阅读说明：本技术 蔗糖异构酶突变体、编码基因及其应用 (Sucrose isomerase mutant, coding gene and application thereof ) 是由 柳志强 张烽 蔡雪 程峰 郑裕国 于 2021-06-24 设计创作，主要内容包括：本发明属于生物技术领域,具体涉及一种来源于Erwiniasp.Ejp617的蔗糖异构酶突变体及其编码基因、含有该突变体基因的重组载体以及蔗糖异构酶突变体生物催化蔗糖制备异麦芽酮糖中的应用。所述蔗糖异构酶突变体氨基酸序列如SEQ ID NO:1所示。本发明构建了一个酶活和催化效率均得到了提高的蔗糖异构酶双重突变体Q209S/R456H,在pH 6.0,30℃的水浴中,蔗糖异构酶突变体Q209S/R456H的酶活达到684U/mg,催化效率是野生型蔗糖异构酶的16倍以上；酶动力学分析显示,Q209S/R456H的K-(m)值分别比天然酶下降了48.7％,k-(cat)值是天然酶的8.3倍；催化效率k-(cat)/K-(m)是WT的16倍以上。催化蔗糖生产异麦芽酮糖时,突变体的异麦芽酮糖最大转化率比天然酶提高了19.3％。(The invention belongs to the technical field of biology, and particularly relates to a sucrose isomerase mutant from Erwiniasp.Ejp617, a coding gene thereof, a recombinant vector containing the mutant gene, and application of the sucrose isomerase mutant in preparing isomaltulose through biocatalysis of sucrose. The amino acid sequence of the sucrose isomerase mutant is shown as SEQ ID NO. 1. The invention constructs a sucrose isomerase double mutant Q209S/R456H with improved enzyme activity and catalytic efficiency, the enzyme activity of the sucrose isomerase mutant Q209S/R456H reaches 684U/mg in water bath with pH6.0 and 30 ℃, and the catalytic efficiency is that wild sucrose16 times or more of isomerase; enzyme kinetic analysis showed K for Q209S/R456H m The value is respectively reduced by 48.7 percent compared with the natural enzyme cat The value was 8.3 times that of the native enzyme; catalytic efficiency k cat /K m Is 16 times or more the WT. When the mutant catalyzes sucrose to produce isomaltulose, the maximum conversion rate of the isomaltulose is increased by 19.3 percent compared with the natural enzyme.)

1. The amino acid sequence of the sucrose isomerase mutant is shown as SEQ ID NO. 1.

2. A gene encoding the sucrose isomerase mutant as claimed in claim 1.

3. The encoding gene of claim 2, wherein the nucleotide sequence of the encoding gene is shown as SEQ ID No. 2.

4. A recombinant vector comprising the coding gene of claim 2.

5. Use of the sucrose isomerase mutant according to claim 1 for biocatalysis of sucrose to isomaltulose.

6. The use according to claim 5, characterized in that said use is: and (3) reacting the sucrose isomerase mutant serving as a catalyst and sucrose serving as a substrate for 3-12 hours at the pH of 6.0-7.0 and at the temperature of 28-32 ℃, and obtaining the isomaltulose in a reaction solution.

7. The method of claim 5, wherein the sucrose concentration in the reaction system is 500-600 g/L.

(I) technical field

The invention belongs to the technical field of biology, and particularly relates to a sucrose isomerase mutant from Erwiniasp.Ejp617, a coding gene thereof, a recombinant vector containing the mutant gene, and application of the sucrose isomerase mutant in preparing isomaltulose through biocatalysis of sucrose.

(II) background of the invention

Isomaltulose (Isomaltulose), also known as Palatinose (Palatinose), is a reducing disaccharide, an isomer of sucrose, and is composed of D-glucose and D-fructose linked by α -1,6 glycosidic linkages, unlike the α -1,2 glycosidic linkages in sucrose. In 1957, it was first discovered by Weidenhagen et al in beet fabrication. Isomaltulose has similar sweetness characteristics and mouthfeel to sucrose, but has a low sweetness, only 52% of sucrose, and its outstanding advantages over sucrose are mainly reflected in: (1) low cariogenic properties; (2) has good therapeutic effect on diabetic patients and patients in the early stage of diabetes; (3) in the human intestinal tract, selectively stimulating the growth of bifidobacteria; (4) extremely low hygroscopicity, strong stability and longer shelf life; (5) is suitable for people who need to continuously and long-term brain work, such as primary and secondary school students, white-collar workers and the like. Isomaltulose has been widely used in japan, usa, western europe and other countries as a promising functional sweetener, and its applications include hard candy, soft candy, chewing gum, chocolate, baked goods, canned fruit, jam, sports drinks, toothpaste and the like. In addition, isomaltulose is also a starting material for Isomalt (Isomalt). Isomalt is a functional sugar alcohol which is newly and internationally emerging in recent years, and is widely applied to the production of products such as sugar-free foods, sugar-free health products, sugar-free medicines and the like.

