, air outlet temperature 80°C). The reducing sugar content in the crude sugar solution was determined by the 3,5-dinitrosalicylic acid method, and the reducing sugar yield, hydrolysis rate and product yield were calculated. The composition of the crude sugar solution was qualitatively analyzed by thin layer chromatography. The calculation method and thin-layer chromatography conditions of reducing sugar yield, hydrolysis rate and product yield are the same as those in Example 3.

The results showed that the hydrolysis rate of RmXEC12A hydrolyzed tamarind gum was 74.3%, the yield of reducing sugar was 35.0%, and the yield of product was 68.7%. The experimental results of thin layer chromatography are shown in Figure 11 (M is the standard control, from top to bottom are glucose, cellobiose, cellotriose, cellotetraose, xyloglucose oligosaccharide XXXG, XXLG/XLXG and XLLG standard products composition of the mixture). The results showed that when tamarind gum xyloglucan was used as the substrate, three high molecular weight xyloglucan oligosaccharides were mainly produced, namely XXXG, XXLG (XLXG) and XLLG.

Example 5. Probiotic proliferation experiment

The xyloglucose oligosaccharide used in this example is the xyloglucose oligosaccharide in Example 3.

1. Experiment on the proliferation of probiotic Lactobacillus oligosaccharides

(1) Seed culture: 9 strains of Lactobacillus (China Agricultural University, School of Food Science and Nutritional Engineering, China Key Laboratory of Food Bioengineering for Light Industry) were preserved and recorded in "Li et al. Preparation, characterization, and prebiotic activity of mannooligosaccharides produced from Cassia gum by aglycoside hydrolase family 134β-mannanase. Food Chemistry, 2020, 309: 125709” article) (Table 3) was inoculated into a commercial MRS medium for activation and culture 3 times (MRS medium formula: peptone 10g/L, beef Cream 10g/L, Yeast Extract 5g/L, Diamine Hydrogen Citrate 2g/L, Glucose 20g/L, Tween 80 1mL/L, Sodium Acetate 5g/L, Dipotassium Hydrogen Phosphate 2g/L, Magnesium Sulfate 0.6 g/L, manganese sulfate 0.25g/L, pH 6.4±0.2), centrifuged at 11510×g for 5min to collect bacterial cells. The bacteria were resuspended in glucose-free MRS liquid medium as seed solution.

(2) The seed liquids of 9 strains of Lactobacillus were inoculated into MRS medium without glucose to serve as a control group. At the same time, the glucose in MRS was replaced with 10g/L xyloglucose oligosaccharide, and it was set as the experimental group, which was placed in an anaerobic incubator at 37°C for 48h, and the absorbance value _OD595 at 595nm wavelength was measured by turbidimetry.

As can be seen from Table 3, the growth amount (OD ₅₉₅ ) of Lactobacillus coryneformis NRRL B-4391 with xyloglucan oligosaccharide as the sole carbon source is 0.25, that of Lactobacillus brevis NRRL B-4527 is 0.12, and that of Lactobacillus casei AS 1.62 is 0.31 , Lactobacillus rhamnosus AS1.2466 was 0.07, Lactobacillus delbrueckii NRRL B-548 was 0.34, significantly higher than the control group. It indicated that xyloglucose oligosaccharide could promote the proliferation of Lactobacillus coryneformis NRRL B-4391, Lactobacillus brevis NRRL B-4527, Lactobacillus casei AS1.62, Lactobacillus rhamnosus AS1.2466 and Lactobacillus delbrueckii NRRL B-548.

Table 3, the proliferation effect of xyloglucan oligosaccharide on 9 strains of lactobacillus pure culture for 48h

Note: Different lowercase letters indicate significant differences (P<0.05).

