CN111484989A - Preparation method of β -mannase and application of β -mannase in preparation of partially hydrolyzed mannan - Google Patents

Preparation method of β -mannase and application of β -mannase in preparation of partially hydrolyzed mannan Download PDF

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CN111484989A
CN111484989A CN202010354187.0A CN202010354187A CN111484989A CN 111484989 A CN111484989 A CN 111484989A CN 202010354187 A CN202010354187 A CN 202010354187A CN 111484989 A CN111484989 A CN 111484989A
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gum
mannan
sequence
protein
mannase
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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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China Agricultural University
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China Agricultural University
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Priority to PCT/CN2020/135515 priority patent/WO2021218171A1/en
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Abstract

The invention discloses a preparation method of β -mannase and application thereof in preparation of partially hydrolyzed mannan.A preparation method of β -mannase disclosed by the invention comprises the steps of introducing an rMan26 gene shown in a sequence 2 in a sequence table into a biological cell, culturing the obtained recombinant cell to enable an rMan26 gene to be expressed, and obtaining a protein with β -mannase activity, wherein the expression of the rMan26 gene is driven by an ATX1 promoter shown in the 6 th to 930 th positions of a sequence 4 in the sequence table.

Description

Preparation method of β -mannase and application of β -mannase in preparation of partially hydrolyzed mannan
Technical Field
The invention relates to the field of biotechnology, in particular to a preparation method of β -mannase and application of the β -mannase in preparation of partially hydrolyzed mannan.
Background
Mannan is a linear polysaccharide formed by connecting mannose by β -1, 4-D-glucopyranoside, some residues of the main chain can be replaced by glucose residues connected by β -1, 4-glycosidic bond and galactose residues connected by α -1, 6-glycosidic bond, mannan is the second major component of hemicellulose, has the content which is only inferior to that of xylan, is widely distributed in nature, and is rich in mannan such as endosperm of leguminous plant seeds, some plant gums (such as locust bean gum, fenugreek gum and the like), coconut meat, konjak bulbs and the like, and the mannan can be divided into linear mannan, galactomannan, glucomannan and galactoglucomannan according to the difference of the structure and the physicochemical properties of the mannan, β -mannase is a key enzyme for hydrolyzing the mannan and can randomly hydrolyze β -1, 4-glycosidic bond in the mannan main chain to generate mannan oligosaccharide.
β -mannase has a complex structure, the complete degradation requires the synergistic effect of a plurality of enzymes, the most important of which is β -mannase β -mannase is widely applied to the industries of food, feed and the like, but the prior mannase has low enzyme activity and high production cost, and the application of β -mannase in industrial production is limited, so that the β -mannase is efficiently expressed and fermented at high density to improve the β -mannase expression level, and the application of the mannase in the industries of food, feed and the like is of great significance.
Based on the homology of the amino acid sequences of the catalytic domains, β -mannanases can be divided into glycoside hydrolase families 5, 26 and 113. most of the β -mannanases belong to the Glycoside Hydrolase (GH)5 family and the 26 family. GH5 family β -mannanase has been used for preparing partially hydrolyzed mannans, such as GH5 family β -mannanase from Arabidopsis thaliana and Rhizomucor miehecau 432. GH26 family β -mannanase has more advantages when the substrate is galactomannan with more galactose substituents at the side chain. to meet the industrial production requirements, some GH26 family β -mannanase has been successfully expressed in yeast but at lower expression levels, such as Bacillus subtilis (Thermophilus subtilis) (TBS2) β -mannanase (Retman26) with an enzyme activity of 5435U/m 5 (Thermophilus Bacillus subtilis) (L. L) and a mannitol release reaction buffer with a mannitol units of the enzyme activity of the industrial mannose release rate of the mannose units of the mannose equivalent of the Aspergillus niger strain U5, 26. mu. min, 200. mu. sup. the industrial production of the industrial mannanase, the industrial production of Bacillus subtilis strain, the industrial production of the.
The partially hydrolyzed mannan as a prebiotic has the characteristics of low calorie, stable property, promotion of proliferation of beneficial flora in organisms, regulation of metabolic cycle and the like, and can be widely applied to the food industry. At present, partially hydrolyzed mannan is prepared mainly by methods such as high-temperature degradation, acid-base hydrolysis, enzymatic hydrolysis and the like. The method for preparing the partially hydrolyzed mannan by the enzyme method has the advantages of easy process control, mild reaction conditions, less side reactions and the like, and becomes the most common method for preparing the partially hydrolyzed mannan. The substrates for the preparation of partially hydrolyzed mannans are mainly vegetable gums rich in mannans. Among the materials commonly used to prepare partially hydrolyzed mannan are konjac flour and guar gum, but the concentrations of the materials used in the hydrolysis are low (< 5%). In addition, mannan-rich vegetable gums such as cassia gum, fenugreek gum and the like are good substrates for preparing partially hydrolyzed mannan.
The yoghourt is a food prepared by fermenting fresh milk serving as a raw material through streptococcus thermophilus, lactobacillus bulgaricus and the like. The yoghourt is fine, smooth, thick in texture, sour, sweet and tasty, and has the advantages of being easy to digest and absorb, promoting intestinal tract peristalsis, adjusting flora balance in intestinal tracts, resisting oxidation and bacteria, reducing blood pressure and the like. The prebiotics is added into the yoghourt to prepare the prebiotics yoghourt, so that the physical and chemical properties of the yoghourt can be improved, the functional activity of the yoghourt is improved, and the prebiotics yoghourt is widely concerned.
Aspergillus niger is a heat-resistant filamentous fungus capable of secreting various glycoside hydrolases, and the secreted β -mannase has various excellent enzymological characteristics.
Disclosure of Invention
The invention provides a preparation method of β -mannase and application of the mannase in preparation of partially hydrolyzed mannan and yoghourt.
The invention firstly provides a preparation method of a protein with β -mannanase activity, which comprises the steps of introducing an rMan26 gene shown in a sequence 2 in a sequence table into a biological cell to obtain a recombinant cell, culturing the recombinant cell to express the rMan26 gene, and obtaining the protein with β -mannanase activity.
In the method, the expression of the rMan26 gene is driven by a promoter named as ATX1, and the ATX1 is a DNA molecule shown in the 6 th to 930 th positions of a sequence 4 in a sequence table.
In the above method, the introducing of the rMan26 gene represented by sequence 2 in the sequence listing into a biological cell may comprise introducing a recombinant vector comprising the rMan26 gene and the ATX1 into the biological cell.
The recombinant vector can be pPAT19K-AnMan26, the pPAT19K-AnMan26 is a recombinant vector obtained by replacing a DNA fragment between Bgl II and BamHI recognition sequences of pPIC9K with a DNA molecule shown in the 6 th to 930 th sites of a sequence 4 in a sequence table, and replacing a DNA fragment between EcoRI and NotI recognition sequences with rMan26 gene.
In the above method, the protein may be a1), a2), or A3) as follows:
A1) the amino acid sequence is the protein of sequence 3;
A2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in an amino acid sequence shown in a sequence 3 in a sequence table and has β -mannanase function;
A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2).
In order to facilitate the purification of the protein in A1), the amino terminal or the carboxyl terminal of the protein consisting of the amino acid sequence shown in the sequence 3 in the sequence table is attached with the tags shown in the following table.
Table: sequence of tags
Label (R) Residue of Sequence of
Poly-Arg 5-6 (typically 5) RRRRR
Poly-His 2-10 (generally 6) HHHHHH
FLAG 8 DYKDDDDK
Strep-tag II 8 WSHPQFEK
c-myc 10 EQKLISEEDL
The protein in A2) above is a protein having 75% or more identity to the amino acid sequence of the protein shown in SEQ ID NO. 3 and having the same function. The identity of 75% or more than 75% is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.
The protein of A2) above may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression.
The gene encoding the protein of A2) above can be obtained by deleting one or several amino acid residues from the DNA sequence shown in SEQ ID No. 2, and/or by carrying out missense mutation of one or several base pairs, and/or by attaching the coding sequence of the tag shown in the above table to the 5 'end and/or 3' end thereof. Wherein, the DNA molecule shown in the sequence 2 encodes the protein shown in the sequence 3.
