A cellobiose 2-epimerase mutant, gene, expression vector, recombinant bacteria, preparation method and application thereof

Abstract

The application discloses a cellobiose 2-epimerase mutant and a gene, an expression vector, a recombinant bacterium, a preparation method and an application thereof. The mutant comprises the following point mutations on the amino acid sequence shown in SEQ ID No. 1: S12A, S24A, G131A, G176A, G224A, K46R, K69R, K72R, K91R, K103R, K201R, K219R, K223R, K241R, K263R, K266R, K273R, K284R, K325R, K335R, K349R, K387R. The application further discloses a gene fragment, a plasmid and a recombinant bacterium for recombinantly expressing the mutant, and is used for preparing the mutant by recombinant expression. The mutant can continuously and stably catalyze the preparation of lactulose at high temperature, has good thermal stability, and has a good industrial application prospect.

Description

Technical Field

[0001] This invention relates to an enzyme protein mutant and its preparation method, and more particularly to a cellobiose 2-epimerase mutant, its gene, expression vector, recombinant bacteria, preparation method, and its application in the catalytic preparation of lactulose. Background Technology

[0002] Lactulose is a high-value derivative of lactose, composed of galactose and fructose linked by a β-1,4 glycosidic bond. Currently, lactulose is mainly produced through chemical or enzymatic methods. Chemical production of lactulose is primarily based on the Lobry de Bruyn-Alberdavan-Ekenstein conversion mechanism; however, it suffers from drawbacks such as numerous reaction steps, harsh reaction conditions, many byproducts, and difficulty in product separation and purification. Enzymatic methods typically offer advantages such as high conversion efficiency, mild reaction conditions, and simple operation steps, making them promising candidates for the catalytic synthesis of lactose from lactose.

[0003] Currently, the enzymes used in the biological preparation of lactulose are classified into β-galactosidase and cellobiose 2-epimerase. β-galactosidase hydrolyzes lactose into galactose and glucose. When fructose is added to the reaction system, the transglycosidic activity of β-galactosidase catalyzes the linkage of galactose and fructose through a β-1,4 glycosidic bond to generate lactulose. However, the limitations of β-galactosidase in synthesizing lactulose lie not only in the need to add fructose as a co-substrate to provide a galactosyl acceptor, but also in the presence of byproducts such as galactose, glucose, 4-lactulose, 1-lactulose, and galactooligosaccharides in the entire enzymatic reaction system, which are detrimental to the subsequent separation and purification of lactulose. Cellobiose 2-epimerases (CEs) can catalyze the one-step structural isomerization of D-glucose residues at the reducing end of β-1,4-glycosidic oligosaccharides such as cellobiose, lactose, and 4-O-β-D-mannosyl-D-glucose to generate D-fructose residues. Currently, CEs are widely used in the catalytic preparation of lactulose.

[0004] Compared to CEs from other sources, glycolytic pyrolytic cellulosic bacteria ( Caldicellulosiruptor saccharolyticus Cellobiose 2-epimerase (CsCE) from which lactulose is derived has higher structural isomerization activity and is widely used in the enzymatic production of lactulose.

[0005] Although CsCE is a commonly used cellobiose 2-epimerase for lactulose production, its structural isomerization activity is hindered by thermodynamic equilibrium, resulting in a low efficiency in the conversion of lactose to lactulose.

[0006] Currently, studies have constructed the CsCE mutant G4-C5 using a random mutation strategy. Although the structural isomerization activity of G4-C5 is 2.8 times that of CsCE, G4-C5 exhibits maximum enzyme activity only at 80°C, and its half-life at 80°C is (…). t 1 / 2 The time taken for G4-C5 was only 18.1 min longer than that of CsCE (141.3 min), indicating that G4-C5 is difficult to react for a long time under the high temperature conditions corresponding to its maximum enzyme activity. Therefore, it cannot continuously and efficiently catalyze the synthesis of lactulose at its maximum enzyme activity, which limits the industrial application prospects of G4-C5. Thus, improving the thermal stability of G4-C5 under high temperature conditions has become an urgent technical problem to be solved. Summary of the Invention