Isomaltulose is produced by Sucrose isomerase EC 5.4.99.22(Sucrose isomerase), or Isomaltulose synthase (Isomaltulose synthases), Sucrose glucosyl mutase (Sucrose glucosyl mutase), alpha-glucosyl transferase (alpha-glucosyl transferase), which rearranges the alpha-1, 2 bond connecting glucose and fructose in Sucrose to produce trehalulose when it rearranges to alpha-1, 4 bond and to alpha-1, 6 bond to produce Isomaltulose. At present, sucrose isomerase used for isomaltulose production is derived from various microorganisms, such as natural enzymes of Erwinia rhapontici NX-5, Erwinia D12(Erwinia sp. D12), Serratia alba ATCC15928(Serratia plymuthica ATCC15928), Klebsiella, Pseudomonas acidophilus MX-45(Pseudomonas mesophila MX-45) and Erwinia rhapontici NCPPB1578(E.rhapontici NCPPB1578), and recombinases from Erwinia rhapontici DSM4484(GenBank accession No. AAK28735.1), Erwinia rhapontici NX-5(ADJ56407.2), Enterobacter FMB-1(Enterobacter sp. FMB-1) (ACF42098.1), Pseudomonas acidophilus MX-45(ACO05018.1), Escherichia erythraea CBS574.77(CAF32985.1), Klebsiella pneumoniae NK33-98-8(Klebsiella pneumoniae pNeumonia NK33-98-8) (AAM96902.1), Pantoea dispersa UQ68J (Pantoea dispersa AAP57083.1) (AAP57083.1), Klebsiella pneumoniae UQ14S (Klebsiella planticola UQ14S) (AAP57085.1) and Klebsiella LX3(AAK 82938.1). Although sucrose isomerase produced by the above bacteria can convert sucrose into isomaltulose, the yield is very unstable and the conversion rate is not high, namely 8-85%. In addition to the main product, a part of trehalulose and a small amount of by-products such as isomaltose, isomaltotriose, glucose and fructose are present in the enzymatic conversion solution, and the product specificity is not high. To increase the enzyme activity of sucrose isomerase to increase the yield of isomaltulose, generation of isomaltulose-producing strains by various mutation methods is also a major focus of current research. For example, Zhangda and the like take a Klebsiella LX3 screened in a laboratory as an original strain, the strain is mutagenized by a normal-pressure room-temperature plasma injection technology, and the strain with high isomaltulose yield and low viscosity (LX3-1) is obtained by measuring the sucrose isomerase activity and the isomaltulose content in a fermentation liquid and detecting the flocculation effect of the strain, compared with a wild strain, the sucrose isomerase activity of a mutant strain is improved by 20.42 percent (P <0.05), the isomaltulose yield is improved by 41.87 percent, and after 6 times of subculture of the mutant strain, the sucrose isomerase activity and the isomaltulose yield in the fermentation liquid are still stable. Liu et al mutated Y296 and Q299 which are close to the substrate binding site of sucrose isomerase derived from Pantoea dispersa UQ68J, and realized the increase of isomaltulose yield.

At present, although many studies on sucrose isomerase are available at home and abroad, most studies are still in the laboratory level, and further research on industrial application of sucrose isomerase is needed.

Disclosure of the invention

The invention aims to provide a sucrose isomerase mutant, a coding gene thereof, a recombinant vector containing the mutant gene and application of the sucrose isomerase mutant in preparing isomaltulose by biologically catalyzing sucrose.

The technical scheme adopted by the invention is as follows:

the amino acid sequence of the sucrose isomerase mutant is shown as SEQ ID NO. 1. The sucrose isomerase mutant is obtained by mutating glutamine to serine and arginine to histidine at position 456 from amino acid 209 of Erwiniasp.Ejp617. The invention utilizes error-prone PCR technology to carry out site-directed mutagenesis on sucrose isomerase for molecular modification, and further improves the enzyme activity of the sucrose isomerase and the yield of isomaltulose.

The invention also relates to a gene for coding the sucrose isomerase mutant.

Specifically, the nucleotide sequence of the coding gene is shown as SEQ ID No. 2.

The invention also relates to a recombinant vector containing the coding gene. These genes, expression cassettes, plasmids, and transformants can be obtained by genetic engineering construction means well known to those skilled in the art.

The invention also relates to the application of the sucrose isomerase mutant in preparing isomaltulose by biocatalysis of sucrose.

When used as a biocatalyst for production, the sucrose isomerase mutant of the present invention may be in the form of an enzyme or in the form of a fungus. The enzyme forms comprise free enzyme and immobilized enzyme, including purified enzyme, crude enzyme, fermentation liquor, carrier-immobilized enzyme, cell disruption product and the like: the form of the thallus comprises a viable thallus cell and a dead thallus cell.