2. Experiment on the proliferation of probiotic Bifidobacterium

(1) Seed culture: 5 strains of Bifidobacterium (China Agricultural University, School of Food Science and Nutrition Engineering, China Key Laboratory of Food Bioengineering for Light Industry) were preserved and recorded in "Li et al. Preparation, characterization, and prebiotic activity of mannooligosaccharides" Produced from cassia gum by aglycoside hydrolase family 134β-mannanase. Food Chemistry, 2020, 309: 125709" article) (Table 4) inoculated into a commercial MRS medium for activation and culture 3 times (MRS medium formula: peptone 10g/L , beef extract 10g/L, yeast extract 5g/L, diamine hydrogen citrate 2g/L, glucose 20g/L, Tween 80 1mL/L, sodium acetate 5g/L, dipotassium hydrogen phosphate 2g/L, sulfuric acid Magnesium 0.6g/L, manganese sulfate 0.25g/L, pH 6.4±0.2), centrifuged at 11510×g for 5min to collect bacteria. The bacteria were resuspended in glucose-free MRS liquid medium as seed solution.

(2) 5 strains of bifidobacteria were inoculated into MRS medium without glucose, respectively, as a control group. At the same time, the glucose in MRS was replaced with 10g/L xyloglucose oligosaccharide, and it was set as the experimental group, which was placed in an anaerobic incubator at 37°C for 48h, and the absorbance value _OD595 at 595nm wavelength was measured by turbidimetry.

It can be seen from Table 4 that the OD ₅₉₅ of xyloglucose oligosaccharide against Bifidobacterium bifidum NRRL B-41410 was 0.23, which was significantly higher than that of the control group -0.06. It shows that xyloglucose oligosaccharide can promote the proliferation of Bifidobacterium bifidum NRRL B-41410.

Table 4. Proliferation effect of xyloglucan oligosaccharide on 5 strains of Bifidobacterium pure culture for 48h

Note: Different lowercase letters indicate significant differences (P<0.05).

Embodiment 6, utilize xyloglucose oligosaccharide to produce prebiotic yoghurt

The xyloglucose oligosaccharide used in this example is the xyloglucose oligosaccharide in Example 3. Production of prebiotic yogurt by xyloglucose oligosaccharide is made from the following raw materials (1000 parts) by weight: 930-910 parts of fresh milk, 0-20 parts of xyloglucose oligosaccharide powder, 70 parts of sucrose (addition of xyloglucose oligosaccharide) Mass percentage (%) was set as: 0, 0.5, 1, 1.5, 2%).

The preparation method of prebiotic yogurt specifically includes the following steps:

(1) Mixing: Mix the fresh milk, xyloglucose oligosaccharide and sucrose uniformly according to the above weight parts, preheat at 65°C until the material is completely dissolved, and perform homogenization treatment at 40MPa.

(2) Sterilization: the feed liquid obtained in step (1) was heated to 95° C. and pasteurized for 5 minutes.

(3) Inoculation: after the above-mentioned feed liquid is cooled to 40° C., add Streptococcus thermophilus (Streptococcus thermophilus) and Lactobacillus bulgaricus (Lactobacillus bulgaricus) to the feed liquid under aseptic environment for inoculation. The contents of viable bacteria in the system were all 1×10 ⁶ CFU/mL.

(4) Fermentation: The inoculated feed liquid was placed in a constant temperature fermentation chamber, and fermented at 42°C for 4 hours (to about pH 4.6).

(5) Demulsification and cooling: the fermented prebiotic yogurt was demulsified at 350 rpm for 1 min, and cooled to 20°C.

(6) Cooking after refrigeration: the prebiotic yoghurt after being refrigerated in step (5) is placed in a refrigerator (4° C.) and cooked for 16 hours to obtain yogurt.

Evaluation

1. Sensory evaluation: 20 persons with experience in sensory evaluation of yogurt were asked to make an overall evaluation on the mouthfeel, flavor, color and viscosity of yogurt.

2. Water holding capacity: Take the yogurt that has been cooked for 16 hours for measurement, weigh the mass of the sterilized centrifuge tube, record it as m1, add 20g of yogurt to each centrifuge tube and weigh the total mass, record it as m2, use a centrifuge at 3000rpm for centrifugation 10min, discard the supernatant, weigh the centrifuge tube and the lower layer material as m3.