[0007] Objectives of this invention: The objective of this invention is to provide a cellobiose 2-epimerase mutant, addressing the problem of improving the thermostability of the cellobiose 2-epimerase mutant G4-C5. The second objective is to propose a gene encoding the cellobiose 2-epimerase mutant. The third objective is to propose an expression vector or cloning vector containing the cellobiose 2-epimerase mutant gene. The fourth objective is to propose a recombinant bacterium containing the expression vector or cloning vector of the cellobiose 2-epimerase mutant gene. The fifth objective is to propose a method for preparing the cellobiose 2-epimerase mutant, addressing the problem of how to prepare the cellobiose 2-epimerase mutant. The sixth objective is to propose an application of the cellobiose 2-epimerase mutant in the catalytic preparation of lactulose, addressing the problem of how to catalyze the preparation of lactulose.

[0008] Technical solution: The cellobiose 2-epimerase mutant of the present invention comprises a core functional sequence obtained by performing at least one of the following point mutations on the amino acid sequence shown in SEQ ID No. 1: S12A, S24A, G131A, G176A, G224A, K46R, K69R, K72R, K91R, K103R, K201R, K21 9R, K223R, K241R, K263R, K266R, K273R, K284R, K325R, K335R, K349R, K387R.

[0009] Preferably, the point mutation is one or two of K72R, K91R, K201R, K266R, K335R, and K387R.

[0010] Preferably, the point mutation is a double point mutation of K266R and K72R.

[0011] Specifically, the amino acid sequence of the above-mentioned cellobiose 2-epimerase mutant is shown in SEQ ID No. 3.

[0012] A second aspect of the present invention discloses a gene encoding the above-mentioned cellobiose 2-epimerase mutant.

[0013] Preferably, the gene encoding the above-mentioned cellobiose 2-epimerase mutant contains the DNA sequence shown in SEQ ID No. 4.

[0014] The third aspect of this invention discloses an expression vector or cloning vector containing the above-mentioned cellobiose 2-epimerase mutant gene.

[0015] In some embodiments, the expression vector or cloning vector is selected from plasmids and viruses.

[0016] The fourth aspect of this invention discloses a recombinant bacterium containing an expression vector or cloning vector of the above-mentioned cellobiose 2-epimerase mutant gene.

[0017] The fifth aspect of this invention discloses a method for preparing the above-mentioned cellobiose 2-epimerase mutant, comprising the following steps: (1) Design point mutation primers for different mutation sites based on the DNA sequence shown in SEQ ID No. 2, wherein the point mutation primers include:

[0018] (2) The first PCR product was obtained by performing a PCR reaction using a plasmid containing the DNA sequence shown in SEQ ID No. 2 and a pair of point mutation primers for any mutation site. Dpn The first PCR product was digested with enzyme I to obtain the first recombinant expression plasmid expressing the single-point mutant protein of cellobiose 2-epimerase. (3) The first recombinant expression plasmid was transferred into the host bacteria to obtain recombinant bacteria, and the recombinant bacteria were cultured and fermented to express the cellobiose 2-epimerase single-point mutant protein; (4) Collect recombinant bacterial cells expressing cellobiose 2-epimerase single-point mutant protein, resuspend the bacterial cells and break the cells, centrifuge the cell lysate and collect the supernatant. (5) Purify the cellobiose 2-epimerase single-point mutant protein in the supernatant; (6) Use the first recombinant expression plasmid and a pair of point mutation primers for another mutation site to perform a full plasmid PCR reaction to obtain a second recombinant expression plasmid expressing cellobiose 2-epimerase double point mutant protein; use the second recombinant expression plasmid to repeat steps (3)-(5) to obtain cellobiose 2-epimerase double point mutant protein.

