CN116555231A - Mutant of beta-galactosidase and application thereof - Google Patents
Mutant of beta-galactosidase and application thereof Download PDFInfo
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- CN116555231A CN116555231A CN202310530242.0A CN202310530242A CN116555231A CN 116555231 A CN116555231 A CN 116555231A CN 202310530242 A CN202310530242 A CN 202310530242A CN 116555231 A CN116555231 A CN 116555231A
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- 108010005774 beta-Galactosidase Proteins 0.000 title claims abstract description 40
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- 150000003271 galactooligosaccharides Chemical class 0.000 claims abstract description 34
- 238000006243 chemical reaction Methods 0.000 claims abstract description 24
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- 108090000790 Enzymes Proteins 0.000 claims abstract description 23
- 108090000623 proteins and genes Proteins 0.000 claims description 18
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
- C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/12—Disaccharides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Abstract
The invention belongs to the technical field of enzyme engineering, and particularly discloses a beta-galactosidase mutant and application thereof. Under the same enzyme adding amount and reaction condition, the galactooligosaccharide conversion rate reaches 67.09 percent, which is 36.73 percent higher than that of the recombinant beta-galactosidase (49.06 percent), and the mutant H542V strain can efficiently synthesize galactooligosaccharide and has a certain potential application prospect.
Description
Technical Field
The invention belongs to the technical field of enzyme engineering, and particularly relates to a beta-galactosidase mutant and application thereof.
Background
Galacto-oligosaccharides (GOS) are formed by linking 1 to 7 galactosyl groups and 1 terminal glucosyl group through glycosidic linkages such as beta- (1-3), beta- (1-4) and beta- (1-6). Natural GOS are found in human and animal milk and fruit vegetables (e.g., onions, bananas, soybeans, etc.). The GOS is semitransparent yellowish or colorless in appearance, low in viscosity, good in water solubility and fresh and cool in taste, the sweetness of the GOS is only 35% of that of the sucrose, and the heat of the GOS is only half of that of the sucrose. GOS has high thermal stability and can be stored at 120deg.C for 30min. Is not easy to decompose under the acidic environment condition (pH=3), has long storage period and no bad flavor, and has higher application value in the food and pharmaceutical industries.
The reported GOS preparation methods are as follows: direct extraction of natural materials (such as dairy products), acid hydrolysis of natural polysaccharides, chemical synthesis, direct fermentation, and enzymatic processes. The content of GOS in the natural raw materials is low, so that the separation and extraction difficulties are high; the product obtained by the acid hydrolysis method of the natural polysaccharide has low yield and complex components, and is difficult to obtain a pure product; the chemical synthesis method has the problems of high catalyst toxicity, easy residue, low yield and environmental pollution; the direct fermentation synthesis by using microorganisms is still not mature. Enzymatic synthesis uses enzyme as biocatalyst and lactose as substrate to synthesize GOS. The method has the advantages of simple reaction, mild condition, sufficient raw materials, lower production cost, no toxicity, environmental protection and the like, and is the main method for producing GOS at present. Among the most commonly used biocatalysts are beta-galactosidases.
Beta-galactosidase has been used in industrial production of GOS, but has still had a problem of low catalytic activity as a biocatalyst.
Disclosure of Invention
The invention aims to obtain a biocatalyst (beta-galactosidase) for efficiently preparing galactooligosaccharides by developing and modifying novel beta-galactosidase so as to meet the industrial production requirement.
In a first aspect, the invention provides a beta-galactosidase mutant, which is obtained by mutating amino acid 542 of beta-galactosidase with an amino acid sequence shown as SEQ ID NO. 2.
In a second aspect, the invention provides a gene encoding a mutant as described above.
In a third aspect, the present invention provides a recombinant expression vector carrying a gene as described above.
In a fourth aspect, the invention provides a recombinant cell expressing a mutant as described above, or carrying a gene as described above, or carrying a recombinant expression vector as described above.