Specifically, the application is as follows: and (3) reacting for 3-12 h at the pH of 6.0-7.0 and the temperature of 28-32 ℃ by taking the sucrose isomerase mutant as a catalyst and sucrose as a substrate to obtain the isomaltulose in a reaction solution.

Preferably, the concentration of sucrose in the reaction system is 500-600 g/L.

Compared with the prior art, the invention has the following beneficial effects: the invention constructs a sucrose isomerase double mutant Q209S/R456H with improved enzyme activity and catalytic efficiency, the enzyme activity of the sucrose isomerase mutant Q209S/R456H reaches 684U/mg in water bath with pH6.0 and 40 ℃, and the catalytic efficiency is more than 16 times of that of wild sucrose isomerase; enzyme kinetic analysis showed K for Q209S/R456H_mThe value is respectively reduced by 48.7 percent compared with the natural enzyme_catThe value was 8.3 times that of the native enzyme; catalytic efficiency k_cat/K_mIs 16 times or more the WT. When the mutant catalyzes sucrose to produce isomaltulose, the maximum conversion rate of the isomaltulose is increased by 19.3 percent compared with the natural enzyme.

(IV) description of the drawings

FIG. 1: a good mutation point color reaction is obtained by utilizing a high-throughput screening method;

FIG. 2: is SDS-PAGE gel electrophoresis of natural sucrose isomerase and five mutant pure enzymes; wherein, M represents a protein molecular weight standard, WT is wild-type sucrose isomerase, and lane 1 is mutant Q209N; lane 2 is mutant R456K; lane 3 is mutant Q209S, lane 4 is mutant R456H, and lane 5 is mutants Q209S-R456H.

FIG. 3: temperature and pH optima for ErSIase _ WT and ErSIase _ Q209S-R456H. (A) Temperature optimum for ErSIase _ WT, (B) pH optimum for ErSIase _ WT, (C) temperature optimum for ErSIase _ Q209S-R456H, and (D) pH optimum for ErSIase _ Q209S-R456H.

FIG. 4: conversion rate for preparing isomaltulose for wild-type sucrose isomerase and its mutant at 40 ℃.

FIG. 5: construction of recombinant E.coli BL21(DE3)/pET28b (+) -ErSIase containing recombinant plasmid of ErSIase gene.

(V) detailed description of the preferred embodiments

The present invention will be described in further detail with reference to specific examples, but the present invention is not limited to the following examples:

the invention relates to the addition amount, content and concentration of various substances, wherein the percentage content refers to the mass percentage content except for special description.

Reagents used for upstream genetic engineering: the one-step cloning kits used in the examples of the present invention were purchased from Vazyme, nuozokenza biotechnology ltd; plasmid extraction kit DNA recovery and purification kit was purchased from Axygen Hangzhou Co., Ltd; plasmids and the like were purchased from Shanghai; DNA marker, Fast Pfu DNA polymerase, low molecular weight standard protein, agarose electrophoresis reagent, primer synthesis and gene sequencing and gene synthesis are completed by Hangzhou Zhikechi catalpi limited Biotechnology. The method of using the above reagent is referred to the commercial specification. Common reagents such as sucrose and isomaltulose are available from the national drug group chemical agents limited.

Example 1: cloning and expression of sucrose isomerase gene in E.coli BL21(DE 3).

Constructing an expression vector: the following sucrose isomerase gene synthesis was performed by Hangzhou Zhikexi Biotechnology Limited. Sucrose isomerase gene (GenBank: G37835) derived from Erwinia (Erwinia. Ejp617) was seamlessly cloned between Nco I and Xho I in pET28b (+) vector by PCR to obtain expression vector pET28b (+) -SI (FIG. 4) carrying sucrose isomerase. The PCR procedure was performed as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 53-58 ℃ for 15s, and extension at 72 ℃ for 1.5 min for 25 cycles; then extended at 72 ℃ for 10 minutes.

The preparation method of the competent cell comprises the following steps: obtaining an E.coli BL21(DE3) strain preserved in a glycerin tube from a refrigerator at the temperature of-80 ℃, streaking the strain on an anti-LB-free plate, and culturing the strain at the temperature of 37 ℃ for 10 hours to obtain a single colony; picking single colony of LB plate, inoculating to test tube containing 5mL LB culture medium, culturing at 37 deg.C and 180rpm for 9 h; 200. mu.L of the cell suspension was taken out from the test tube, inoculated into 50mL of LB medium, cultured at 37 ℃ and 180rpmOD ₆₀₀To 0.4-0.6; precooling the bacterial liquid on ice, taking the bacterial liquid to a sterilized centrifugal tube, placing the bacterial liquid on ice for 10min, and centrifuging the bacterial liquid at 4 ℃ and 5000rpm for 10 min; pouring out the supernatant, taking care to prevent contamination, using precooled 0.1mol/L CaCl₂Resuspending the precipitated cells in an aqueous solution and placing on ice for 30 min; centrifuging at 4 deg.C and 5000rpm for 10min, discarding supernatant, and adding pre-cooled 0.1mol/L CaCl containing 15% glycerol₂Resuspending the precipitated cells in aqueous solution, 100. mu.L of the resuspended cells were dispensed into sterilized 1.5mL centrifuge tubes, stored in a-80 ℃ freezer, and removed if necessary.