The calculation formula of water holding capacity is: water holding capacity (%)=(m3-m1)/(m2-m1)×100%.

3. Viable count

According to the national standard GB4789.35-2016 "Food Microbiology Inspection Lactic Acid Bacteria Inspection" method, the number of viable bacteria is detected, as follows:

Counting Streptococcus thermophilus: Choose 2-3 consecutive suitable dilutions, and draw 1 mL of sample homogenate solution for each dilution into a sterile plate. After the diluent was transferred to the plate, pour about 15 mL of the MC medium cooled to 48°C into the plate, and rotate the plate to mix evenly. Incubate aerobic at 36℃±1℃ for 72h±2h, and count after culturing.

Lactobacillus count: select 2-3 consecutive suitable dilutions, and draw 1 mL of sample homogenate for each dilution into a sterile plate. After the diluent is transferred to the plate, pour about 15 mL of the MRS agar medium cooled to 48°C into the plate, and rotate the plate to mix evenly. Anaerobic incubation at 36℃±1℃ for 72h±2h, and counting after incubation.

4. Results

The xyloglucose oligosaccharide yogurt was uniform in texture, smooth in surface, shiny, white in color without yellowing, which was consistent with the control group. The fermentation aroma is strong and has no bad odor, which is consistent with the control group. It shows that xyloglucose oligosaccharide can be used in the production of prebiotic yogurt.

Table 5 is the result of the water-holding capacity of yogurt with xyloglucose oligosaccharide. It can be seen from Table 5 that the water-holding capacity (46.7%-48.8%) of yogurt after adding xyloglucose oligosaccharide is higher than that of yogurt without xyloglucose oligosaccharide added. (43.2%). In the range of 0-2% xyloglucose oligosaccharide addition, the water holding capacity of yogurt increases with the increase of xyloglucan oligosaccharide addition, indicating that adding xyloglucan oligosaccharide can increase the water holding capacity of yogurt. After the xyloglucose oligosaccharide is added to the yogurt, the gel structure of the yogurt is enhanced, thereby enhancing its water retention effect, preventing the milk from washing out, and improving the tissue state of the yogurt.

Table 5. Water holding capacity of xyloglucose oligosaccharide yogurt

Table 6 is the effect of xyloglucosaccharide yogurt on the viable count of Lactobacillus and Streptococcus thermophilus. As can be seen from Table 6, the number of viable bacteria in the yogurt without xyloglucose oligosaccharide was 2.78×10 ⁸ CFU/mL, while the number of viable bacteria in the yogurt after adding xyloglucan oligosaccharide was 3.38×10 ⁸ CFU/mL- 4.56×10 ⁸ CFU/mL, which was significantly higher than the number of viable bacteria in yogurt without xyloglucose oligosaccharide. It shows that the addition of xyloglucose oligosaccharide can effectively increase the viable counts of Lactobacillus and Streptococcus thermophilus in yogurt and improve the quality of yogurt.

Table 6. Effects of xyloglucose oligosaccharide yogurt on viable bacteria

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experimentation, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. While the invention has been given particular embodiments, it should be understood that the invention can be further modified. In conclusion, in accordance with the principles of the present invention, this application is intended to cover any alterations, uses or improvements of the invention, including changes made using conventional techniques known in the art, departing from the scope disclosed in this application. The application of some of the essential features can be made within the scope of the following appended claims.

<110> China Agricultural University

<120> Preparation method and application of 12 family xyloglucanase of Rhizomucor militaris glycoside hydrolase

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Claims (10)

1. A method of producing xyloglucanase comprising the steps of:

(A) Introducing a coding gene of xyloglucanase XEG12A derived from Rhizomucor miehei CAU432, a coding gene of FLD1 protein derived from pichia pastoris and a coding gene of Sso1 protein derived from saccharomyces cerevisiae into a receptor pichia pastoris to obtain recombinant pichia pastoris;