Specifically, the protein A3) can be a protein shown as a sequence 6 in a sequence table.
In the above method, the biological cell may be a microbial cell. Further, the microbial cell is a fungus. Still further, the fungus may be a yeast. The yeast can be Pichia pastoris (such as Pichia pastoris GS 115).
In one embodiment of the invention, the recombinant cell is GS115-pPAT19K-AnMan26, and the GS115-pPAT19K-AnMan26 is a recombinant bacterium obtained by introducing the pPAT19K-AnMan26 into Pichia pastoris GS 115.
The invention also provides a protein with β -mannanase activity prepared by the preparation method of the protein with β -mannanase activity.
The invention also provides any one of the following products:
x1, the recombinant cell;
x2, the rMan26 gene;
x3, the ATX 1;
x4, a kit consisting of said rMan26 gene and said ATX 1;
x5, a recombinant vector containing said rMan26 gene and/or said ATX 1.
The kit of X4 can be used for preparing β -mannanase.
The recombinant vector X5 can be the pPAT19K-AnMan 26.
The invention also provides any one of the following applications of the protein:
a1) use as β -mannanase;
a2) the application in preparing products with β -mannase activity;
a3) the application in preparing mannan oligosaccharide;
a4) the application in preparing the product for preparing the mannan oligosaccharide;
a5) the application in degrading mannan;
a6) the application in preparing products for degrading mannan;
a7) use in the preparation of partially hydrolysed mannans;
a8) use in the preparation of a product for the preparation of a partially hydrolysed mannan;
a9) the application in degrading vegetable gum;
a10) the application in the preparation of products for degrading vegetable gums.
In the above application, the vegetable gum may be b1) or b2) or b3) or b4) or b 5): b1) locust bean gum; b2) cassia seed gum; b3) fenugreek gum; b4) konjaku flour; b5) a mannan-rich vegetable gum.
The mannooligosaccharide can be a mannooligosaccharide formed by polymerizing 2-10 mannose.
The "production of mannooligosaccharides" and "production of partially hydrolyzed mannans" can use mannan or vegetable gums as substrates.
The plant gum is fenugreek gum, the proportion of the fenugreek gum to the protein with the activity of β -mannanase is that 100-200U of the protein with the activity of β -mannanase is added into each gram of the fenugreek gum, the hydrolysis temperature is 30-45 ℃, and the solvent of the fenugreek gum is citric acid buffer solution or water.
The plant gum can be cassia seed gum, the proportion of the cassia seed gum and the protein with β -mannase activity is that the protein with β -mannase activity is added into each gram of cassia seed gum at 100-800U, the hydrolysis temperature is 30-45 ℃, and the solvent of the cassia seed gum is citric acid buffer solution or water.
The application of the partially hydrolyzed mannan prepared by the protein in the preparation of prebiotics yoghourt also belongs to the protection scope of the invention.
In the above application, the vegetable gum from which the partially hydrolyzed mannan is produced may be c1) or c2) or c3) or c4) or c 5): c1) locust bean gum; c2) cassia seed gum; c3) fenugreek gum; c4) konjaku flour; c5) a mannan-rich vegetable gum.
The preparation of the prebiotics yoghourt also comprises the addition of streptococcus thermophilus (streptococcus thermophilus) and lactobacillus bulgaricus (L actinobacillus bulgaricus) into the system.
According to the invention, Aspergillus niger β -mannase is subjected to codon optimization, and a pAOX1 promoter is modified to obtain a pATX1 promoter, an expression vector pATX19K is constructed and is converted into Pichia pastoris GS115 for high-density expression, so that β -mannase with high enzyme activity is obtained, the enzyme activity can reach 22100U/m L, the optimum pH of β -mannase obtained by the invention is 5.0, the optimum temperature of the enzyme is 45 ℃, plant gums rich in mannan such as cassia seed gum and fenugreek gum and the like can be hydrolyzed, and partially hydrolyzed mannan containing mannan oligosaccharides with different polymerization degrees is obtained.
Drawings
FIG. 1 shows the enzyme production history (A) and SDS-PAGE electrophoresis (B) of high density fermentation of recombinant β -mannanase in 5L fermenter (■), enzyme activity, (▲) protein concentration, (●) wet weight of the strain; lane M in B is the protein molecular weight standard, and lanes 1-8 are the fermentation supernatants inducing 0, 24, 48, 72, 96, 120, 144, and 168h, respectively.
FIG. 2 shows the purification of recombinant β -mannanase, wherein lane 1 is crude enzyme, lane 2 is purified enzyme by Sephacryl S-100HR gel column chromatography, and lane 3 is deglycosylated with N-deglycosylase.
FIG. 3 is a graph showing the determination of the optimum pH of recombinant β -mannanase, wherein (◆) citrate buffer (pH3.0-6.0) and (▲) phosphate buffer (pH 6.0-8.0).
FIG. 4 is a graph showing the pH stability assay of recombinant β -mannanase, wherein (●) glycine-hydrochloric acid buffer (pH 2.0-3.0), (◆) citric acid buffer (pH3.0-6.0), and (▲) phosphoric acid buffer (pH 6.0-8.0).
FIG. 5 is a graph showing the optimum temperature determination of recombinant β -mannanase.
FIG. 6 is a graph of the temperature stability assay of recombinant β -mannanase.
FIG. 7 is a thin layer chromatography analysis chart of AnMan26 as β -mannase hydrolysis product of fenugreek gum, M, M2, M3, M4, M5 and M6 are mannose, mannose disaccharide, mannose trisaccharide, mannose tetrasaccharide, mannose pentasaccharide and mannose hexasaccharide respectively, and 1 is partial hydrolysis fenugreek gum crude sugar solution.
FIG. 8 is a gel permeation chromatography analysis of AnMan26 as a product of β -mannanase hydrolysis of fenugreek gum, 0min representing unhydrolyzed fenugreek gum.
FIG. 9 is a TLC (thin layer chromatography) analysis chart of a product of hydrolyzing cassia gum by using AnMan26 as β -mannase, M, M2, M3, M4, M5 and M6 are respectively mannose, mannose disaccharide, mannose trisaccharide, mannose tetrasaccharide, mannose pentasaccharide and mannose hexasaccharide, and 1 is a crude sugar solution of partially hydrolyzed cassia gum (cassia oligosaccharide).
FIG. 10 is a gel permeation chromatography analysis chart of AnMan26 as a product of β -mannanase hydrolysis of cassia gum 0min represents unhydrolyzed cassia gum.
Fig. 11 is a graph of the effect of partially hydrolyzed fenugreek gum addition on prebiotic yogurt water retention.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.
Streptococcus thermophilus (Streptococcus thermophilus) in the following examples is a product of Chr-Hansen, Denmark.
The Lactobacillus bulgaricus strain (L Acobacillus bulgaricus) in the examples described below is a product of Chr-Hansen, Denmark.
The expression vector pPIC9K in the examples described below was the product of Invitrogen, USA, cat # V17520.
Pichia pastoris GS115 in the following examples is the product of U.S. Invitrogen, cat # C18100.
β -mannanase activity is measured by 3, 5-dinitrosalicylic acid (DNS) method, if not specially indicated, the measuring steps are (1) taking 0.9m L0.5.5 g/100m L locust bean gum solution (prepared by locust bean gum and citric acid buffer solution with pH5.0 and 50 mmol/L), adding 0.1m L appropriately diluted enzyme solution to be measured, placing the enzyme solution in a constant temperature water bath at 45 ℃ for reaction for 10min, (2) after the reaction of the step (1), stopping the reaction by using DNS reagent and reacting with released reducing sugar, and measuring the amount of the reducing sugar (taking mannose as a standard), drawing a standard curve by using the mannose standard solution, wherein the activity unit of β -mannanase is defined as one enzyme activity unit (1U) required by generating 1 mu mol of mannose per minute under the reaction conditions.
The specific enzyme activity was defined as the unit of enzyme activity (U/mg) possessed by 1mg of the protein.
Locust bean gum: galactomannan, available from Sigma-Aldrich, USA under the product number G0753.
Mannose: is a product of Sigma-Aldrich company in the United states, and has the product number of M2069.
