Sucrose isomerase mutant, high-yield isomaltulose engineering bacterium, and construction method and application of sucrose isomerase mutant and high-yield isomaltulose engineering bacterium

Abstract

The invention belongs to the field of gene engineering, and discloses a sucrose isomerase mutant, a high-yield isomaltulose engineering bacterium and a construction method and application thereof. According to the sucrose isomerase mutant obtained through rational design of an enzyme molecular structure, the enzyme activity and the isomaltulose conversion rate of the sucrose isomerase mutant are remarkably improved, compared with a wild enzyme, the specific enzyme activity of the sucrose isomerase mutant is improved by 2.29 times, the isomaltulose conversion rate is improved to 98.9% from 91.1%, the impurity content is lower, and therefore the sucrose isomerase mutant is more suitable for synthesis of isomaltulose. A recombinant engineering bacterium of the high-yield isomaltulose engineering bacterium is further constructed; the recombinant engineering bacterium capable of efficiently expressing the sucrose isomerase is constructed by utilizing ubiquitin-like protein modified molecular genes to be fused with sucrose isomerase mutant coding genes, the enzyme activity of the prepared sucrose isomerase can reach 40.5 U / mg wet cells, and the recombinant engineering bacterium has a great application prospect in industrial production.

Description

Technical Field

[0001] The present invention belongs to the technical fields of genetic engineering and bioengineering, and particularly relates to a sucrose isomerase mutant, a construction method and application of an engineering bacterium with high yield of isomaltulose. Background Art

[0002] Isomaltulose ( isomaltulose , α-D-glucopyranosyl-1,6-D-fructose), also known as palatinose, is a functional reducing disaccharide formed by linking glucose and fructose through an α-1,6-glycosidic bond, and is an isomer of sucrose. Isomaltulose has high safety, a pure taste similar to sucrose, and a sweetness only 52% of that of sucrose, and is non-cariogenic, so it can be used as a new sweetener substitute for sucrose; at the same time, since the rate of releasing monosaccharides of isomaltulose in human blood is very slow and does not stimulate insulin secretion, it is especially suitable for diabetics and obese people. Given the excellent physical and chemical properties and physiological functions of isomaltulose, the US FDA has approved it as a safe food additive without intake limit, and it has extremely high application value in many fields such as food, medicine, and health products. In addition, as a reducing sugar, isomaltulose can also be used as a precursor for the production of new functional edible sugar alcohols.

[0003] Natural isomaltulose only exists in small amounts in foods such as beets and honey. Its natural abundance is low and extraction is difficult, making it difficult to meet market demand. At present, the preparation methods of isomaltulose mainly include chemical synthesis method and biotransformation method. The chemical synthesis method for preparing isomaltulose has high cost and large pollution, and cannot achieve large-scale production of isomaltulose. While using microbial transformation to prepare isomaltulose has problems such as difficult product separation and extraction and weak production intensity. At present, the biocatalytic method for converting and producing isomaltulose has become the most effective method.