(B) Fermenting and culturing the recombinant pichia pastoris according to the following steps, and obtaining xyloglucanase XEG12A from a fermentation product:

b1 Basic fermentation culture: inoculating the recombinant pichia pastoris into a BSM culture medium for culture, and performing a glycerol feeding fermentation stage when glycerol is completely consumed;

b2 Glycerol fed-batch fermentation stage: on the basis of the step B1), glycerol is fed into a fermentation system at the speed of 15-30mL/h/L initial fermentation liquid, the feeding of the glycerol is stopped when the wet weight of the thalli reaches 180-220g/L, starvation is carried out for about 0.5-1h, and then a methanol feeding induction expression stage is carried out;

b3 Methanol feed induced expression phase: on the basis of the step B2), adding the methanol flow into the fermentation system for 4 hours to increase the flow rate from 3.6mL/h/L of the initial fermentation broth to 10.9mL/h/L of the initial fermentation broth, and then adding the methanol flow at the speed of maintaining 10.9mL/h/L of the initial fermentation broth until the end of the fermentation.

2. The method of claim 1, wherein: in the step (A), the xyloglucanase XEG12A derived from Rhizomucor miehei CAU432 is any one of the following proteins:

a1 Protein consisting of 21 st to 242 nd amino acid residues from the N-terminal of SEQ ID No. 1;

a2 A protein consisting of the amino acid sequence shown in SEQ ID No. 1;

a3 A protein with xyloglucanase activity is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein of a 1) or a 2);

a4 A protein having 99% or more, 95% or more, 90% or more, 85% or more or 80% or more homology with the amino acid sequence defined in any of a 1) to a 3) and having xyloglucanase activity;

a5 A fusion protein obtained by connecting a label to the N-terminal and/or C-terminal of the protein defined in any one of a 1) to a 4);

and/or

In the step (A), the FLD1 protein derived from Pichia pastoris is any one of the following proteins:

b1 A protein consisting of the amino acid sequence shown in SEQ ID No. 2;

b2 Protein with the same activity is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein in b 1);

b3 A protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology and the same activity as the amino acid sequence defined in any of b 1) to b 2);

b4 A fusion protein obtained by connecting a label to the N-terminal and/or C-terminal of the protein defined in any one of b 1) to b 3);

and/or

In the step (A), the Sso1 protein derived from the saccharomyces cerevisiae is any one of the following proteins:

c1 A protein consisting of the amino acid sequence shown in SEQ ID No. 3;

c2 Protein with the same activity is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein in c 1);

c3 Protein with 99% or more, 95% or more, 90% or more, 85% or more or 80% or more homology and the same activity as the amino acid sequence defined in any of c 1) to c 2);

c4 C) a tag is attached to the N-and/or C-terminus of a protein as defined under C1) to C3).

3. The method according to claim 1 or 2, characterized in that: in the step (A), the coding gene of xyloglucanase XEG12A derived from Rhizomucor miehei CAU432 is introduced into the receptor Pichia pastoris in the form of a recombinant vector 1;

further, the recombinant vector 1 is a recombinant plasmid obtained by inserting the coding gene of the xyloglucanase XEG12A derived from the Rhizomucor miehei CAU432 into a multiple cloning site of a pPIC9K vector;

and/or

In the step (A), the coding gene of the FLD1 protein derived from the pichia pastoris is introduced into the receptor pichia pastoris in a form of a recombinant vector 2;

further, the recombinant vector 2 is a recombinant plasmid obtained by inserting the coding gene of the FLD1 protein from pichia pastoris into a multiple cloning site of a pPICZA vector;

and/or

In the step (A), the coding gene of the Sso1 protein derived from the saccharomyces cerevisiae is introduced into the receptor pichia pastoris in a form of a recombinant vector 3;

further, the recombinant vector 3 is a recombinant plasmid obtained by inserting the coding gene of the Sso1 protein from Saccharomyces cerevisiae into the multiple cloning site of the pGAPZB vector;

and/or

The pichia is pichia GS115.