In a fifth aspect, the invention provides the use of a mutant as described above, or a gene as described above, or a recombinant expression vector as described above, or a recombinant cell as described above, in the production of galactooligosaccharides.
In a sixth aspect, the present invention provides a method for producing galacto-oligosaccharides as defined above, comprising: lactose is used as a substrate and the mutant is used to convert lactose into galactooligosaccharides.
The invention has the beneficial effects that: according to the invention, the beta-galactosidase from enterobacter cloacae Enterobacter cloacae Zjut HJ2001 is subjected to molecular transformation, and mutant strains with excellent properties are obtained by screening, so that the obtained mutant still has the performance of converting lactose into galactooligosaccharide, the conversion rate is obviously improved, the galactooligosaccharide conversion rate can reach 67.09%, the yield is improved by 36.73% compared with that of the recombinant beta-galactosidase, the yield of galactooligosaccharide prepared by microorganisms is generally 30-50%, and compared with the conventional galactooligosaccharide, the mutant has good industrial application prospect.
Drawings
FIG. 1 shows a plasmid PCR amplification and SDS-PAGE electrophoresis of alanine scanning mutants: m is Marker;1 is N104A;2 is D203A;3 is R390A;4 is H393A;5 is E418A;6 is H420A;7 is N462A;8 is M504A;9 is Y505A;10 is H542A;11 is Y570A;12 is F603A;13 is N606A;14 is W1004A.
FIG. 2 shows a plasmid PCR amplification and SDS-PAGE electrophoresis of saturated mutants at position H542: m is Marker;1 is H542C;2 is H542D;3 is H542E;4 is H542F;5 is H542G;6 is H542I;7 is H542L;8 is H542M;9 is H542N;10 is H542P;11 is H542Q;12 is H542S;13 is H542T;14 is H542V;15 is H542M;16 is H542Y;17 is H542K;18 is H542R.
FIG. 3 is an enzyme catalyzed HPLC chromatogram: 1, beta-gal or H542V crude enzyme solution; 2: beta-gal conversion solution; 3: H542V conversion solution; 4:3mg/mL commercial GOS standard (G909325, macklin).
Detailed Description
In a first aspect, the invention provides a beta-galactosidase mutant, which is obtained by mutating amino acid 542 of beta-galactosidase with an amino acid sequence shown as SEQ ID NO. 2.
In one embodiment of the present invention, the β -galactosidase-derived microorganism Enterobacter cloacae (GenBank accession No. OQ 579159.1).
In the research process, the inventor discovers that mutation of the 542 th amino acid of the beta-galactosidase shown in SEQ ID NO.2 can not reduce or even improve the catalytic activity of the beta-galactosidase through multi-site single mutation, so that the variety of the beta-galactosidase is enriched on the one hand, and on the other hand, the improvement of the catalytic activity of the beta-galactosidase can effectively improve the yield of a catalytic product GOS (galactooligosaccharide).
In the present invention, the term "without decreasing the catalytic activity of β -galactosidase" means that the mutant provided by the present invention has a catalytic activity within.+ -. 2% compared to the β -galactosidase shown in SEQ ID NO. 2.
In the present invention, the term "increase the catalytic activity of β -galactosidase" means that the mutant provided by the present invention has a catalytic activity of 1.02 times or more, for example, 1.025, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times as compared to the β -galactosidase shown in SEQ ID NO. 2.
In the present invention, the catalytic activity of the beta-galactosidase can be obtained by measuring the amount of the catalytic product GOS thereof. In one embodiment of the invention, the assay method comprises adding 5U/mL of enzyme into 2mL of reaction system, reacting at 40 ℃ for 36h with 380g/L of initial lactose concentration (100 mM, prepared by pH7.0 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer), boiling in boiling water for 3min, and performing HPLC detection to obtain GOS yield.
In a preferred embodiment of the invention, the mutant is H542G.