[0004] Sucrose isomerase ( Sucrose isomerase , EC.5.4.99.11), also known as isomaltulose synthase and sucrose mutase, belongs to glycoside hydrolase family 13, and it can efficiently catalyze the isomerization of sucrose to produce isomaltulose and trehalulose, and at the same time produce a part of glucose and fructose. Sucrose isomerase mainly comes from microorganisms, such as Polyangium cellulosum ( Pantoea dispersa ), Erwinia rhapontici ( Erwinia rhapontici ), Serratia plymuthica ( Serratia plymuthica ), Klebsiella ( Klebsiella sp. ), Enterobacter ( Enterobacter sp.) etc. The enzymatic properties and catalytic abilities of sucrose isomerases from different microorganisms vary greatly, and they generally have problems such as poor stability, low conversion rate, and accumulation of byproducts. The conversion rate of isomaltulose produced by enzymatic isomerization of sucrose is between 65-92%. Using sucrose isomerase to achieve high yield, high purity and low cost in industrial production of isomaltulose is an important research direction of sucrose isomerase. A higher isomaltulose conversion rate means that in the production process, the yield of isomaltulose can be higher, or isomaltulose products with higher purity can be produced. At the same time, improving the enzymatic activity of sucrose isomerase can improve production efficiency and reduce production costs. At present, neither the wild-type sucrose isomerase nor the reported modified enzyme can take into account the catalytic characteristics of high activity and high conversion rate. Because of these two factors, the cost of enzymatic preparation of isomaltulose remains high, which seriously limits the pace of isomaltulose replacing sucrose. Therefore, obtaining a more efficient biocatalyst and developing a sucrose isomerase mutant that takes into account both activity and conversion rate for large-scale application in the industrial production of isomaltulose has become a key task at present. Summary of the invention The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the above background technology, and to provide a sucrose isomerase mutant with high activity and high conversion rate by using genetic engineering and protein engineering, to provide a recombinant plasmid and an engineered bacterium expressing the sucrose isomerase mutant and a construction method thereof, and to provide the application of the sucrose isomerase mutant and the engineered bacterium thereof in the production of isomaltulose.

[0005] In order to solve the above technical problems, the technical solution proposed by the present invention is: The present invention uses wild-type sucrose isomerase (PdSIase) derived from strain Pantoea dispersa UQ68J (accession number: AY223549.1) as a parent (the amino acid sequence and gene sequence of which are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively), and obtains a sucrose isomerase mutant with both high activity and high conversion rate by knocking out some amino acid sites.

[0006] A sucrose isomerase mutant, whose amino acid sequence is shown in SEQ ID NO: 3. The mutant is obtained by deleting the first 32 amino acids (MFLNGFKTVIALTMASSFYLAASPLTKPSTPI) from the amino acid sequence shown in SEQ ID NO: 1.

[0007] A gene encoding the sucrose isomerase mutant, the nucleotide sequence of which is shown in SEQ ID NO:4.

[0008] A recombinant plasmid carrying the gene of the nucleotide sequence.

[0009] The above recombinant plasmid, preferably, any one of pET series vectors, pMA5 vector, pPICZαA vector, pPIC9k vector and pPIC3.5k vector is used as the expression vector. More preferably, the expression vector is pET-22b.

[0010] A high-yield isomaltulose engineering bacterium carrying the said gene or the said recombinant plasmid.

[0011] The above high-yield isomaltulose engineering bacterium, preferably, the host cell of the high-yield isomaltulose engineering bacterium is Escherichia coli ( Escherichia coli ), Bacillus subtilis ( Bacillus subtilis ), and yeast ( Saccharomyces ), any one of them. More preferably, the host cell is Escherichia coli BL21 ( Escherichia coliB21).

[0012] Preferably, the high-yield isomaltulose engineering bacterium also carries a solubilizing protein SUMO tag or SSO tag, and their amino acid sequences are shown in SEQ ID NO:5 and SEQ ID NO:7 respectively. More preferably, it carries a solubilizing protein SUMO tag, and its amino acid sequence is shown in SEQ ID NO:5.

[0013] A method for preparing the sucrose isomerase mutant includes the following steps: 1) Connect the gene shown in SEQ ID NO:2 between the restriction enzyme sites of the expression vector to obtain a recombinant plasmid; 2) Using the recombinant plasmid as a template, design mutant primers, perform PCR amplification reaction, and then connect the reaction product between the restriction enzyme sites of the expression vector to construct a mutant expression vector; 3) Introduce the successfully constructed mutant expression vector into the engineering strain, and select the verified positive monoclonal for induced expression culture; 4) Collect the cultured bacteria, resuspend them in a solution and break the bacterial cells, centrifuge to remove cell debris, and the obtained supernatant is purified by nickel column (Ni-NTA) affinity chromatography to obtain the pure enzyme of sucrose isomerase and its mutant.

[0014] A method for constructing the above high-yield isomaltulose engineering bacterium includes the following steps: Connect the said gene to an expression vector to obtain a recombinant plasmid, and transfer it into a host cell to obtain the high-yield isomaltulose engineering bacterium.