Constructing an engineering bacterium library: carrying out ice bath on competent cells of escherichia coli BL21(DE3) (Invitrogen) stored at-80 ℃ for 10min at 0 ℃, then respectively adding 5 mu L of pET28b (+) -SI with sucrose isomerase gene expression vectors into a super clean bench, carrying out ice bath at 0 ℃ for 30min, carrying out heat shock on 90s in water bath at 42 ℃, carrying out ice bath at 0 ℃ for 4min, adding 600 mu L of LB culture medium, and carrying out shake culture at 37 ℃ and 200rpm for 1 h; spread on LB plate containing 50 ug/ml kanamycin resistance, and cultured at 37 deg.C for 8-12h to obtain recombinant E.coli BL21(DE3)/pET28b (+) -SI engineering bacteria containing recombinant plasmid.

Example 2: induced expression and purification of sucrose isomerase recombinant bacteria

Sucrose isomerase-containing wet cells: the recombinant E.coli BL21(DE3)/pET28b (+) -SI obtained in example 2 was inoculated into LB liquid medium containing 50. mu.g/mL kanamycin resistance, cultured at 37 ℃ and 200rpm for 12 hours, further inoculated into fresh LB liquid medium containing 50. mu.g/mL kanamycin resistance in an inoculum size of 1% (v/v), and cultured at 37 ℃ and 150rpm until the microbial cells OD₆₀₀Reaching 0.6-0.8, adding IPTG with the final concentration of 0.1mM, carrying out induction culture at 25 ℃ for 10h, centrifuging at 4 ℃ and 8000rpm for 20min, discarding the supernatant, collecting the precipitate, washing twice by using Tris HCl buffer solution with the pH of 7.0 and 50mM to obtain the wet thallus of the recombinant strain E.coli BL21(DE3)/pET28b (+) -SI containing sucrose isomerase; adding the wet thalli into a Tris HCl buffer solution with the pH value of 7.0 and the concentration of 50mM for resuspension, and carrying out ultrasonic disruption on an ice-water mixture for 20min under the ultrasonic disruption conditions: the power is 400W, the crushing is carried out for 1s, and the suspension is suspended for 5s, so as to obtain a crude enzyme solution.

Purification of sucrose isomerase: due to the fact that6 histidine tag is expressed together with sucrose isomerase gene, so that peptide fragment and bivalent Ni can be expressed by the histidine tag²⁺The protein was purified by affinity chromatography using a Ni affinity column (40X 12.6mm, Bio-Rad, USA). The purification process is mainly used and carried out. The specific operation is as follows: [ solution ] equilibration of buffer A (50mM NaH) with 5 column volumes of Ni column₂PO₄·2H₂O +300mM NaCl +50mM imidazole) equilibrated with Ni column until baseline was stable; sample loading, wherein the flow rate is 1mL/min, and the sample loading amount is 25-40mg/mL protein, so that the target protein is adsorbed on the Ni column; flushing the foreign protein by using buffer solution A with 6 times of column volume, wherein the flow rate is 1mL/min until the base line is stable; (iv) eluent B (50mM NaH)₂PO₄·2H₂O +300mM NaCl +500mM imidazole) at a flow rate of 1mL/min, and collecting the target protein. The objective protein was dialyzed overnight in 20mM phosphate buffer at pH 7.5 to obtain a purified enzyme, and the SDS-PAGE electrophoresis is shown in FIG. 2.

Example 3 construction and screening of sucrose isomerase Gene library.

(one) introduction of beneficial mutations by error-prone PCR

Plasmid DNA was isolated from E.coli BL21(DE3)/pET28b (+) -SI recombinant E.coli using the MiniBEST plasmid purification kit (TaKaRa, Dalian, China). 60pmol of each mutagenic primer (E2-F, E2-R, Table S1), 0.4mM MnCl in the presence of 60ng of plasmid (pET28b-SI) as a template₂Amplification of DNA fragments was performed in 2 X50. mu.L reaction mix Taq PCR StarMix and Loding Dye (TaKaRa, Chinese Dalian). The amplification process of the gene encoding the SIase is as follows: 94 ℃ for 2min, then 30 cycles of 94 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 2min, respectively, and finally at 72 ℃ for 10 min. This protocol resulted in an average mutation frequency of every 1000bpA single base substitution. The PCR product was digested with QuickCut Dpn I at 37 ℃ for 3 hours to remove the template. Then, the library gene fragment was purified using MiniBEST DNA fragment purification kit (TaKaRa, da lian, china). The purified gene fragment was then ligated into pET28b between the Nco I and Xho I sitesTo form recombinant plasmid pET28 b-SI. pET28b-SI was then transformed into E.coli and all transformed cells were grown overnight. The best variants were screened and sequenced.