Example 1 preparation of recombinant β -mannanase
PCR amplification of β -mannanase gene from Aspergillus niger
β -mannanase gene (MAN26 gene) XM _001397260.1 has a coding region of 954bp, the sequence of which is sequence 1 in the sequence table, and encodes β -mannanase MAN26 shown as sequence 3 in the sequence table, the MAN26 gene is subjected to codon optimization to obtain an optimized gene, which is marked as rMan26 gene, and the sequence of which is sequence 2 in the sequence table.
The rMan26 gene is artificially synthesized, and PCR amplification is carried out by using the rMan26 gene as a template and using specific primers rMan26AF and rMan26AR to obtain a PCR product. The primer sequences are as follows:
rMan26AF：5′-CCGGAATTCGCTTCTAACCAAACTTTGTCTTACG-3' (the restriction sites of the restriction enzyme EcoRI are underlined);
rMan26 AR: 5'-GAATGCGGCCGCTTAAGCACCTTCCCAATTCAAAG-3' (restriction sites for the restriction enzyme NotI are underlined).
Secondly, expression of recombinant β -mannase (AnMan26)
1. The TATA box sequence in the alcohol oxidase promoter AOX1 gene sequence of the pichia pastoris is removed, the modified new promoter is named as ATX1, and the sequence of the modified new promoter is 6 th to 930 th of the sequence 4 in the sequence table. Artificially synthesizing a DNA fragment shown in the sequence 4, recovering a large enzyme digestion fragment by utilizing BglII and BamHI double enzyme digestion, connecting a recovered product with a vector framework obtained by BglII and BamHI double enzyme digestion of an expression vector pPIC9K, and recording an obtained recombinant vector with a correct sequence as pATX 19K.
2. And (3) carrying out double digestion on the PCR product obtained in the first step and the pPAT19K by using EcoRI and NotI respectively, recovering the digestion large fragment of the PCR product and the vector skeleton, connecting, and marking the obtained recombinant vector with the correct sequence as pPAT19K-AnMan 26.
pPAT19K-AnMan26 is a recombinant vector obtained by replacing a DNA fragment between BglII and BamHI recognition sequences of pPIC9K with DNA molecules shown in the 6 th to 930 th sites of the sequence 4 in the sequence table and replacing a DNA fragment between EcoRI and NotI recognition sequences with rMan26 gene pPAT19K-AnMan26 contains a DNA molecule shown in the sequence 5 in the sequence table (namely a fusion gene formed by the rMan26 gene and a partial sequence on the vector and marked as AnMan26 gene), can express a fusion protein shown in the sequence 6 in the sequence table and marked as recombinant β -mannanase (AnMan26), and the expression of the AnMan26 gene is driven by ATX 1.
Wherein, the 280 th-1233 th site of the sequence 5 in the sequence table is the sequence of rMan26 gene, and the 1 st-279 th sites are the sequence on the carrier; the 94 th to 410 th positions of the sequence 6 are the amino acid sequence of MAN26, and the 1 st to 93 th positions are polypeptides encoded by the sequence on the carrier.
3. The SalI linearized recombinant vector pPAT19K-AnMan26 is subjected to alcohol precipitation recovery, is electrically shocked into Pichia pastoris GS115, is coated with an MD (methyl methacrylate) plate, is subjected to inverted culture in an incubator at 30 ℃ for 2-3d until a bacterial colony grows out, and is obtained as a recombinant bacterium, and the recombinant bacterium is marked as GS115-pPAT19K-AnMan 26.
4. The recombinant bacterial cells of step 3 were collected with sterile water, and the appropriately diluted cells were spread on YPD plates (1G/100m L yeast extract, 2G/100m L tryptone, 2G/100m L glucose) containing G418 at concentrations of 1mg/m L, 2mg/m L, 3mg/m L0, 4mg/m L1, respectively, to select single colonies that grew well at different G418 concentrations, and inoculated into 5m L BMGY medium (composed of solvent and solute, solvent 100 mmol/100 m L pH 6.0 phosphate buffer, solute and its concentration in the medium of 1G/100m L yeast extract, 2G/100m L peptone, 1.34G/100m L YNB (Biomol products of USA), 4L 210-5g/100m L biotin and 1g/100m L glycerol), placed in a shaker at 30 ℃ and 200rpm, and cultured with shaking until OD is reached600At 2-6 days, centrifugation was carried out, the supernatant was discarded, and the collected cells were transferred to 10m L BMMY medium (a medium obtained by replacing glycerol in BMGY medium with methanol, which was cultured in the medium)0.5 percent of the total concentration in the base), placing the mixture in a shaker at 30 ℃ and 200rpm for induction expression, adding methanol every 24 hours until the final concentration is 0.5 percent, inducing for 3d, collecting fermentation liquor, centrifuging for 5min at 11510 × g, collecting supernatant, filling the supernatant into a dialysis bag (the cut-off molecular weight is 3.50kDa) and putting the dialysis bag into a 20 mmol/L citric acid buffer solution (pH 5.0) for dialysis overnight, and measuring the activity of the supernatant β -mannanase after dialysis, wherein the yield of the GS115-pPAT19K-AnMan26 enzyme can reach 467U/m L.
5. High density fermentation
The method referred to in "Pica Fermentation Process Guidelines (Version B,053002, Invitrogen)" utilizes the recombinant bacteria obtained in step 4 to perform high density Fermentation, the Fermentation tank is a 5L Fermentation tank, the seed culture medium BMGY, the minimal medium BSM, the glycerol fed-batch medium and the 100% methanol induction medium are all configured according to the method in the reference document, the whole Fermentation Process is divided into four stages of seed liquid culture, basal culture, glycerol fed-batch culture and 100% methanol induction culture.
1) Seed culture, inoculating GS115-pPAT19K-AnMan26 into 500m L triangular flask containing 150m L BMGY medium, culturing at 30 deg.C and 200rpm in shaker for more than 24 hr to obtain OD600Is seed liquid of 2-6.
2) Basic culture, inoculating the seed liquid in 1) into a 5L sterilized fermentation tank (containing 1.5L BSM fermentation basic culture medium), adjusting the pH of the culture medium to 4.0 with ammonia water, adding PTM 14.35m L/L initial fermentation liquid, inoculating the seed liquid by 10%, culturing at 30 deg.C and 600rpm, allowing the DO value to rise rapidly when the dissolved oxygen is exhausted, and allowing glycerol to flow and add into glycerol culture stage when the glycerol is exhausted, wherein PTM1 (CuSO) is added4·7H2O 6.0g，NaI 0.08g，MnSO4·H2O 3.0g，Na2MoO4·2H20.2g of O, 0.02g of boric acid, CoCl20.5g，ZnCl220.0g，FeSO4·7H2O65.0 g, biotin 0.2g, concentrated sulfuric acid 5.0m L, and water 1L.).
3) And (3) glycerol feeding culture, namely feeding an aqueous glycerol solution (the concentration of the glycerol is 500 g/L) at a feeding speed of 30m L/h/L to start fermentation broth, monitoring dissolved oxygen all the time in the stage, keeping DO greater than 20% of the dissolved oxygen by adjusting the feeding speed of the glycerol, controlling the temperature at 30 ℃ and the pH value of the fermentation broth to be 4.0, stopping feeding when the wet weight of the thalli reaches 220 g/L, and fermenting for 4-6h in the whole process.
4) And (3) a 100% methanol feeding stage, namely after stopping feeding the glycerol, starving for about 30min to exhaust the glycerol in the tank, adjusting the pH of the fermentation liquor to 6.0, rotating the speed to 800rpm, starting feeding the 100% methanol to induce enzyme production, increasing the methanol feeding from 3.6m L/h/L initial fermentation liquor to 10.9m L/h/L initial fermentation liquor, monitoring the dissolved oxygen DO in the tank to be more than 20% (if the dissolved oxygen DO is less than 20%, properly reducing the feeding speed), and fermenting for 6-7 days at the temperature of 30 ℃.