[0019] The sixth aspect of this invention discloses the application of the above-mentioned cellobiose 2-epimerase mutant in the catalytic preparation of lactulose.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: This invention constructed multiple single-point and double-point mutants of G4-C5 using a surface amino acid modification strategy. Comparative screening revealed that the single-point mutants K72R, K91R, K201R, K266R, K335R, and K387R exhibited higher thermal stability compared to G4-C5. Further screening showed that the K266R / K72R double-point mutant possessed even higher thermal stability than the K266R and K72R single-point mutants.

[0021] This invention utilizes an E. coli expression system to achieve active expression of the K266R / K72R double-point mutant. Its optimal pH and temperature are 7.5 and 80℃, respectively, the same as G4-C5. The specific enzyme activity and catalytic efficiency are 1.26 times and 1.71 times that of G4-C5, respectively. After incubation at 80℃ for 4 h, 4.41 U·mg remained. -1 Enzyme activity was higher than that of G4-C5 (1.86 U·mg). -1 ) and single mutant K266R (4.03 U·mg -1 ) and K72R (3.77 U·mg -1 This indicates that K266R / K72R has higher thermal stability. The mutant K266R / K72R can be used as a biocatalyst for the preparation of lactulose and has promising prospects for industrial application. Attached Figure Description

[0022] Figure 1 Figure 1 shows the SDS-PAGE analysis results of purified wild-type G4-C5 and its single-point mutants. Figure 2 The image shows the SDS-PAGE analysis results of the purified G4-C5 double-point mutant. Figure 3 Analysis of the structural isomerization activity and residual enzyme activity of wild-type G4-C5 and its single-point mutants after incubation at 80℃ for 4 h; Figure 4 Analysis of the structural isomerization activity and residual enzyme activity of the two-point mutant after incubation at 80℃ for 4 h; Figure 5 The optimal pH for wild-type G4-C5 and the two-point mutant K266R / K72R; Figure 6 The optimal reaction temperature is for wild-type G4-C5 and the double-point mutant K266R / K72R. Detailed Implementation

[0023] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0024] Example 1: The construction and screening method of a single-point mutant of cellobiose 2-epimerase is as follows: (1) Construct an expression vector containing the G4-C5 recombinant coding gene, as follows: The amino acid sequence of the cellobiose 2-epimerase mutant G4-C5 (abbreviated as G4-C5) derived from pyrolytic cellulosic bacteria is shown in SEQ ID No. 1: MDITMFKEDLKSHLEEKIIPFWQSLKDDEFGGYYGYMDFNLNIDRKAQKGCVLNSRILWFFSACYNVLKSEKCKEMAFHAFEFLKNKFWDKEYEGLFWSVSHKGVPVDVTKHVYVQAFGIYGLSEYYEASGDEEALHMAKRLFEILETKCKRENGYTEQFERNWQEKENRFLSENGVIASKTMNTHLHVLESYTN LYRLLKLDDVYEALEWIVRLFVDKIYKKGTGHFKVLCDDNWNELIKAVSYGHDIEASWLLDQAAKYLKDEKLKEEVEKLALEVAQITLKEAFDGQSLI NEMIEDRIDRSKIWWVEAETVVGFFNAYQKTKEEIYLDAAIKTWEFIKEHLVDRRKNSEWLWKVNEDLEAVNMPIVEQWKCPYHNGRMCLEIIKRVD* The nucleotide sequence of the G4-C5 encoding gene was optimized according to the codon preference of E. coli to obtain the G4-C5 recombinant encoding gene, the sequence of which is shown in SEQ ID No. 2: The G4-C5 recombinant coding gene fragment was obtained by total synthesis according to the sequence shown in SEQ ID No. 2, and the G4-C5 recombinant coding gene fragment was cloned into the pET-28a(+) vector. Bam HI and Eco The RI restriction site was used to obtain the recombinant expression plasmid pET-28a(+)-G4-C5.