Error-prone PCR primer sequences

(II) establishment of high-throughput screening method and detailed steps

1. Culturing a 96 deep-hole plate to prepare a seed solution: to each well of the 96-deep well plate was added 500. mu.L of LB liquid medium containing 0.1% (v/v)100g/L Kana, and then a single colony was picked up in the corresponding well with a sterile toothpick, and the remaining 4 wells in each 96-deep well plate were picked up and added with WT single colony as a positive control. Then, the cells were sealed with a cap and cultured at 37 ℃ for 5 hours with shaking at 180 rpm.

2. Transferring and inducing seed liquid: mu.L of the transformant seed solution cultured in the previous step was taken out and put into another 96-well plate, 900. mu.L of LB liquid medium containing 0.1% (v/v)100g/L Kan was added, and then sealed with a lid, and cultured with shaking at 37 ℃ and 180rpm for 2 hours, and then 50. mu.L of LB liquid medium containing 0.8% (v/v)120g/L IPTG and 0.1% (v/v)100g/L Kana was added to each well, and cultured with shaking at 25 ℃ and 180rpm for 10 hours or more after sealed with a lid. The remaining transformant seed solution was stored in a refrigerator at 4 ℃ until use.

3. And (3) detecting enzyme activity by using a DNS method: 50. mu.L of the elicitor was placed in a 96-well plate, and 50. mu.L of 50g/L sucrose (prepared with 50mM citric acid-disodium hydrogenphosphate buffer solution (pH 6.0)) was added thereto, followed by a reaction at 40 ℃ for 15 min. Then 50 μ L DNS reagent is added, the mixture is heated by middle fire for about 25s for color development in a microwave oven, the experimental result is observed, and the absorbance of the developing solution at 540nm is detected by a microplate reader.

4. Preserving high-enzyme-activity mutant bacterial liquid and determining a sequence: according to the DNS method determination result, marking samples with higher absorbance than the positive control, taking 200 mu L of corresponding seed liquid in a glycerol tube, adding 200 mu L of 50% glycerol, mixing uniformly, and storing in a refrigerator at-20 ℃. Then 10 mul of seed liquid is taken to be put into 10mL of LB liquid culture medium containing 0.1% (v/v)100g/L Kan, shaking culture is carried out for more than 7h at 37 ℃ and 180rpm, more than 200 mul of bacterial liquid is taken to be tested, and proper mutants are reserved according to the sequencing result.

(III) High Performance Liquid Chromatography (HPLC) for re-screening

Determining enzyme activity by high performance liquid chromatography: 200. mu.L of the crude enzyme solution (supernatant after ultrasonication of wet cells at 100 g/L) was added to 800. mu.L of a 50mM citric acid-disodium hydrogenphosphate buffer solution (pH 6.0) containing sucrose at a final sucrose concentration of 50 g/L. After 30min of reaction at 40 ℃, the product is treated in boiling water bath for 10min for inactivation. Taking the supernatant after centrifugation, diluting the supernatant by 5 times by using a mobile phase, then filtering the supernatant by using a filter membrane, and measuring the amount of isomaltulose in the diluent by using a high performance liquid chromatograph. The liquid chromatograph used is waters 2414; the mobile phase is a mixed solution of acetonitrile and water with the volume ratio of 4:1, and the flow rate is 1.5 mL/min; the chromatographic column is a chromatographic column special for Agilent ZORBAX sugar analysis (specification 4.6 × 250mm, carbon carrying capacity 3.5%, pore diameter 70 angstroms, particle size 5 μm, and specific surface area 300m²(iv)/g, pH range 2.0-8.0); the detector is a differential refraction detector; the external temperature and the column temperature are 35 ℃ and 30 ℃ respectively; the amount of sample was 10. mu.L. Calibrating a standard curve of the high performance liquid chromatography: sucrose solutions and isomaltulose solutions were prepared at different concentrations (1mM, 10mM, 20mM, 40mM, 80mM and 120mM), the peak areas thereof were measured by the HPLC method, respectively, and the relevant standard curves were plotted with the sucrose solution (or isomaltulose solution) as abscissa and the peak area as ordinate.

Definition of enzyme Activity: the enzyme amount required for catalyzing sucrose isomerization to generate 1 mu mol of isomaltulose within 1min under the conditions of pH6.0 and 40 ℃, namely 1 enzyme activity unit (U).

The calculation formula of the specific activity of the pure enzyme is as follows:

after 4500 mutants are screened, two optimal mutants Q209N, R456K, E.coli BL21(DE3)/pET28b (+) -SI-Q209N and E.coli BL21(DE3)/pET28b (+) -SI R456K are obtained, the enzyme activities respectively reach 59U/mg and 165U/mg, the enzyme activity of the wild type E.coli BL21(DE3)/pET28b (+) -SI is 39U/mg protein, and the specific activities of the mutants Q209N and R456K are respectively improved by 1.5 times and 4.2 times.