In the fermentation process, the wet weight, the protein content and the β -mannase activity of the thalli are measured by sampling, the measuring conditions are 50 mmol/L citric acid buffer solution (pH 5.0), the reaction temperature is 45 ℃, and the measuring steps are as follows:
1) wet weight of the thalli: weighing the mass of the centrifugal tube, and recording the mass as m1Adding 1m L fermentation liquid into a centrifuge tube, centrifuging for 3min by using a centrifuge 11510 × g, discarding the supernatant, and weighing the centrifuge tube and the lower layer substances as m2。
The wet weight calculation formula of the thalli is as follows: wet weight of the strain is m2-m1。
2) Protein content (mg/m L) was determined by L wry method (L wry et al the journal of Biological Chemistry,1951,193(1): 265. sup. 275) using bovine serum albumin as standard protein.
The changes of the wet weight of the thallus, the protein content and the enzyme activity of β -mannase in the fermentation process are shown as A in figure 1, and the SDS-PAGE electrophoresis chart of the protein in the fermentation process is shown as B in figure 1. the enzyme activity reaches the highest when the high-density fermentation is carried out for 168h, the enzyme activity of β -mannase in the supernatant of the fermentation liquid is 22100U/m L, the protein content is 12.0mg/m L, and the wet weight of the thallus is 355.8 g/L.
Thirdly, purifying the recombinant β -mannase
The fermentation supernatant (i.e., crude enzyme solution) obtained by high density fermentation of 10m L for 168 hours was packed in a dialysis bag (cut-off molecular weight: 3.50kDa) and dialyzed overnight in 20 mmol/L citric acid buffer solution (pH 5.0), the dialyzed enzyme solution was concentrated to 0.6m L with a10 kDa ultrafiltration membrane, and the concentrated sample 11510 × g was centrifuged for 5min to collect the supernatant.
The obtained supernatant was applied to Sephacryl S-100HR (1 × 100cm) column equilibrated with buffer (the solvent of the buffer was 20 mmol/L citrate buffer (pH5.0, solute and its concentration in the buffer was 150 mmol/L NaCl), the flow rate during the gel column separation was 0.3m L/min. the fractions with enzyme activity were collected, the obtained purified enzymes were combined and dialyzed against 20 mmol/L citrate buffer (pH 5.0), and the purified recombinant β -mannanase solution (purified enzyme solution) was obtained, the whole purification process is shown in Table 1, and the SDS-PAGE result is shown in FIG. 2, wherein lane 1 is crude enzyme solution, lane 2 is purified enzyme by Sephacryl S-100HR gel column chromatography, and lane 3 is deglycosylated with N-deglycosylase.
The result shows that both the crude enzyme solution and the purified enzyme are single molecular weight bands, the corresponding molecular weight is 65.3kDa, the corresponding molecular weight is recombinant β -mannase, the specific enzyme activity is 2869.0U/mg, whether the recombinant β -mannase is glycosylated or not is detected, the obtained purified enzyme is treated by N-deglycosylation enzyme, the deglycosylated recombinant β -mannase solution shows a single molecular weight band, the corresponding molecular weight is 38.7kDa, the size is consistent with the expected size, and the specific enzyme activity of the recombinant β -mannase is 191.2U/mg after deglycosylation enzyme deglycosylation.
The deglycosylation of the N-deglycosylation enzyme comprises the following steps:
1) mu.g of pure enzyme was added to the centrifuge tube, 1. mu. L glycoprotein denaturation buffer (10 ×) was added, water was added to the total reaction volume to 10. mu. L, and the pure enzyme was denatured in a boiling water bath for 10 min.
2) 1) adding 2 μ L GlycoBuffer3(10 ×) and 2 μ L deglycosylation enzyme (product of NEB company, USA, product No. P0702S), supplementing the reaction volume to 20 μ L with water, placing in 37 deg.C water bath for 60min, and detecting protein by SDS-PAGE electrophoresis.
Among them, glycoprotein denaturation buffer (10 ×) (product of NEB, USA, No. P0702S) GlycoBuffer3(10 ×) (product of NEB, USA, No. P0702S).
TABLE 1 purification of recombinant β -mannanase
Purification step Total enzyme activity (U) Total protein (mg) Specific activity (U/mg) Recovery (%) Multiple of purification
Crude enzyme solution 221000 120 1841.7 100 1.0
Pure enzyme liquid 65700 22.9 2869.0 29.7 1.6
In Table 1, the recovery rate is the percentage of the total enzyme activity of the pure enzyme to the total enzyme activity of the crude enzyme solution. The purification fold 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 the recombinant protein AnMan26 as β -mannanase
The buffers used were specifically glycine-hydrochloric acid buffers (pH 2.0, 2.5 and 3.0), citric acid buffers (pH3.0, 3.5, 4.0, 4.5, 5.0, 5.5 and 6.0) and phosphate buffers (pH 6.0, 6.5, 7.0, 7.5 and 8.0), all of which had a concentration of 50 mmol/L.
Firstly, the optimum pH value of the recombinant β -mannase
The purified recombinant β -mannase solution prepared in example 1 was used as a sample solution to be tested, and the enzyme activity was measured by taking 0.9m L0.5 g/100m L locust bean gum solution (prepared from locust bean gum and different buffer systems to be tested with a pH range of 3.0-8.0), adding 0.1m L diluted enzyme solution to be tested, placing the obtained reaction systems at 30 ℃ for reaction for 10 minutes, measuring the enzyme activity, recording the highest enzyme activity as 100%, calculating the relative activity of β -mannase under each pH condition, and the optimal pH of the recombinant β -mannase was 5.0 as shown in FIG. 3.
The recombinant β -mannanase prepared in example 1 has relative enzyme activities of 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% in a citrate buffer at pH3.0, 3.5, 4.0, 4.5, 5.0, 5.5 and 6.0, respectively, and a phosphate buffer at pH 6.0, 6, 7.0, 7.0.5% and 8.0, respectively.
II, pH stability of recombinant β -mannase
Diluting the purified recombinant β -mannase solution prepared in example 1 by proper times with the buffer solutions to enable the recombinant β -mannase to be in the buffer solutions with the pH values respectively, then placing the diluted enzyme solution in a water bath at 30 ℃ for heat preservation for 30min, then quickly taking out and immediately carrying out ice bath for 30min to determine the residual β -mannase activity, wherein the contrast is the dilution of the recombinant β -mannase solution (namely the dilution of the recombinant β -mannase pure enzyme solution obtained in example 1) which is not subjected to the treatment (the treatment refers to the water bath at 30 ℃ for 30min and then carrying out ice bath for 30min), and the relative activity of the enzyme treated by the buffer solutions with different pH values is calculated by taking the enzyme activity of the contrast as 100%, and the result is shown in figure 4, wherein the enzyme is stable within the pH range of 2.5-6.0, and the residual enzyme activity is still kept above 80% after the treatment for 30 min.
The recombinant β -mannanase prepared in example 1 has relative enzyme activities of 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%, 59.59.5% + -2.0% and 26.0% after treatment with glycine-hydrochloric acid buffer at pH 2.0, 2.5, 4.0, 3.0, 4.0, 4.5, 5, 5.3.3.3.3.0, 6% + -2.1.3.3.3.3.3.0, and 8.0.
Thirdly, the optimum temperature of the recombinant β -mannase
The recombinant β -mannase solution prepared in example 1 was used as an enzyme solution to be tested, a citric acid buffer system with pH of 5.0 and 50 mmol/L was used to react at different temperatures within the range of 30 ℃ to 80 ℃, the enzyme activities at different temperatures were measured, the highest enzyme activity was taken as 100%, and the relative activities at each reaction temperature were calculated, the results are shown in FIG. 5, and the optimum temperature of the recombinant β -mannase was taken as 45 ℃.
The recombinant β -mannanase prepared in example 1 has relative enzyme activities of 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% at 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃ and 80 ℃, respectively.
Fourthly, temperature stability of recombinant β -mannase
Diluting the recombinant β -mannase solution prepared in example 1 to a proper multiple by using a citric acid buffer solution with pH of 5.0 and 50 mmol/L, placing the solution in water bath at different temperatures for 30min (the water bath temperature is 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃ or 80 ℃), quickly taking out and carrying out ice bath for 30min, and determining the residual β -mannase activity, wherein the comparison is the recombinant β -mannase solution diluent which is not treated in the steps (the treatment refers to the water bath for 30min, and the quick ice bath for 30min), the enzyme activity of the comparison is taken as 100%, the relative activity result of the enzyme solution to be detected after the treatment at each temperature is shown in figure 6, and the activity of the recombinant β -mannase at 40 ℃ or higher than 80% is kept.