[0025] (2) Recombinant expression of G4-C5 single-point mutant: Select mutation sites: by Caldicellulosiruptor saccharolyticus Using the crystal structure of cellobiose 2-epimerase (PDB: 4Z4J) as a template, a three-dimensional structural model of G4-C5 was constructed using SWISS-MODEL (https: / / www.swissmodel.expasy.org). Then, the surface amino acids of G4-C5 were analyzed using the bioinformatics analysis tool GetArea (https: / / curie.utmb.edu / getarea.html) (where a ratio value greater than 50% was considered a surface amino acid). The surface lysine (K) was mutated to arginine (R), glycine (G) to alanine (A), and serine (S) to alanine (A) to improve the thermostability of G4-C5. Specific mutation sites include: Table 1. Screening results of point mutation sites

[0026] Using the recombinant expression plasmid pET-28a(+)-G4-C5 as a template, point mutation primers were designed according to the mutation sites in Table 1 for PCR reactions to construct the corresponding single mutants. The designed point mutation primers are as follows: Table 2. Sequences of primer pairs with different point mutations

[0027] In Table 2, the underlined bases are mutant bases.

[0028] The PCR reaction system and PCR amplification conditions are shown in Tables 3 and 4: Table 3 PCR reaction system and components

[0029] Table 4 PCR reaction procedure

[0030] Take 8 μL of PCR product and add 1 μL of each product. Dpn I and 1 μL of 10×QuickCut buffer were added and incubated at 37℃ for 2 h to construct a series of single-point mutant expression vectors, including pET-28a(+)-K387R, pET-28a(+)-K335R, pET-28a(+)-K266R, pET-28a(+)-K201R, pET-28a(+)-K91R and pET-28a(+)-K72R. Dpn I enzyme digestion is used to digest the methylated template plasmid in the PCR product. The resulting digested PCR product is a circular recombinant plasmid, which can be directly used for subsequent transformation.

[0031] Then, the enzyme-digested reaction solution was added to 100 μL of ice bath. Escherichia co1i DH5α competent cells were incubated on ice for 30 min, then heat-shocked at 42℃ for 90 s, and rapidly cooled on ice for 2 min. 700 μL of LB liquid medium was added, and the cells were incubated at 37℃ and 200 r / min for 2 h. After centrifugation at 5000 r / min for 1 min, the supernatant was discarded, and the bacterial slurry was mixed with the small amount of liquid remaining at the bottom of the tube. The mixture was then spread onto LB agar plates containing a final concentration of 50 μg / mL kanamycin and incubated upside down at 37℃ for 12 h. Single colonies were picked and inoculated into 2 mL of LB liquid medium containing a final concentration of 50 μg / mL kanamycin and incubated at 37℃ for 12 h. The bacterial culture was sent to Suzhou Genewise Biotechnology Co., Ltd. for gene sequencing. The correctly sequenced recombinant plasmid was transformed into *E. coli* BL21, yielding recombinant bacteria expressing the cellobiose 2-epimerase mutant protein.

[0032] A single colony of the recombinant bacteria was picked and inoculated into 2 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured overnight at 37°C to prepare a seed culture. The seed culture was then diluted with 1% ( v / v The inoculum was injected into 50 mL of LB liquid medium containing 50 μg / mL kanamycin and incubated at 37°C and 200 r / min until OD500. 600 =0.6, add a final concentration of 0.4 mM IPTG to the culture medium, induce expression at 25℃ for 16 h, centrifuge at 8000 r / min and 4℃ for 10 min, discard the supernatant, wash the remaining cells three times with 20 mL of 50 mM PIPES (pH 7.5), then resuspend in 20 mL of 50 mM PIPES (pH 7.5), and disrupt the cells using an ultrasonic cell disruptor with the following parameters: power 300 W, time 50 min, with a 3 s working pause and 7 s pause as one cycle. Centrifuge at 4℃ and 12000 r / min for 1 h to collect the cell disruption supernatant for recombinant protein purification.