4. A method according to any one of claims 1-3, characterized in that: in the step (A), the coding gene of xyloglucanase XEG12A derived from Mucor miehei CAU432 is a DNA molecule shown in any one of the following:

d1 A DNA molecule shown from position 61-729 of the 5' end of SEQ ID No. 4;

d2 A DNA molecule shown as SEQ ID No. 4;

d3 A DNA molecule which hybridizes under stringent conditions with a DNA molecule defined under d 1) or d 2) and which codes for the xyloglucanase XEG12A from Mucor miehei CAU 432;

d4 A DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more or 80% or more homology to the DNA sequence defined in any of d 1) to d 3) and encoding the xyloglucanase XEG12A derived from Mucor miehei CAU 432;

and/or

In the step (A), the coding gene of the FLD1 protein derived from Pichia pastoris is a DNA molecule shown in any one of the following sequences:

e1 A DNA molecule shown as SEQ ID No. 5;

e2 A DNA molecule which is hybridized with the DNA molecule defined by the e 1) under strict conditions and codes the FLD1 protein derived from the pichia pastoris;

e3 A DNA molecule which has more than 99 percent, more than 95 percent, more than 90 percent, more than 85 percent or more than 80 percent of homology with the DNA sequence defined by any one of e 1) to e 2) and codes the FLD1 protein derived from pichia pastoris;

and/or

In the step (A), the coding gene of the Sso1 protein derived from the saccharomyces cerevisiae is a DNA molecule shown in any one of the following steps:

f1 A DNA molecule shown as SEQ ID No. 6;

f2 A DNA molecule hybridizing under stringent conditions with the DNA molecule defined in f 1) and encoding the Sso1 protein from Saccharomyces cerevisiae;

f3 A DNA molecule which has more than 99 percent, more than 95 percent, more than 90 percent, more than 85 percent or more than 80 percent of homology with the DNA sequence limited by any one of f 1) and f 2) and codes the Sso1 protein derived from the saccharomyces cerevisiae.

5. The method according to any one of claims 1-4, wherein: in the step B1), the recombinant Pichia pastoris inoculated in the BSM culture medium is a seed solution of the recombinant Pichia pastoris;

further, the seed liquid is prepared according to a method comprising the following steps: inoculating the recombinant pichia pastoris obtained in the step (A) into a BMGY culture medium, and carrying out shaking culture at 30 ℃ and 200rpm until the light absorption value of the fermentation liquor at 600nm reaches 2-6;

and/or

In the step B1), the seed solution is inoculated in the BSM culture medium and is inoculated in a fermentation tank filled with the BSM culture medium, and the liquid filling amount of the BSM culture medium in the fermentation tank is 1.5/5 of the volume of the fermentation tank;

and/or

In the step B1), a PTM1 solution is added;

further, the addition amount of the PTM1 solution is 4.35mL of the PTM1 solution added in each 1L of the initial fermentation broth;

and/or

In the step B1), the temperature of the culture is 30 ℃, the pH value is 4.0, and the rotating speed is 600rpm;

and/or

In the step B2), the glycerol is a 500g/L aqueous glycerol solution formed by dissolving in water; the culture temperature of the glycerol feeding fermentation stage is 30 ℃, the pH is 4.0, and the dissolved oxygen is controlled to be more than 20%;

and/or

In step B3), the methanol is anhydrous methanol; the culture temperature of the methanol feeding induction expression stage is 30 ℃, the pH value is 6.0, the dissolved oxygen is controlled to be more than 20%, and the fermentation time is 6-7 days.

6. The method according to any one of claims 1-5, wherein: in the step (B), the fermentation culture of the recombinant pichia pastoris is carried out, and the method also comprises the following steps: dialyzing the supernatant of the fermentation liquor to remove salt, then performing anion exchange chromatography, performing linear elution by using a2 mM MOPS buffer solution with pH7.0 and containing 0-500mM NaCl, and collecting to obtain purified xyloglucanase XEG12A;

further, the anion exchange chromatography is Q-sepharose FF ion exchange chromatography; and/or

The elution procedure for the linear elution with 20mM MOPS buffer, pH7.0, containing 0-500mM NaCl was as follows: the concentration of NaCl in the eluate increased linearly from 0 to 500mM within 50 min.