The recombinant β -mannanase prepared in example 1 has relative enzyme activities of 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% after treatment at 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, 60 deg.C, 70 deg.C, 75 deg.C and 80 deg..
Fifth, determination of substrate specificity
The recombinant β -mannase solution prepared in example 1 was used as an enzyme solution to be tested to determine β -mannase activities of different substrates, wherein the substrates were locust bean gum, konjac flour, cassia gum, fenugreek gum and carboxymethyl cellulose, and the concentrations of the substrates in the reaction system were all 0.5g/100m L.
Wherein the rhizoma Amorphophalli powder is produced by KJ-30 by Kjen Konjac science and technology Limited in Hubei province; the cassia gum is a product of Beijing guar technologies, Inc.; the fenugreek gum is a product of Beijing guar technologies, Inc.; the carboxymethyl cellulose is manufactured by Sigma-Aldrich company in the United states, and the product number is C4888-500G.
The enzyme activity of the recombinant β -mannase on locust bean gum is 100%, and the relative activity and specific enzyme activity of the enzyme on the locust bean gum, konjac flour, cassia gum, fenugreek gum and carboxymethyl cellulose are calculated.
The result is shown in Table 2, the recombinant β -mannase has the highest specific enzyme activity to locust bean gum of 2869.0U/mg, the second is konjaku flour of 1905.0U/mg, the lowest specific enzyme activity to fenugreek gum of 341.1U/mg, and has no hydrolysis capacity to carboxymethyl cellulose.
TABLE 2 substrate specificity of recombinant β -mannanase
Figure BDA0002472913750000121
"-" indicates no detectable activity
Example 3 production of partially hydrolyzed fenugreek Gum by hydrolysis of fenugreek Gum with recombinant β -mannanase (AnMan26)
Weighing 10g of fenugreek gum, stirring at 200rpm, dissolving in a citric acid buffer solution with the pH value of 100m L of 5.0 and 50 mmol/L to prepare 10g/100m L of a fenugreek gum solution, adding the recombinant β -mannase of example 1 into the fenugreek gum solution to obtain a reaction system, adding 200U of recombinant β -mannase into each gram of fenugreek gum, placing the reaction system at 40 ℃ for hydrolysis for 8 hours to obtain a hydrolysate, after the hydrolysis is completed, carrying out water bath enzyme deactivation on the reaction system for 20 minutes, centrifuging for 10 minutes at 11510 g of 11510 ×, collecting precipitates and supernate, wherein the precipitates are hydrolysate residues, and the supernate is a part hydrolyzed fenugreek gum crude sugar solution.
And (3) determining the content of reducing sugar in the crude sugar solution by using a3, 5-dinitrosalicylic acid method, and calculating the yield of the reducing sugar, the hydrolysis rate and the product yield. The composition of the crude sugar solution was qualitatively analyzed by thin layer chromatography. The weight average molecular weight of the partially hydrolyzed fenugreek gum was analyzed by gel permeation chromatography.
The calculation formulas of the reducing sugar yield, the hydrolysis rate and the product yield are respectively as follows:
the yield (%) of reducing sugar is equal to the total reducing sugar mass in the system after hydrolysis/dry weight × 100% of the raw material added before hydrolysis;
the hydrolysis rate (%) - (adding the dry weight of the raw material before hydrolysis-the dry weight of the hydrolysate residue)/adding the dry weight of the raw material before hydrolysis × 100%, wherein the dry weight of the hydrolysate residue is obtained by drying the hydrolysate residue in an oven at 105 ℃ until the constant weight is measured;
the yield (%) of the product, which is determined by placing the partially hydrolyzed fenugreek gum crude sugar solution in an oven at 105 ℃ and drying to constant weight, was × 100% based on the dry weight of the starting material added before hydrolysis.
Thin layer chromatography detection conditions: the spreading agent is n-butyl alcohol: ethanol: water 2: 1: 1, the color developing agent is methanol: sulfuric acid 95: 5. the standard control is a mixture of mannose, mannose disaccharide, mannose trisaccharide, mannose tetrasaccharide, mannose pentasaccharide and mannose hexasaccharide. A proper amount of samples to be detected are spotted on a thin-layer chromatography chromatographic analysis plate, a spreading agent is put into the samples to be spread for two times, then the samples are completely soaked and dried by a color developing agent, and the samples are baked and developed at the temperature of 130 ℃.
Wherein, the mannose is produced by Sigma-Aldrich in America, and the product number is M2069; the mannose disaccharide, the mannose trisaccharide, the mannose tetrasaccharide, the mannose pentasaccharide and the mannose hexasaccharide are all Ireland Megazyme products, and the product numbers are O-MBI, O-MTR, O-MTE, O-MPE and O-MHE respectively.
Gel permeation chromatography detection conditions: TSKgel GMPWXLWater phase gel chromatographic column (7.8 × 300mM), column temperature 60 deg.C, RID detector, sample size 20 μ L, and mobile phase 100mM NaNO3The flow rate was 0.6m L/min, and a calibration curve was prepared using a polyoxyethylene standard (molecular weight: 300-500000Da, available from Tosoh corporation, Japan).
The result shows that the hydrolysis rate of AnMan26 hydrolysis fenugreek gum is 82.2%, the yield of reducing sugar is 43.8%, and the yield of the product is 73.5%, the result of thin layer chromatography analysis is shown in figure 7(Mn is a standard control, and mannose, mannose tetraose, mannose pentaose, and mannose hexaose are sequentially arranged from top to bottom), the result shows that the recombinant β -mannase hydrolysis fenugreek gum generates mannan oligosaccharide with the polymerization degree of 2-6, and a large amount of mannan with the polymerization degree of more than 6.
The gel permeation chromatography analysis result is shown in FIG. 8, and the weight average molecular weight of fenugreek gum is 4.9 × 106Da, weight average molecular weight of partially hydrolyzed fenugreek gum rapidly decreased after AnMan26 was added, and weight average molecular weight was 1.8 × 10 after hydrolysis was completed3Da, the average polymerization degree of the partially hydrolyzed fenugreek gum is less than or equal to 10.
Therefore, the partially hydrolyzed fenugreek gum with the yield of 73.5 percent can be produced by hydrolyzing the fenugreek gum by adopting the recombinant β -mannase, the components of the partially hydrolyzed fenugreek gum are mannan oligosaccharide with the polymerization degree of between 2 and 6, a large amount of mannan with the polymerization degree of more than 6, the average polymerization degree of the partially hydrolyzed fenugreek gum is less than or equal to 10, and the weight-average molecular weight is 1.8 × 103Da。
EXAMPLE 4 hydrolysis of Cassia Torae Gum with recombinant β -mannase (AnMan26) to produce partially hydrolyzed Cassia Torae Gum (Cassia Torae oligosaccharide)
Weighing 10g of cassia seed gum, dissolving the cassia seed gum in a citric acid buffer solution with the pH value of 100m L of 5.0 and 50 mmol/L under the stirring of 200rpm, preparing a cassia seed gum solution with the concentration of 10g/100m L, placing the cassia seed gum solution in the citric acid buffer solution at 40 ℃, preserving the temperature for 30min to ensure that the cassia seed gum is uniformly dispersed in the buffer solution, then adding recombinant β -mannase (AnMan26) into the reaction system, adding 800U/g of cassia seed gum, hydrolyzing at 40 ℃, continuously stirring the hydrolysis system at the stirring speed of 600rpm in the hydrolysis process, hydrolyzing for 8h to obtain a hydrolysis solution, after the hydrolysis is finished, inactivating enzymes in a boiling water bath of the reaction system for 20min to obtain an enzymatic hydrolysis solution, centrifuging the enzymatic hydrolysis solution for 10min in 11510 × g, and collecting a supernatant liquid sugar, namely.