[0033] All the recombinant mutant proteins had a His6 tag at their N-terminus, therefore nickel column purification was used. Take 1 mL of a well-mixed 50% ( w / v BevoGold TM His-tag Purification Resin (reduction-resistant synergist): Centrifuge at 4°C, 1000 r / min for 10 s and discard the stock solution. Add 0.5 mL of non-denaturing lysis buffer to the gel and mix well to equilibrate the gel. Centrifuge at 4°C, 1000 r / min for 10 s and discard the liquid. Repeat this step twice. Add 4 mL of cell lysis supernatant and shake at 4°C, 50 r / min for 1 h. Then, load the mixture into an affinity chromatography column, open the bottom cap of the purification column, and allow the liquid to flow out under gravity. Collect approximately 20 μL of the flow-through for subsequent analysis. Wash the column 5 times, adding 1 mL of non-denaturing wash buffer each time, collecting approximately 20 μL of the flow-through wash each time for subsequent analysis. Elute 5 times, adding 1 mL of non-denaturing eluent each time, and collect the pure enzyme solution. Dialyze the pure enzyme solution into 50 mM PIPES (pH 7.5) buffer for 12 h, and analyze by SDS-PAGE. Results are as follows. Figure 1 As shown, Figure 1 M: Marker; Lane 1: G4-C5; Lane 2: S12A; Lane 3: S24A; Lane 4: G131A; Lane 5: G176A; Lane 6: G224A; Lane 7: K46R; Lane 8: K69R; Lane 9: K72R; Lane 10: K91R; Lane 11: K103R; Lane 12: K201R; Lane 13: K219R; Lane 14: K223R; Lane 15: K241R; Lane 16: K263R; Lane 17: K266R; Lane 18: K273R; Lane 19: K284R; Lane 20: K325R; Lane 21: K335R; Lane 22: K349R; Lane 23: K387R.

[0034] Structural isomerization activity assay: 100 g / L lactose was dissolved in 50 mM PIPES buffer (pH 7.5), and the pure enzyme solution was mixed with the above substrate lactose solution at a volume ratio of 1:1 (…). v / v Mix well and react at 80℃ for 4 h, then terminate the reaction by adding 200 mM HCl. The lactulose content in the reaction solution was determined using high-performance liquid chromatography (HPLC). One activity unit (U) is defined as the amount of enzyme required to produce 1 μmol of lactulose per minute.

[0035] Sample preparation and HPLC detection conditions: The reaction solution was centrifuged at 4℃ and 10000 r / min for 10 min. The supernatant was diluted 20 times and filtered through a 0.22 μm aqueous filter membrane. The filtrate was used for HPLC detection: Asahipak NH2P-50 4E column (250 mm × 4.6 mm, 5 μm), column temperature 40℃, mobile phase acetonitrile:water = 75:25 (… v / v The injection volume was 10 μL, the flow rate was 1.0 mL / min, the detector was an evaporative light scattering detector, and the drift tube temperature was 95 °C. The lactulose content in the reaction solution was determined based on the retention time and peak area of ​​the lactulose standard.

[0036] The results are as follows Figure 3 As shown, Figure 3 The structural isomerization activities of wild-type G4-C5 and 22 single mutants were measured, along with the residual enzyme activity after incubation at 80°C for 4 h. The structural isomerization activities of K72R, K91R, K201R, K266R, K335R, and K387R were 5.19 U·mg. -1 5.39 U·mg -1 5.31 U·mg -1 5.72 U·mg -1 5.11 U·mg -1 and 3.86 U·mg -1 The concentrations were 1.39, 1.44, 1.42, 1.53, 1.37, and 1.03 times higher than those of wild-type G4-C5, respectively; after incubation at 80℃ for 4 h, K72R, K91R, K201R, K266R, K335R, and K387R still retained 3.77 U·mg. -1 4.33 U·mg -1 4.07 U·mg -1 4.03 U·mg -1 4.04 U·mg -1 and 3.37 U·mg -1 Activity was significantly higher than that of wild type (1.86 U·mg). -1 This indicates that these six single mutants have higher thermostability compared to the wild-type G4-C5. Subsequently, these six single mutants were combined with other mutants to obtain 15 double mutants, whose enzyme activity and thermostability were further analyzed.