7. Xyloglucanase XEG12A prepared by the method of any of claims 1-6.

8. The recombinant Pichia pastoris, according to any one of claims 1 to 4, in step (A).

9. Use as shown in any one of:

use of the xyloglucanase XEG12A according to P1, claim 7 for the preparation of xylo-oligosaccharides;

p2, use of the xyloglucanase XEG12A according to claim 7 for enzymatic hydrolysis of xyloglucan-rich plants;

use of P3, the xyloglucanase XEG12A according to claim 7, for the preparation of a product for enzymatic hydrolysis of a xyloglucan-rich plant;

p4, use of xyloglucanase XEG12A of claim 7 in the preparation of prebiotic yoghurt;

use of the xyloglucanase XEG12A according to claim 7 in the food industry;

the use of P6, the recombinant Pichia pastoris of claim 8 for the production of xyloglucanase XEG12A of claim 7;

further, in P1, the xylo-oligosaccharide is a xylo-oligosaccharide polymerized by 2-10 glucose, xylose and/or galactose;

and/or

Further, in P1, the xyloglucan oligosaccharide preparation takes xyloglucan or a xyloglucan-rich plant as a substrate;

and/or

Further, the plant is tamarind, apple and/or blueberry.

10. Preparing the obtained product by any one of the following methods or by using the method:

q1, a method for hydrolyzing tamarind powder, comprising the steps of: hydrolyzing tamarind powder with xyloglucanase XEG12A as claimed in claim 7, wherein the ratio of tamarind powder to said xyloglucanase XEG12A is 1g tamarind powder: 200-1000U of xyloglucanase XEG12A, hydrolyzing for 8h at 30-60 ℃ to obtain the following three xylo-oligosaccharides with high molecular weight: XXXXKG, XXLG (XLXG) and XLG;

q2, a method for hydrolyzing tamarind gum, comprising the steps of: hydrolyzing tamarind gum with xyloglucanase XEG12A as claimed in claim 7, wherein the ratio of tamarind gum to said xyloglucanase XEG12A is 1g tamarind gum: 200-1000U of xyloglucanase XEG12A, hydrolyzing for 8h at 30-60 ℃ to obtain the following three xylo-oligosaccharides with high molecular weight: XXXXKG, XXLG (XLXG) and XLG;

q3, a method for promoting the proliferation of Lactobacillus coryniformis NRRL B-4391, lactobacillus brevis NRRL B-4527, lactobacillus casei AS1.62, lactobacillus delbrueckii NRRL B-548, lactobacillus rhamnosus AS 1.2466 and/or Bifidobacterium bifidum NRRL B-41410, comprising the steps of: production of xylo-oligosaccharides by the xyloglucanase XEG12A according to claim 7, followed by the use of said xylo-oligosaccharides to promote the proliferation of Lactobacillus coryniformis NRRL B-4391, lactobacillus brevis NRRL B-4527, lactobacillus casei AS1.62, lactobacillus delbrueckii NRRL B-548, lactobacillus rhamnosus AS 1.2466 and/or Bifidobacterium bifidum NRRL B-41410;

q4, a method for preparing prebiotics yoghourt, which comprises the following steps: producing xylo-oligosaccharides from xyloglucanase XEG12A according to claim 7, and then preparing prebiotic yoghurt using said xylo-oligosaccharides, fresh milk, sucrose and Streptococcus thermophilus (Streptococcus thermophilus) and Lactobacillus bulgaricus (Lactobacillus bulgaricus);

further, the method comprises the steps of: mixing 93-91 wt% of fresh milk, 0-2 wt% (without 0 point) of xylo-oligosaccharide and 7 wt% of sucrose, sterilizing, inoculating Streptococcus thermophilus (Streptococcus thermophilus) and Lactobacillus bulgaricus (Lactobacillus bulgaricus), wherein the viable bacteria content of the two added bacteria in the system is 1 × 10 ⁶ And (5) CFU/mL, and then sequentially fermenting, demulsifying, chilling, refrigerating and maturing to obtain the prebiotics yoghourt.

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