Example 4: site-directed saturation mutation of sucrose isomerase

In order to further screen the potential activity-improving strains, site-directed saturation mutagenesis was performed on two beneficial mutation sites of Q209 and R456 of the sucrose isomerase obtained in example 3, and further screening was performed, wherein PCR primers were designed as shown in Table 1, and a PCR system (50. mu.L) was: 2. mu.L Phanta Max buffer 25. mu.L, 1. mu.L dNTPs, 1. mu.L each of the mutant upper and lower primers, 1. mu.L template (original strain), 0.5. mu.L Pfu DNA polymerase, and complement ddH₂O to 50. mu.L. The PCR conditions were: pre-denaturation at 95 ℃ for 3 min: denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 7min for 20s, 30 cycles; final extension at 72 ℃ for 10 min. And verifying the PCR product through DNA agarose gel electrophoresis, digesting a template through DpnI, transforming the PCR product into a competent cell of Escherichia coli E.coli BL21(DE3), coating the transformed product on an LB (LB) plate containing 50 mu g/mL ampicillin resistance, performing inverted culture at 37 ℃ overnight, screening the obtained mutant for dominant mutants, sending the obtained dominant strains to Hangzhou Okagaku Biotechnology Limited for sequencing confirmation, and storing.

Table 1: design of sucrose isomerase site-directed saturation mutation primer

The method for calculating the enzyme activity by liquid phase detection was the same as in example 3 above.

After the site-directed saturation mutation is carried out on two beneficial mutation sites of Q209 and R456 of sucrose isomerase, the enzyme activities of the mutants Q209S and R456H are respectively improved by 7.3 times and 11.5 times except that Q209N and R456K are used as the beneficial mutation sites, and the enzyme activities are improved by a larger range than that of the mutants Q209N and R456K. Then, the Q209N and the R456K mutants are combined to obtain a double mutant E.coli BL21(DE3)/pET28b (+) -SI-Q209S/R456H, the enzyme activity reaches up to 684U/mg, and the catalytic efficiency also reaches up to 22.67s^-1mM^-1Is 16 times or more the WT.

Table 2: enzymatic characterization of wild-type and different mutant sucrose isomerases

Example 5: the recombinant Escherichia coli E.coli BL21(DE3)/pET28b (+) -SI-Q209S/R456H sucrose isomerase mutant is used for catalyzing substrate sucrose to synthesize isomaltulose.

The temperature and pH values of the original thallus and the mutant E.coli BL21(DE3)/pET28b (+) -SI-Q209S/R456H are optimized. The reaction was carried out at different pH (from 4.6 to 10.0) conditions to determine the optimum pH, using the following buffers: 100mM disodium hydrogen phosphate buffer (pH 4.6-7.5), 100mM Tris-HCl buffer (pH 7.5-8.6), and 100mM Gly medium-NaOH buffer (pH 8.6-10.0). At the same time, the optimum temperature of ErSIase was investigated by measuring the enzyme activity at different temperatures (20-55 ℃).

The results show that the pH optima for ErSIase _ WT and ErSIase _ Q209S-R456H are 6.5 and 6.0, respectively (FIGS. 3b and 3 d). As shown in FIGS. 3a and 3c, both the purified ErSIase _ WT and ErSIase _ Q209S-R456H had the highest enzymatic activity around 40 ℃.

Finally, the whole cell is utilized to carry out catalytic reaction under the reaction condition of the optimal temperature and pH. The reaction system is 600g/L sucrose, recombinant Escherichia coli E.coli BL21(DE3)/pET28b (+) -SI-Q209S/R456H (high expression amount) (0.1g/L stem cells). Reaction conditions are as follows: constant temperature water bath at 40 deg.c and rotation speed of 600 rpm. Samples (100. mu.L) were taken at 30min intervals throughout the reaction, and the reaction was stopped by adding 5. mu.L of 6M concentrated hydrochloric acid, and then the samples were processed to determine the conversion by measuring the sucrose or isomaltulose concentration using HPLC, and the reaction progress curve is shown in FIG. 4.

When the concentration of sucrose serving as a substrate is 600g/L, the conversion rate can reach 80.0% at 60min by using 0.1g/L catalyst, and finally the reaction is finished in 3 hours, and the conversion rate reaches 93.6%. Under the same reaction conditions, the conversion rate can only reach 74.3% after 3 hours of reaction completion by using the initial thallus (sucrose isomerase WT). Compared with the wild sucrose isomerase, the E.coli BL21(DE3)/pET28b (+) -SI-Q209S/R456H sucrose isomerase mutant has the conversion rate improved by 19.3 percent when catalyzing 600g/L sucrose substrate.