In another preferred embodiment of the invention, the mutant is selected from H542M, H542R, H542D, H542S, H542A, H542E, H542K, H542M or H542P.
In a further preferred embodiment of the invention, the mutant is selected from H542I, H542N, H L or H542Y.
In a still further preferred embodiment of the invention, the mutant is selected from H542V, H542C, H T or H542Q.
In a particularly preferred embodiment of the invention, the mutant is H542V. In one embodiment of the present invention, the amino acid sequence of the mutant H542V is shown in SEQ ID NO. 4.
In a particularly preferred embodiment of the present invention, the above-mentioned method for producing a beta-galactosidase mutant H542V comprises the following specific steps:
(1) Docking is carried out by utilizing a three-dimensional model of the recombinant beta-galactosidase and the ligand galactose to determine mutation sites; recombinant beta-galactosidase is heterologously expressed in E.coli BL21 (DE 3) by beta-galactosidase derived from Enterobacter cloacae Zjut 2001, a specific preparation process has been disclosed in Chinese patent CN 202211511049.4;
(2) Designing alanine scanning mutation primers of mutants, taking a vector pET-28a (+) -bga carrying a beta-galactosidase gene as a template to obtain 14 mutants, and determining H542 as an optimal reconstruction site;
(3) Designing a saturation mutation primer of the mutant, and carrying out saturation mutation by taking a vector pET-28a (+) -bga carrying a beta-galactosidase gene as a template to construct a mutation plasmid pET-28a (+) -H542V;
(4) Transforming the mutant plasmid pET-28b (+) -H542V into E.coli BL21 (DE 3), and selecting positive clones which are successfully verified to perform fermentation culture;
(5) Centrifuging the thalli, carrying out ultrasonic crushing after resuspension, and taking the supernatant to obtain mutant enzyme H542V.
In a second aspect, the invention provides a gene encoding a mutant as described above.
In one embodiment of the invention, the nucleotide sequence encoding the beta-galactosidase shown as SEQ ID NO.2 is shown as SEQ ID NO. 1.
It will be appreciated that, based on the codon corresponding to the amino acid, the skilled person will be able to select the appropriate nucleotide sequence of the gene encoding the mutant, based on the degeneracy of the codon and the characteristics of the expression system, in the knowledge of the mutant according to the invention.
Once the relevant nucleotide sequence is obtained, the relevant amino acid sequence can be obtained in large quantities by recombinant methods. The obtained nucleotide sequence is cloned into a vector, transferred into genetically engineered bacteria, and then separated from the proliferated host cells by a conventional method to obtain the related nucleotide sequence.
In addition, the nucleotide sequence of interest can be synthesized by well-known methods of artificial chemical synthesis.
In one embodiment of the present invention, the nucleotide sequence encoding the mutant H542V gene is shown in SEQ ID NO. 3.
In a third aspect, the present invention also provides a recombinant expression vector carrying a gene as described above.
As the "vector" used in the recombinant expression vector, various vectors known in the art, such as various plasmids, cosmids, phages, retroviruses and the like, can be used, and in one embodiment of the present invention, pET-28a (+) is used as the expression vector.
The recombinant expression vector can be constructed by performing enzyme digestion with various endonucleases capable of having cleavage sites at the multiple cloning sites of the vector to obtain a linear plasmid, and ligating the linear plasmid with the gene fragments cleaved with the same endonucleases to obtain a recombinant plasmid.
The recombinant expression vector may be transformed, transduced or transfected into a host cell (strain) by methods conventional in the art, such as calcium chloride chemical transformation, high voltage shock transformation, preferably shock transformation.
In a fourth aspect, the invention provides a recombinant cell expressing a mutant as described above, or carrying a gene as described above, or carrying a recombinant expression vector as described above.
In one embodiment of the invention, the host cell may be a prokaryotic cell or a eukaryotic cell, preferably a prokaryotic cell.
In one embodiment of the invention, the recombinant cell uses bacteria as expression hosts, preferably a coryneform bacterium, such as E.coli (Escherichia coli) or Bacillus subtilis (Bacillus subtilis).