And (3) determining the content of reducing sugar in the crude sugar solution by using a3, 5-dinitrosalicylic acid method, and calculating the yield of the reducing sugar, the hydrolysis rate and the product yield. The composition of the crude sugar solution was qualitatively analyzed by thin layer chromatography. The weight average molecular weight of the partially hydrolyzed cassia gum (cassia oligosaccharide) was analyzed by gel permeation chromatography. The calculation methods of the reducing sugar yield, hydrolysis rate and product yield, thin layer chromatography conditions and gel permeation chromatography analysis conditions were the same as in example 3.
The result shows that AnMan26 has the hydrolysis rate of 83.3%, the reducing sugar yield of 43.9% and the product yield of 74.7% when the cassia gum is hydrolyzed partially by thin-layer chromatography, the result is shown in figure 9(Mn is a standard control, and mannose, mannose trisaccharide, mannose tetrasaccharide, mannose pentasaccharide and mannose hexasaccharide are sequentially arranged from top to bottom), and the result shows that the recombinant β -mannase hydrolyzes the cassia gum to mainly generate mannose, mannose disaccharide, mannose trisaccharide, mannose tetrasaccharide, mannose oligosaccharide with the polymerization degree of 4-6 and a small amount of mannan with the polymerization degree of more than 6.
The gel permeation chromatography analysis result is shown in FIG. 10, and the weight average molecular weight of the cassia gum is 1.7 × 106Da, after AnMan26 is added, part of water solves the problem that the weight average molecular weight of the gelatin is rapidly reduced, and after hydrolysis is finished, the weight average molecular weight is 1188 Da.
Therefore, the recombinant β -mannase can hydrolyze cassia seed gum to produce 74.7% yield of partial hydrolyzed gelatin (cassia seed oligosaccharide), the components of the partial hydrolyzed gelatin are mainly mannan oligosaccharide with the polymerization degree of 1-6 and a small amount of mannan with the polymerization degree of more than 6, the average polymerization degree of the partial hydrolyzed cassia seed gum is less than 7, and the weight average molecular weight is 1188 Da.
Example 5 production of prebiotic yogurt Using partially hydrolyzed mannan
The partially hydrolyzed mannan used in this example was the partially hydrolyzed fenugreek gum obtained in example 3. And (3) spray-drying the partially hydrolyzed fenugreek gum liquid to obtain partially hydrolyzed fenugreek gum powder (the spray-drying conditions are that the air inlet temperature is 180 ℃ and the air outlet temperature is 80 ℃). The prebiotics yoghourt produced by partially hydrolyzing mannan is prepared from the following raw materials (1000 parts by weight): 925-910 parts of fresh milk, 5-20 parts of partially hydrolyzed tonka bean gum powder and 70 parts of cane sugar.
The preparation method of the prebiotics yoghourt comprises the following steps:
the first step is as follows: mixing fresh milk, partially hydrolyzed tonka-bean gum and sucrose uniformly according to the weight parts, preheating at 65 ℃ until the materials are completely dissolved, homogenizing under 40MPa, and pasteurizing at 95 ℃ for 5min to obtain a mixture.
Secondly, after the mixture is cooled to 40 ℃, streptococcus thermophilus (streptococcus thermophilus) and lactobacillus bulgaricus (L actinobacillus bulgaricus) are added for inoculation, and the viable bacteria content of the two added bacteria in the system is 1 × 106cfu/ml。
The third step: placing the inoculated mixture in a constant temperature fermentation chamber, and fermenting at 42 deg.C for 4h (to pH of about 4.6).
The fourth step: demulsifying the fermented prebiotics yoghourt for 1min at 350rpm, cooling to 20 ℃, putting into a refrigerator, and aging for 16h to obtain the yoghourt.
Evaluation of the effects:
1. sensory evaluation: please refer to 20 persons who have experience in sensory evaluation of yogurt to perform sensory evaluation on the mouthfeel, flavor, color, viscosity and overall evaluation of yogurt. The grading range is 0-10 points, wherein the taste is smooth and high (7-10 points), and the granular sensation is low (0-3 points); the fermentation flavor is light and low (0-3 min), and the fermentation flavor is rich and high (7-10 min); the color is white at 0 point and yellow at 10 points; the stronger the sticky feeling, the higher the score; the overall evaluation is scored according to personal preferences.
2. Water holding capacity: measuring yogurt after aging for 16h, weighing sterilized centrifuge tube, and recording as m1Adding 20g of yoghourt into each centrifugal tube, and weighing the total mass as m2Centrifuging at 3000rpm for 10min, discarding supernatant, and weighing the centrifuge tube and lower layer material as m3。
The water holding capacity calculation formula is as follows: water holding capacity (%) - (m)3-m1)/(m2-m1)×100％。
3. Viable count
The viable count detection is carried out according to the Chinese method of the national standard GB4789.35-2016 lactic acid bacteria detection in food microbiology, which comprises the following steps:
and counting the streptococcus thermophilus, namely selecting 2-3 continuous proper dilutions, sucking 1m L sample uniform liquid into a sterilization plate for each dilution, pouring the MC culture medium cooled to 48 ℃ into the plate by about 15m L after the dilutions are moved into the plate, rotating the plate to mix uniformly, carrying out aerobic culture at 36 +/-1 ℃ for 72 +/-2 h, and counting after culture.
And (3) counting the lactobacilli, namely selecting 2-3 continuous proper dilutions, sucking 1m L sample uniform liquid in each dilution, pouring the MRS agar culture medium cooled to 48 ℃ into a dish about 15m L after the dilutions are moved into the dish, rotating the dish to uniformly mix, carrying out anaerobic culture at 36 +/-1 ℃ for 72 +/-2 h, and counting after culture.
4. Results
Table 3 shows sensory evaluation data of partially hydrolyzed fenugreek gum yogurt, and it can be seen from the weight of table 3 that the total score of yogurt with the addition of partially hydrolyzed fenugreek gum is between 5.3 and 6.3, which is lower than the total score of yogurt without the addition of partially hydrolyzed fenugreek gum by 6.6, and the main difference is that the flavor and viscosity scores of yogurt with the addition of partially hydrolyzed fenugreek gum are lower than those of yogurt without the addition of partially hydrolyzed fenugreek gum. However, the total score of the yoghourt added with 1% of the partially hydrolyzed fenugreek gum is 6.3, which is close to the total score of the yoghourt without the partially hydrolyzed fenugreek gum, and the yoghourt can be applied to the preparation of prebiotics yoghourt.
TABLE 3 organoleptic evaluation of partially hydrolyzed fenugreek gum yogurt
Figure BDA0002472913750000151
Note: the mix in table 1 was made from the following raw materials (1000 parts) by weight: 925-910 parts of fresh milk, 5-20 parts of partially hydrolyzed tonka bean gum powder and 70 parts of cane sugar.
Fig. 11 shows the water holding capacity of the partially hydrolyzed fenugreek gum yogurt, and it can be seen from fig. 11 that the water holding capacity of the yogurt with the partially hydrolyzed fenugreek gum added is between 36.8% and 38.6%, which is higher than the water holding capacity of the yogurt without the partially hydrolyzed fenugreek gum added by 34.7%. The water holding capacity of the yoghourt is increased along with the increase of the adding amount of the partially hydrolyzed fenugreek gum within the range of 0-2.0% of the adding amount of the partially hydrolyzed fenugreek gum, which shows that the adding amount of the partially hydrolyzed fenugreek gum is beneficial to the increase of the water holding capacity of the yoghourt, the adding amount of the partially hydrolyzed fenugreek gum can effectively enhance the gel structure of the yoghourt, the yoghourt can effectively retain water, whey is prevented from being separated out, and the tissue state of the yoghourt is improved.