[0015] For the above construction method, preferably, the expression vector is further ligated with the coding genes of the solubilizing proteins SUMO tag or SSO tag, and their nucleotide sequences are shown in SEQ ID NO: 6 or SEQ ID NO: 8, respectively. More preferably, the expression vector is further ligated with the coding gene of the solubilizing protein SUMO tag, and its nucleotide sequence is shown in SEQ ID NO: 6.

[0016] More preferably, the construction method of the high-yield isomaltulose engineering bacteria specifically includes the following steps: Design primers according to the coding gene sequence of the sucrose isomerase mutant (the upstream and downstream primers shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively), introduce BamH I and HindⅢ restriction enzyme sites through the primers, perform PCR amplification, and then recover the target gene fragment after double digestion of the PCR product. At the same time, double digest and recover the pET22b-SUMO vector fragment. This expression vector is obtained by modifying pET22b to fuse a SUMO tag (the amino acid sequence and gene sequence are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively) between the Nde I and BamH I restriction enzyme sites at the N-terminus. After ligation with T4 DNA ligase, it is transferred into Escherichia coli JM109 competent cells, and the plasmid is extracted, verified by double digestion, and sequenced. After correct sequencing, it is obtained. Transfer this recombinant expression vector into Escherichia coli BL21(DE3) competent cells to obtain the sucrose isomerase recombinant engineering strain. The biotransformation system includes 500 - 800 g / L of sucrose and 0.2% (v / v) of crude sucrose isomerase solution, the pH value of the transformation system is 4.0 - 7.0, and the transformation temperature is 30 - 40°C. Preferably, the transformation temperature is 40°C.

[0017] Use of a sucrose isomerase mutant, gene, recombinant vector or high-yield isomaltulose engineering bacteria as described above in the preparation of sucrose isomerase or the conversion of isomaltulose.

[0018] For the above use, preferably, the method for converting isomaltulose includes the following steps: Using sucrose as a substrate, adding the sucrose isomerase prepared from the sucrose isomerase mutant, gene, recombinant vector or high-yield isomaltulose engineering bacteria, and isomaltulose can be obtained after the reaction.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The sucrose isomerase mutant obtained by rational design of the enzyme molecular structure in the present invention has significantly improved enzyme activity and isomaltulose conversion rate. Compared with the wild-type enzyme, the specific enzyme activity of the sucrose isomerase mutant is increased by 2.29 times, and the isomaltulose conversion rate is increased from 91.1% to 98.9%, with lower impurity content. Therefore, it is more suitable for the synthesis of isomaltulose.

[0020] 2. The present invention uses the above-mentioned sucrose isomerase mutant as the target enzyme to construct a recombinant engineering bacterium for high-yield production of isomaltulose; and constructs a recombinant engineering bacterium capable of highly expressing sucrose isomerase by fusing the gene of the ubiquitin-like protein modification molecule with the gene encoding the sucrose isomerase mutant. The sucrose isomerase obtained by preparing has an enzyme activity of up to 40.5 U / mg wet cells, showing great application prospects in industrial production.

[0021] 3. The present invention uses the recombinant engineering bacterium to ferment and produce the enzyme. After obtaining the crude enzyme solution of sucrose isomerase, the crude enzyme solution is used to isomerize sucrose to prepare isomaltulose. By using this method to convert and produce isomaltulose for 4 h, 800 g / L of sucrose in the reaction system can be converted into 791.2 g / L of isomaltulose, and the yield of isomaltulose is as high as 98.9%. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0023] Figure 1 It is the relative activity of the wild-type and mutant of sucrose isomerase in Example 1.

[0024] Figure 2 It is the conversion rate of isomaltulose of the wild-type and mutant of sucrose isomerase in Example 1. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] To facilitate the understanding of the present invention, the following will describe the present invention more comprehensively and meticulously in combination with the accompanying drawings of the specification and the preferred embodiments. However, the protection scope of the present invention is not limited to the following specific embodiments.