The invention also provides a recombinant escherichia coli which expresses the beta-galactosidase mutant.
In one embodiment of the invention, the recombinant E.coli takes E.coli BL21 (DE 3) as an expression host and pET-28a (+) as an expression vector.
In a fifth aspect, the invention provides the use of a mutant as described above, or a gene as described above, or a recombinant expression vector as described above, or a recombinant cell as described above, in the production of galactooligosaccharides.
In a sixth aspect, the present invention also provides a method for producing, in particular for increasing, the yield of galactooligosaccharides, by adding the above-mentioned β -galactosidase mutant, or the above-mentioned recombinant cell, to a reaction system comprising lactose for reaction.
In one embodiment of the present invention, the lactose concentration in the above reaction system is 350 to 400g/L, for example, 350, 360, 370, 380, 390, 400g/L, preferably 370 to 390g/L.
In one embodiment of the present invention, the beta-galactosidase mutant is added in an amount of 3 to 10U/mL, for example, 3U/mL, 4U/mL, 5U/mL, 6U/mL, 7U/mL, 8U/mL, 9U/mL, 10U/mL, and preferably 4 to 6U/mL.
In one embodiment of the invention, the reaction conditions of the reaction system are: the reaction is carried out at 30-45deg.C (for example, 30 ℃, 32 ℃, 34 ℃, 36 ℃, 38 ℃,40 ℃, 42 ℃, 44 ℃ and 45 ℃) for 30-45 hours (for example, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours and 45 hours).
Examples
The media referred to in the following examples are as follows:
cloacae Zjut HJ2001 medium (g/L): lactose 15, yeast extract 10, peptone 5, naCl 4, deionized water as solvent, and initial pH7.0. Agarose 20 was added to the solid medium.
LB medium (g/L): tryptone 10,Yeast Extract 5,NaCl 10,pH7.0. Agarose 20 was added to the solid medium.
LB/Kan Medium (g/L): LB liquid medium was supplemented with kanamycin to a final concentration of 50. Mu.g/mL before use. Agar 20 was added to the solid medium, and kanamycin was added to a final concentration of 50. Mu.g/mL before plating.
The following examples relate to the detection methods as follows:
enzyme activity determination:
an oNPG solution of 1mg/mL was prepared using 0.1M, pH 7.0.7.0 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer as a solvent. Taking 480 mu L of oNPG solution with the concentration of 1mg/mL, placing the solution in a 1.5mL centrifuge tube, preserving the temperature at 40 ℃ for 10min, adding 20 mu L of enzyme solution with proper dilution, uniformly shaking and then reacting at 40 ℃ for 10min, and then adding 0.5mL of 0.15M Na 2 CO 3 The reaction was terminated and the absorbance of the sample was measured at a wavelength of 420 nm. Enzyme activity was calculated according to oNP standard curve. Definition of enzyme Activity Unit (U): under the conditions, the amount of enzyme required for enzyme catalysis of oNPG reaction for 1min to produce 1 mu mol of oNP is defined as 1 enzyme activity unit U。
The HPLC conditions were: agilent1290 series, equipped with ELSD 3300 evaporative light detector (Alltech 3300ELSD, america). Chromatographic column: NH2P-504E (4.6X105 mm, shodex, japan) amino column; mobile phase: acetonitrile: water=70:30 (v/v), flow rate: 1.0mL/min; temperature: 30 ℃; sample injection amount: 10 mu L.