Sequence listing

<110> Zhejiang industrial university

<120> sucrose isomerase mutant, coding gene and application thereof

<160> 8

<170> SIPOSequenceListing 1.0

<210> 2

<211> 599

<212> PRT

<213> Unknown (Unknown)

<400> 2

Met Ser Arg Phe Thr Leu Ser Thr Val Met Ala Leu Leu Val Ala Pro

1 5 10 15

Val Leu Ser Val Leu Pro Gly Gln Val Met Ala Gly Lys Met Asp Met

20 25 30

Ala Thr Thr Gln Leu Ala Gln Lys Ser Asp Asp Phe Pro Ala Trp Trp

35 40 45

Lys Gln Ala Val Phe Tyr Gln Val Tyr Pro Arg Ser Phe Lys Asp Thr

50 55 60

Asn Gly Asp Gly Ile Gly Asp Leu Lys Gly Ile Ile Glu Lys Leu Asp

65 70 75 80

Tyr Leu Lys Arg Leu Gly Val Asp Ala Ile Trp Ile Asn Pro His Tyr

85 90 95

Asp Ser Pro Asn Thr Asp Asn Gly Tyr Asp Ile Arg Asp Tyr Arg Lys

100 105 110

Ile Met Lys Glu Tyr Gly Thr Met Glu Asp Phe Asp Arg Leu Ile Ala

115 120 125

Glu Met Asn Lys Arg Asn Met Arg Leu Met Ile Asp Ile Val Ile Asn

130 135 140

His Thr Ser Asp Gln His Ser Trp Phe Val Gln Ser Lys Gly Ser Lys

145 150 155 160

Asp Asn Pro Tyr Arg Asp Tyr Tyr Phe Trp Arg Asp Gly Lys Asn Gly

165 170 175

Gln Pro Pro Asn Asn Tyr Pro Ser Phe Phe Gly Gly Ser Ala Trp Lys

180 185 190

Lys Glu Asp Asn Ser Gly Gln Tyr Tyr Leu His Tyr Phe Ala Thr Gln

195 200 205

Ser Pro Asp Leu Asn Trp Asp Asn Pro Lys Val Arg Glu Asp Leu Tyr

210 215 220

Ala Met Leu Arg Phe Trp Leu Asp Lys Gly Val Ala Gly Leu Arg Phe

225 230 235 240

Asp Thr Val Ala Thr Tyr Ala Lys Ile Pro Gly Phe Pro Asp Leu Thr

245 250 255

Pro Gln Gln Arg Lys Asn Phe Ala Arg Thr Tyr Thr Glu Gly Pro Ser

260 265 270

Ile His Arg Tyr Ile Lys Glu Met His Gln Gln Val Phe Ser His Tyr

275 280 285

Asn Ile Ala Thr Ala Gly Glu Ile Phe Gly Val Pro Leu Glu Lys Ser

290 295 300

Ile Asp Tyr Phe Asp Arg Arg Arg Gly Glu Leu Asn Ile Ala Phe Thr

305 310 315 320

Phe Asp Leu Ile Arg Leu Asp Arg Gly Val Glu Glu Arg Trp Arg Gln

325 330 335

Lys Ala Trp Ser Leu Thr Asp Phe Arg Gln Thr Ile Asp Lys Val Asp

340 345 350

Arg Val Ala Gly Lys Tyr Gly Trp Asn Ala Phe Phe Leu Asp Asn His

355 360 365

Asp Asn Pro Arg Ala Val Ser His Phe Gly Asp Asp Arg Pro Gln Trp

370 375 380

Arg Gln Ala Ser Ala Lys Ala Leu Ala Thr Leu Met Ile Thr Gln Arg

385 390 395 400

Ala Thr Pro Phe Ile Tyr Gln Gly Ser Glu Leu Gly Met Thr Asn Tyr

405 410 415

Pro Phe Lys Ser Ile Ala Asp Phe Asp Asp Ile Glu Val Lys Gly Phe

420 425 430

Trp Gln Asp Tyr Val Ser Ser Gly Lys Val Asp Pro Glu Glu Phe Met

435 440 445

Arg Asn Val Arg Leu Thr Ser His Asp Asn Ser Arg Thr Pro Phe Gln

450 455 460

Trp Asp Glu Ser Ala Asn Ala Gly Phe Thr Ser Gly Thr Pro Trp Phe

465 470 475 480

Asn Val Asn Pro Asn Tyr Lys Leu Ile Asn Ala Ala Asp Gln Thr Arg

485 490 495

Asp Pro Asp Ser Val Phe Asn Tyr Tyr Arg Lys Leu Ile Gly Leu Arg

500 505 510

His Ala Ile Pro Ala Leu Thr Tyr Gly Glu Tyr Lys Asp Leu Asp Pro

515 520 525

Asn Asn Asp Thr Val Tyr Ala Phe Thr Arg Thr His Gly Asp Lys Arg

530 535 540

Tyr Leu Val Val Ile Asn Phe Lys Glu Asn Val Val Asn Tyr Arg Leu

545 550 555 560

Pro Asp Gln Leu Thr Ile Arg Gln Thr Leu Ser Glu Ser Ser Ala Ile

565 570 575

Gln Pro Val Ala Glu Asn Ala Arg Glu Leu Leu Leu Gln Pro Trp Gln

580 585 590

Ser Gly Ile Tyr Gln Leu Asn

595

<210> 2

<211> 1797

<212> DNA

<213> Unknown (Unknown)