Table 4 shows the effect of partially hydrolyzed fenugreek gum yogurt on the viable count of Lactobacillus and Streptococcus thermophilus from Table 4, it can be seen that the viable count of Lactobacillus and Streptococcus thermophilus in yogurt without partially hydrolyzed fenugreek gum added thereto is 1.1 × 108CFU/m L and 2.75 × 108CFU/m L, and the viable count of lactobacillus in yogurt added with partially hydrolyzed fenugreek gum is 4.05 × 108CFU/mL-1.075×109The number of viable streptococcus thermophilus is between CFU/m L and 2.82 × 108CFU/mL-6.9×108Within the range of 0-2.0% of the added amount of the partial hydrolysis fenugreek gum, the number of the viable bacteria of the lactobacillus in the yoghurt is increased and then decreased along with the increase of the added amount of the partial hydrolysis fenugreek gum, the number of the viable bacteria of the streptococcus thermophilus in the yoghurt is increased along with the increase of the added amount of the partial hydrolysis fenugreek gum, and the number of the viable bacteria of the lactobacillus in the yoghurt is up to 1.075 × 10 when 1.5% of the partial hydrolysis fenugreek gum is added9CFU/m L, Activity of Streptococcus thermophilus in yogurt with addition of 2.0% partially hydrolyzed fenugreek gumThe maximum number of bacteria is 6.9 × 108CFU/m L, the number of live bacteria of lactobacilli and the number of live bacteria of streptococcus thermophilus in the partially hydrolyzed fenugreek gum yoghourt are higher than the number of live bacteria of lactobacilli and the number of live bacteria of streptococcus thermophilus in the yoghourt without the partially hydrolyzed fenugreek gum, and therefore the number of live bacteria of lactobacilli and streptococcus thermophilus in the yoghourt is effectively increased by adding the partially hydrolyzed fenugreek gum, and the quality of the yoghourt is improved.
TABLE 4 Effect of partially hydrolyzed fenugreek gum yogurt on Lactobacillus and Streptococcus thermophilus
Figure BDA0002472913750000161
<110> university of agriculture in China
<120> preparation method of β -mannase and application of β -mannase in preparation of partially hydrolyzed mannan
<160>6
<170>PatentIn version 3.5
<210>1
<211>954
<212>DNA
<213> Aspergillus niger (Aspergillus niger)
<400>1
gcttccaacc agactctgtc ctatggcaac attgacaagt cggccacccc cgaagccagg 60
gcgctcctga agtacatcca gcttcagtac ggctcgcact acatctcggg ccagcaggac 120
attgacagct ggaactgggt cgagaagaac attggtgtgg cccctgcgat tctcggcagt 180
gacttcacct actactcgcc ttcggctgtt gctcacggcg gcaagtctca cgccgtcgag 240
gatgtgattc agcacgccgg ccgcaatgga atcaatgccc tggtgtggca ttggtacgct 300
cccacctgcc tgctcgatac cgccaaggag ccgtggtaca aggggttcta caccgaagcc 360
acctgcttca acgtgtctga agccgtcaac gaccatggca acggcaccaa ctacaagctt 420
ctgctgcgtg atatcgacgc catcgctgct cagatcaagc gtctggatca ggccaaagtg 480
cccatcctct tccgcccgct ccacgagccc gagggtggct ggttctggtg gggtgcccag 540
ggtcctgctc ccttcaagaa gctgtgggat atcctctacg accgcatcac tcgctaccac 600
aacctccaca acatggtctg ggtttgcaac actgctgatc cagcgtggta tcccggaaac 660
gacaagtgtg acattgccac catcgatcac tatcccgccg ttggtgacca cggagtcgcg 720
gccgaccagt acaagaagct ccagaccctt accaacaacg agagagtttt ggctatggca 780
gaagtcggtc ccattccgga ccccgataag caggctagtg agaacgtcaa ctgggcttac 840
tggatggttt ggtctggtga cttcatcgag gatggcaagc agaaccctaa ccagttcctg 900
cacaaggtgt acaacgacac ccgtgttgtg gctctgaact gggagggggc ttaa 954
<210>2
<211>954
<212>DNA
<213> Artificial sequence (Artificial sequence)
<400>2
gcttctaacc aaactttgtc ttacggtaac atcgataagt ctgctactcc tgaagctaga 60
gctttgttga agtacatcca attgcaatac ggttctcatt acatctctgg tcaacaagat 120
atcgattctt ggaactgggt tgagaagaat attggtgttg ctcctgctat cttgggttct 180
gatttcactt actactctcc atctgctgtt gctcatggtg gtaaatctca cgctgttgaa 240
gatgttattc aacatgctgg tagaaacggt attaatgctt tggtttggca ctggtacgct 300
cctacttgtt tgttggatac tgctaaggag ccatggtaca aaggttttta tactgaagct 360
acttgtttca acgtttctga ggctgttaac gatcatggta acggtactaa ctacaagttg 420
ttgttgagag atattgatgc tattgctgct caaattaaga gattggatca agctaaagtt 480
ccaattttgt tcagaccttt gcacgaacca gagggtggtt ggttttggtg gggtgctcaa 540
ggtccagctc ctttcaagaa attgtgggat atcttgtacg atagaatcac tagataccat 600
aacttgcaca acatggtttg ggtttgtaac actgctgatc ctgcttggta ccctggtaac 660
gataagtgtg atatcgctac tatcgatcat taccctgctg ttggagatca cggtgttgct 720
gctgatcaat acaagaaatt gcaaactttg actaacaacg aaagagtttt ggctatggct 780
gaggttggtc caattcctga tccagataaa caagcttctg aaaacgttaa ttgggcttac 840
tggatggttt ggtctggaga ttttattgag gatggtaaac aaaacccaaa ccaattcttg 900
cacaaggttt acaacgatac tagagttgtt gctttgaatt gggaaggtgc ttaa 954
<210>3
<211>317
<212>PRT
<213> Artificial sequence (Artificial sequence)
<400>3
Ala Ser Asn Gln Thr Leu Ser Tyr Gly Asn Ile Asp Lys Ser Ala Thr
1 5 10 15
Pro Glu Ala Arg Ala Leu Leu Lys Tyr Ile Gln Leu Gln Tyr Gly Ser
20 25 30
His Tyr Ile Ser Gly Gln Gln Asp Ile Asp Ser Trp Asn Trp Val Glu
35 40 45
Lys Asn Ile Gly Val Ala Pro Ala Ile Leu Gly Ser Asp Phe Thr Tyr
50 55 60
Tyr Ser Pro Ser Ala Val Ala His Gly Gly Lys Ser HisAla Val Glu
65 70 75 80
Asp Val Ile Gln His Ala Gly Arg Asn Gly Ile Asn Ala Leu Val Trp
85 90 95
His Trp Tyr Ala Pro Thr Cys Leu Leu Asp Thr Ala Lys Glu Pro Trp
100 105 110
Tyr Lys Gly Phe Tyr Thr Glu Ala Thr Cys Phe Asn Val Ser Glu Ala
115 120 125
Val Asn Asp His Gly Asn Gly Thr Asn Tyr Lys Leu Leu Leu Arg Asp
130 135 140
Ile Asp Ala Ile Ala Ala Gln Ile Lys Arg Leu Asp Gln Ala Lys Val
145 150 155 160
Pro Ile Leu Phe Arg Pro Leu His Glu Pro Glu Gly Gly Trp Phe Trp
165 170 175
Trp Gly Ala Gln Gly Pro Ala Pro Phe Lys Lys Leu Trp Asp Ile Leu
180 185 190
Tyr Asp Arg Ile Thr Arg Tyr His Asn Leu His Asn Met Val Trp Val
195 200 205
Cys Asn Thr Ala Asp Pro Ala Trp Tyr Pro Gly Asn Asp Lys Cys Asp
210 215 220
Ile Ala Thr Ile Asp His Tyr Pro Ala Val Gly Asp His Gly Val Ala
225 230 235 240
Ala Asp Gln Tyr Lys Lys Leu Gln Thr Leu Thr Asn Asn Glu Arg Val
245 250 255
Leu Ala Met Ala Glu Val Gly Pro Ile Pro Asp Pro Asp Lys Gln Ala
260 265 270
Ser Glu Asn Val Asn Trp Ala Tyr Trp Met Val Trp Ser Gly Asp Phe
275 280 285
Ile Glu Asp Gly Lys Gln Asn Pro Asn Gln Phe Leu His Lys Val Tyr
290 295 300
Asn Asp Thr Arg Val Val Ala Leu Asn Trp Glu Gly Ala
305 310 315
<210>4
<211>941
<212>DNA
<213> Artificial sequence (Artificial sequence)
<400>4
agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 60
gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 120
tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 180
agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 240
acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 300
tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 360
agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 420
gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 480
ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcgcca taccgtttgt 540
cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 600
ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 660
ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 720
gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaaa 780
cagaaggaag ctgccctgtc ttaaaccttt ttttttatca tcattattag cttactttca 840
taattgcgac tggttccaat tgacaagctt ttgattttaa cgacttttaa cgacaacttg 900
agaagatcaa aaaacaacta attattcgaa ggatccaaac g 941
<210>5
<211>1233
<212>DNA
<213> Artificial sequence (Artificial sequence)
<400>5
atgagatttc cttcaatttt tactgcagtt ttattcgcag catcctccgc attagctgct 60
ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120
tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180
aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240
tctctcgaga aaagagaggc tgaagcttac gtagaattcg cttctaacca aactttgtct 300
tacggtaaca tcgataagtc tgctactcct gaagctagag ctttgttgaa gtacatccaa 360
ttgcaatacg gttctcatta catctctggt caacaagata tcgattcttg gaactgggtt 420
gagaagaata ttggtgttgc tcctgctatc ttgggttctg atttcactta ctactctcca 480
tctgctgttg ctcatggtgg taaatctcac gctgttgaag atgttattca acatgctggt 540
agaaacggta ttaatgcttt ggtttggcac tggtacgctc ctacttgttt gttggatact 600
gctaaggagc catggtacaa aggtttttat actgaagcta cttgtttcaa cgtttctgag 660
gctgttaacg atcatggtaa cggtactaac tacaagttgt tgttgagaga tattgatgct 720
attgctgctc aaattaagag attggatcaa gctaaagttc caattttgtt cagacctttg 780
cacgaaccag agggtggttg gttttggtgg ggtgctcaag gtccagctcc tttcaagaaa 840
ttgtgggata tcttgtacga tagaatcact agataccata acttgcacaa catggtttgg 900
gtttgtaaca ctgctgatcc tgcttggtac cctggtaacg ataagtgtga tatcgctact 960
atcgatcatt accctgctgt tggagatcac ggtgttgctg ctgatcaata caagaaattg 1020
caaactttga ctaacaacga aagagttttg gctatggctg aggttggtcc aattcctgat 1080
ccagataaac aagcttctga aaacgttaat tgggcttact ggatggtttg gtctggagat 1140
tttattgagg atggtaaaca aaacccaaac caattcttgc acaaggttta caacgatact 1200
agagttgttg ctttgaattg ggaaggtgct taa 1233
<210>6
<211>410
<212>PRT
<213> Artificial sequence (Artificial sequence)
<400>6
Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser
1 5 10 15
Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln
20 25 30
Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe
35 40 45
Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu
50 55 60
Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val
65 70 75 80
Ser Leu Glu Lys Arg Glu Ala Glu Ala Tyr Val Glu Phe Ala Ser Asn
85 90 95
Gln Thr Leu Ser Tyr Gly Asn Ile Asp Lys Ser Ala Thr Pro Glu Ala
100 105 110
Arg Ala Leu Leu Lys Tyr Ile Gln Leu Gln Tyr Gly Ser His Tyr Ile
115 120 125
Ser Gly Gln Gln Asp Ile Asp Ser Trp Asn Trp Val Glu Lys Asn Ile
130 135 140
Gly Val Ala Pro Ala Ile Leu Gly Ser Asp Phe Thr Tyr Tyr Ser Pro
145 150 155 160
Ser Ala Val Ala His Gly Gly Lys Ser His Ala Val Glu Asp Val Ile
165 170 175
Gln His Ala Gly Arg Asn Gly Ile Asn Ala Leu Val Trp His Trp Tyr
180 185 190
Ala Pro Thr Cys Leu Leu Asp Thr Ala Lys Glu Pro Trp Tyr Lys Gly
195 200 205
Phe Tyr Thr Glu Ala Thr Cys Phe Asn Val Ser Glu Ala Val Asn Asp
210 215 220
His Gly Asn Gly Thr Asn Tyr Lys Leu Leu Leu Arg Asp Ile Asp Ala
225 230 235 240
Ile Ala Ala Gln Ile Lys Arg Leu Asp Gln Ala Lys Val Pro Ile Leu
245 250 255
Phe Arg Pro Leu His Glu Pro Glu Gly Gly Trp Phe Trp Trp Gly Ala
260 265 270
Gln Gly Pro Ala Pro Phe Lys Lys Leu Trp Asp Ile Leu Tyr Asp Arg
275 280 285
Ile Thr Arg Tyr His Asn Leu His Asn Met Val Trp Val Cys Asn Thr
290 295 300
Ala Asp Pro Ala Trp Tyr Pro Gly Asn Asp Lys Cys Asp Ile Ala Thr
305 310 315 320
Ile Asp His Tyr Pro Ala Val Gly Asp His Gly Val Ala Ala Asp Gln
325 330 335
Tyr Lys Lys Leu Gln Thr Leu Thr Asn Asn Glu Arg Val Leu Ala Met
340 345 350
Ala Glu Val Gly Pro Ile Pro Asp Pro Asp Lys Gln Ala Ser Glu Asn
355 360 365
Val Asn Trp Ala Tyr Trp Met Val Trp Ser Gly Asp Phe Ile Glu Asp
370 375 380
Gly Lys Gln Asn Pro Asn Gln Phe Leu His Lys Val Tyr Asn Asp Thr
385 390 395 400
Arg Val Val Ala Leu Asn Trp Glu Gly Ala
405 410

Claims (10)

1. A process for preparing the protein with β -mannanase activity includes such steps as introducing the rMan26 gene shown in sequence 2 in sequence table into biological cell to obtain recombinant cell, culturing said recombinant cell, and expressing the rMan26 gene to obtain the protein with β -mannanase activity.
2. The method of claim 1, wherein: the expression of the rMan26 gene is driven by a promoter named as ATX1, and the ATX1 is a DNA molecule shown in the 6 th to 930 th positions of a sequence 4 in a sequence table.
3. The method according to claim 1 or 2, characterized in that: the protein is A1), A2) or A3) as follows:
A1) the amino acid sequence is the protein of sequence 3;
A2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in an amino acid sequence shown in a sequence 3 in a sequence table and has β -mannanase function;
A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2).
4. A method according to any one of claims 1-3, characterized in that: the biological cells are microbial cells;
further, the microbial cell is a fungus;
still further, the fungus is a yeast.
5. A protein having β -mannanase activity prepared by the method of any one of claims 1-4.
6. Any of the following products:
x1, the recombinant cell of any one of claims 1-4;
x2, the rMan26 gene of any one of claims 1-4;
x3, the ATX1 of any one of claims 1-4;
x4, a kit consisting of the rMan26 gene of any one of claims 1-4 and the ATX 1;
x5, a recombinant vector comprising the rMan26 gene of any one of claims 1-4 and/or the ATX 1.
7. Use of the protein of any one of claims 1-4 for any one of:
a1) use as β -mannanase;
a2) the application in preparing products with β -mannase activity;
a3) the application in preparing mannan oligosaccharide;
a4) the application in preparing the product for preparing the mannan oligosaccharide;
a5) the application in degrading mannan;
a6) the application in preparing products for degrading mannan;
a7) use in the preparation of partially hydrolysed mannans;
a8) use in the preparation of a product for the preparation of a partially hydrolysed mannan;
a9) the application in degrading vegetable gum;
a10) the application in the preparation of products for degrading vegetable gums.
8. Use according to claim 7, characterized in that: the vegetable gum is b1) or b2) or b3) or b4) or b 5): b1) locust bean gum; b2) cassia seed gum; b3) fenugreek gum; b4) konjaku flour; b5) a mannan-rich vegetable gum.
9. Use of the protein according to any one of claims 1 to 4 for the preparation of partially hydrolysed mannans for the preparation of prebiotic yoghurt.
10. Use according to claim 9, characterized in that: the vegetable gum from which the partially hydrolyzed mannan is produced is c1) or c2) or c3) or c4) or c 5): c1) locust bean gum; c2) cassia seed gum; c3) fenugreek gum; c4) konjaku flour; c5) a mannan-rich vegetable gum;
and/or, the prebiotics yoghourt preparation also comprises the step of adding streptococcus thermophilus (streptococcus thermophilus) and lactobacillus bulgaricus (L actinobacillus bulgaricus) into the system.
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