[0037] Example 2: A method for recombination expression and screening of G4-C5 double-point mutants is as follows: Using the gene recombination, induced expression, and protein purification methods described in Example 1, pET-28a(+)-K387R, pET-28a(+)-K335R, pET-28a(+)-K266R, pET-28a(+)-K201R, pET-28a(+)-K91R, and pET-28a(+)-K72R were used as templates. Using the corresponding mutation primers at another mutation position, the combined mutants were constructed by whole plasmid PCR technology. The methods and steps are as follows (2). For example, for the double-point mutant K387R / K335R, pET-28a(+)-K387R was used as a template, and K335R-F and K335R-R were used as upstream and downstream primers, respectively. The PCR reaction system and conditions in Tables 3 and 4 were used for amplification, which is whole plasmid PCR. Finally, recombinant bacteria expressing cellobiose 2-epimerase two-point mutant proteins K387R / K335R, K387R / K266R, K387R / K201R, K387R / K91R, K387R / K72R, K335R / K266R, K335R / K201R, K335R / K91R, K335R / K72R, K266R / K201R, K266R / K91R, K266R / K72R, K201R / K91R, K201R / K72R, or K91R / K72R were obtained.

[0038] The recombinant bacteria expressing the double-point mutant were cultured, the supernatant was removed by centrifugation, the bacterial cells were collected and lysed, and the recombinant protein was purified by nickel column affinity chromatography. The SDS-PAGE analysis results of the purified double-point mutant protein are shown below. Figure 2 As shown, Figure 2 M: Marker; Lane 1: K387R / K335R; Lane 2: K387R / K266R; Lane 3: K387R / K201R; Lane 4: K387R / K91R; Lane 5: K387R / K72R; Lane 6: K335R / K266R; Lane 7: K335R / K201R; Lane 8: K335R / K91R; Lane 9: K335R / K72R; Lane 10: K266R / K201R; Lane 11: K266R / K91R; Lane 12: K266R / K72R; Lane 13: K201R / K91R; Lane 14: K201R / K72R; Lane 15: K91R / K72R.

[0039] Enzyme activity and thermostability analysis of G4-C5 and its double-point mutants, results as follows: Figure 4 As shown, Figure 4 The structural isomerization activities and residual enzyme activities after incubation at 80℃ for 4 h were measured for 15 double mutants. Among them, the structural isomerization activity of K266R / K72R was 4.72 U·mg. -1After incubation at 80℃ for 4 hours, the remaining amount was 4.41 U·mg. -1 The enzyme activity and residual activity were higher than those of the single mutants K266R and K72R, indicating that K266R / K72R has higher thermal stability than K266R and K72R.

[0040] The amino acid sequence of the K266R / K72R double-point mutant is shown in SEQ ID No. 3: MDITMFKEDLKSHLEEKIIPFWQSLKDDEFGGYYGYMDFNLNIDRKAQKGCVLNSRILWFFSACYNVLKSERCKEMAFHAFEFLKNKFWDKEYEGLFWSVSHKGVPVDVTKHVYVQAFGIYGLSEYYEASGDEEALHMAKRLFEILETKCKRENGYTEQFERNWQEKENRFLSENGVIASKTMNTHLHVLESYTN LYRLLKLDDVYEALEWIVRLFVDKIYKKGTGHFKVLCDDNWNELIKAVSYGHDIEASWLLDQAAKYLKDERLKEEVEKLALEVAQITLKEAFDGQSLI NEMIEDRIDRSKIWWVEAETVVGFFNAYQKTKEEIYLDAAIKTWEFIKEHLVDRRKNSEWLWKVNEDLEAVNMPIVEQWKCPYHNGRMCLEIIKRVD* The gene sequence of the K266R / K72R double-point mutant is shown in SEQ ID No. 4: Example 3: Determination of the optimal pH for wild-type G4-C5 and mutants K266R / K72R Dissolve 100 g / L lactose in 50 mM PIPES (pH 6.0-8.5) buffer. Use the purified enzyme solution prepared in Example 1 as the enzyme for the experiment and determine the optimal pH of the enzyme. Mix the enzyme and substrate at a volume ratio of 1:1 (… v / v Mix well and incubate at 80℃ for 4 h. Detect enzyme activity in wild-type and mutant K266R / K72R under different pH conditions. Figure 5 It can be seen that the optimal pH for both the wild type and the mutant K266R / K72R is 7.5, and there is no significant difference in enzyme activity between the two under the same pH environment.