Example 1: construction and screening of alanine scanning mutant library
The method comprises the following specific steps:
the Alpha Fold2 is utilized to carry out homologous modeling on the three-dimensional structure of beta-Gal, the SAVES v6.0 (http:// SAVES. Mbi. Ucla. Edu) is used for scoring the modeled model, and the optimal model is selected as the butt joint model. The acceptor proteins were pre-treated (removal of salt ions, water molecules and small molecules) using autodock vina software followed by molecular docking. And selecting the highest scoring docking conformation output during docking, and importing the docking result into PyMol software for visual analysis. The following fourteen engineering sites were obtained: asn104, asp203, arg390, his393, glu418, his420, asn462, met504, tyr505, tyr570, phe603, asn606, his542, trp1004, alanine scanning mutation primers (as shown in table 1) were designed, which were subjected to alanine scanning mutation, and mutant hydrolysis and transglycosidation activity were examined. The reaction system was 5mL, the enzyme amount was 5U/mL, the initial lactose concentration was 400g/L (100 mM, pH7.0 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer) and the reaction was carried out at 40℃for 96 hours, and the mixture was boiled in boiling water for 3 minutes, followed by HPLC detection. The mutant H542A with the highest transglycosidation activity is obtained, and the conversion rate is up to 52.87% (shown in Table 2).
TABLE 1 alanine mutant primers
Table 2 examination of transglycosylation Activity of mutants
Example 2: construction and screening of H542 site saturated mutant library
The method comprises the following specific steps:
according to the alanine scanning mutation result, selecting the H542 site with the best transglycosylation activity mutation effect, taking plasmid pET-28a (+) -bga as a template to design a saturation mutation primer (shown in table 3) for the H542 site, and carrying out saturation mutation. Mutant hydrolysis and transglycosylation activity were examined. The reaction system is 2mL, the enzyme amount is 5U/mL, the initial lactose concentration is 380g/L (100 mM, pH7.0 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution is prepared), the reaction is carried out for 36H at 40 ℃, boiling is carried out for 3min, and HPLC detection is carried out to obtain the mutant H542V with the highest transglycosylation activity. The yield is up to 67.09% (shown in Table 4), which is improved by 1.81 times compared with the recombinant beta-galactosidase. The catalytic products were examined and contained monosaccharides (galactose 6.6min, glucose 7.0 min), unreacted lactose (9.6 min), galacto-oligosaccharides (10.9 min), trisaccharides (14.14, 14.8 min) as shown in FIG. 3.
TABLE 3H542 saturation mutagenesis primer
Table 4 examination of transglycosylation Activity of H542 mutant
Claims (10)
1. A beta-galactosidase mutant is characterized in that the mutant is obtained by taking an amino acid sequence shown in SEQ ID NO.2 as a parent enzyme and mutating 542 position of the parent enzyme.
2. The mutant according to claim 1, wherein the mutant is selected from H542V, H542C, H T or H542Q; or (b)
The mutant is selected from H542I, H542N, H L or H542Y; or (b)
The mutant is selected from H542M, H542R, H542D, H542S, H542A, H542E, H542K, H542M or H542P. Or (b)
The mutant was H542G.
3. The mutant according to claim 1, wherein the mutant is H542V.
4. A gene encoding the mutant according to any one of claims 1 to 3.
5. The gene according to claim 4, wherein the nucleotide sequence of the gene is shown in SEQ ID NO. 3.
6. A recombinant expression vector carrying the gene according to claim 4 or 5.
7. A recombinant cell expressing the mutant according to any one of claims 1 to 3, or carrying the gene according to claim 4 or 5, or carrying the recombinant expression vector according to claim 6.
8. Use of a mutant according to any one of claims 1 to 3, or a gene according to claim 4 or 5, or a recombinant expression vector according to claim 6, or a recombinant cell according to claim 7, for the production of galactooligosaccharides.
9. A method for producing galacto-oligosaccharides, comprising: lactose is used as a substrate for converting lactose into galactooligosaccharides by using the mutant according to claim 1 or 2.
10. The method of claim 9, wherein the reaction conditions comprise:
the addition amount of galacto-oligosaccharide is 3-10U relative to 1g of initial lactose; and/or
The concentration of the initial lactose is 350-400g/L; and/or
The reaction temperature is 30-45 ℃; and/or
The reaction time is 30-45h.
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