<400> 2

atgtctcgtt tcaccctgtc taccgttatg gctctgctgg ttgctccggt tctgtctgtt 60

ctgccgggtc aggttatggc tggtaaaatg gacatggcta ccacccagct ggctcagaaa 120

tctgacgact tcccggcgtg gtggaaacag gcggtcttct accaggtgta cccgcgctct 180

ttcaaagaca ccaacggtga cggtatcggt gacctgaaag gtatcatcga aaaactggac 240

tacctgaaac gtctgggtgt tgacgctatc tggatcaacc cgcactacga ctctccgaac 300

accgacaacg gttacgacat ccgtgactac cgtaaaatca tgaaagaata cggtaccatg 360

gaagacttcg accgtctgat cgctgaaatg aacaaacgta acatgcgtct gatgatcgac 420

atcgttatca accacacctc tgaccagcac tcttggttcg ttcagtctaa aggttctaaa 480

gacaacccgt accgtgacta ctacttctgg cgtgacggta aaaacggtca gccgccgaac 540

aactacccgt ctttcttcgg tggttctgct tggaaaaaag aagacaactc tggtcagtac 600

tacctgcact acttcgctac ccagagtccg gacctgaact gggacaaccc gaaagttcgt 660

gaagacctgt acgctatgct gcgtttctgg ctggacaaag gtgttgctgg tctgcgtttc 720

gacaccgttg ctacctacgc taaaatcccg ggtttcccgg acctgacccc gcagcagcgt 780

aaaaacttcg ctcgtaccta caccgaaggt ccgtctatcc accgttacat caaagaaatg 840

caccagcagg ttttctctca ctacaacatc gctaccgctg gtgaaatctt cggtgttccg 900

ctggaaaaat ctatcgacta cttcgaccgt cgtcgtggtg aactgaacat cgctttcacc 960

ttcgacctga tccgtctgga ccgtggtgtt gaagaacgtt ggcgtcagaa agcttggtct 1020

ctgaccgact tccgtcagac catcgacaaa gttgaccgtg ttgctggtaa atacggttgg 1080

aacgctttct tcctggacaa ccacgacaac ccgcgtgctg tttctcactt cggtgacgac 1140

cgtccgcagt ggcgtcaggc ttctgctaaa gctctggcta ccctgatgat cacccagcgt 1200

gctaccccgt tcatctacca gggttctgaa ctgggtatga ccaactaccc gttcaaatct 1260

atcgctgact tcgacgacat cgaggttaaa ggcttctggc aggactacgt ttcttctggt 1320

aaagttgacc cggaagaatt catgcgtaac gttcgtctga cctctcatga caactctcgt 1380

accccgttcc agtgggacga atctgctaac gctggtttca cctctggtac cccgtggttc 1440

aacgttaacc cgaactacaa actgatcaac gctgctgacc agacccgtga cccggactct 1500

gttttcaact actaccgtaa actgatcggt ctgcgtcacg ctatcccggc tctgacctac 1560

ggtgaataca aagacctgga cccgaacaac gacaccgttt acgctttcac ccgtacccac 1620

ggtgacaaac gttacctggt tgttatcaac ttcaaagaaa acgttgttaa ctaccgtctg 1680

ccggaccagc tgaccatccg tcagaccctg tctgaatctt ctgctatcca gccggttgct 1740

gaaaacgctc gtgaactgct gctgcagccg tggcagtctg gtatctacca gctgaac 1797

<210> 3

<211> 39

<212> DNA

<213> Unknown (Unknown)

<400> 3

gaaggagata taccatgtct cgtttcaccc tgtctaccg 39

<210> 4

<211> 35

<212> DNA

<213> Unknown (Unknown)

<400> 4

tggtgctcga gttggttcag ctggtagata ccaga 35

<210> 5

<211> 33

<212> DNA

<213> Unknown (Unknown)

<400> 5

tacttcgcta cccagnnkcc ggacctgaac tgg 33

<210> 6

<211> 33

<212> DNA

<213> Unknown (Unknown)

<400> 6

ccagttcagg tccggmnnct gggtagcgaa gta 33

<210> 7

<211> 33

<212> DNA

<213> Unknown (Unknown)

<400> 7

gttcgtctga cctctnnkga caactctcgt acc 33

<210> 8

<211> 33

<212> DNA

<213> Unknown (Unknown)

<400> 8

ggtacgagag ttgtcmnnag aggtcagacg aac 33