[0041] Example 4: Determination of the optimal reaction temperature for wild-type G4-C5 and mutants K266R / K72R 100 g / L lactose was dissolved in 50 mM PIPES (pH 7.5) buffer. The purified enzyme solution prepared in Example 1 was used as the enzyme for the experiment, and the optimal reaction temperature of the enzyme was determined. The enzyme and substrate were mixed at a volume ratio of 1:1 (…). v / v Mix well, and incubate the enzyme reaction system at 60℃, 65℃, 70℃, 75℃, 80℃, 85℃, and 90℃ for 4 h, respectively. Detect the enzyme activity of wild-type and mutant K266R / K72R at different temperatures. Figure 6 It can be seen that the optimal reaction temperature for both the wild type and the mutant K266R / K72R is 80℃. Under the condition of 85℃-90℃, the enzyme activity of the mutant K266R / K72R is slightly higher than that of the wild type.

[0042] Example 5: Kinetic parameter analysis of wild-type G4-C5 and mutants K266R / K72R Prepare 25g·L of each -1 50 g·L -1 75 g·L -1 100 g·L -1 125 g·L -1 150 g·L -1 Lactose substrate solution: Lactose solutions of different concentrations were mixed with the wild-type and mutant K266R / K72R pure enzyme solutions prepared in Example 1 at a volume ratio of 1:1. v / v Mix well, react at 80℃ for 4 h, determine the enzyme activity of wild type and mutant, plot the nonlinear regression equation between substrate concentration and specific enzyme activity, and calculate the Michaelis constant. K m Value, catalytic constant kcat and catalytic efficiency k cat / K m Numerical values. The results are shown in Table 5: Table 5 Kinetic parameter analysis of wild-type G4-C5 and mutant K266R / K72R

[0043] As shown in Table 5, the mutant K266R / K72R... K m The value was 179.61 mM, lower than the wild type (225.04 mM), indicating that the mutant has a higher affinity for the substrate; the catalytic efficiency of the mutant K266R / K72R was [not specified]. k cat / K m (1.39×10) -2 s -1 ·mM -1 ) is wild-type G4-C5 (8.13×10 -3 s -1 ·mM -1 1.71 times that of ).

Claims

1. A cellobiose 2-epimerase mutant, characterized in that, The amino acid sequence shown in SEQ ID No. 1 was obtained by performing only the following point mutations, namely, the K266R and K72R double point mutations.

2. The cellobiose 2-epimerase mutant according to claim 1, characterized in that, The amino acid sequence of the cellobiose 2-epimerase mutant is shown in SEQ ID No.

3.

3. A gene encoding a cellobiose 2-epimerase mutant according to any one of claims 1-2, wherein the gene of the cellobiose 2-epimerase mutant is shown in SEQ ID No.

4.

4. An expression vector or cloning vector containing the cellobiose 2-epimerase mutant gene as described in claim 3.

5. A recombinant bacterium containing the expression vector or cloning vector of claim 4.

6. The use of the cellobiose 2-epimerase mutant according to any one of claims 1-2 in the catalytic preparation of lactulose.

Images