[0026] Unless otherwise defined, all the technical terms used hereinafter have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present invention.

[0027] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through the market or can be prepared by existing methods.

[0028] The detection methods involved in the following examples are as follows: Method for detecting the enzyme activity of sucrose isomerase: Using 200 g / L sucrose as the substrate, adding an appropriate amount of enzyme solution, mixing well by shaking, reacting at 40 °C and pH 7.0 for 10 min, and then boiling for 10 min to inactivate. Centrifuge and filter the reaction product, dilute it to a certain concentration, and then perform HPLC detection. Detection conditions: Chromatograph: Agilent1260; Detector: Refractive index detector; Chromatographic column: Amide-80 (5 μm, 4.6 × 250 mm); Column temperature: 75 °C; Mobile phase: 75% acetonitrile; Flow rate: 1.0 mL / min.

[0029] Enzyme activity definition (U): The amount of enzyme required to produce 1 μmol of isomaltulose per unit time (min) under standard conditions.

[0030] Example 1: Preparation of sucrose isomerase PdSIase mutant (1) Construction of recombinant plasmid PdSIase-pET22b and recombinant engineering strain PdSIase-BL21 According to the wild-type PdSIase amino acid sequence (shown in SEQ ID NO:1) of Pantoea dispersa UQ68J (accession number: AY223549.1), its gene sequence (shown in SEQ ID NO:2) was synthesized and ligated between the restriction enzyme sites Nde I and BamH I of the Escherichia coli expression vector pET-22b(+), obtaining the recombinant plasmid PdSIase-pET22b. This recombinant plasmid PdSIase-pET22b was transformed into Escherichia coli BL21(DE3) competent cells by chemical transformation to obtain the recombinant engineering strain PdSIase-BL21.

[0031] (2) Construction of mutant expression vector Using the recombinant plasmid PdSIase-pET22b obtained in step (1) as a template, designing mutant primers for the mutant, and performing PCR amplification reaction to construct a mutant expression vector.

[0032] The primers include the following 4 pairs of primers: PdSIaseΔ13-F / R, PdSIaseΔ21-F / R, PdSIaseΔ32-F / R, and PdSIaseΔ40-F / R, as shown in Table 1.

[0033] Table 1: Mutant primer sequences (the following primer sequences are shown in SEQ ID NO:9-13 in sequence)

[0034] Perform PCR amplification. The total volume of the PCR reaction system is 50 μL, and the components of the reaction system are referred to Table 2.

[0035] Table 2: PCR Amplification Reaction System

[0036] The PCR reaction program is as follows: 98 °C, 1 min (pre-denaturation); 98 °C, 15 s (denaturation); 68 °C, 15 s (annealing); 72 °C, 20 s (extension); cycle 30 times; 72 °C, 5 min (final extension); 4 °C, ∞ (storage).

[0037] After the PCR amplification reaction is completed, the PCR product is digested with two enzymes and the target gene fragment is recovered. At the same time, the pET22b vector fragment is digested with two enzymes. The total volume of the double digestion system of the target gene and the vector is 50 μL. The components of the reaction system are referred to Table 3.

[0038] Table 3: Double Digestion System of Target Gene and Vector

[0039] Reaction conditions: React at 37 °C for 1.5 h.

[0040] After the double digestion reaction is completed, the target gene fragment and the pET22b vector fragment are recovered and ligated using Ligation Solution I. The total volume of the ligation system of the target gene and the vector is 8 μL. The components of the reaction system are referred to Table 4.

[0041] Table 4: Ligation System of Solution Ⅰ

[0042] Reaction conditions: 16 °C, overnight reaction.

[0043] After the ligation reaction of the target gene and the vector is completed, the ligation product is transformed into Escherichia coli JM109 competent cells by chemical transformation method, and the transformants are screened through ampicillin-resistant plates. The transformants are picked and the recombinant plasmid is extracted and verified by double digestion and then sequenced. The total volume of the double digestion verification system of the recombinant plasmid is 10 μL. The components of the reaction system are referred to Table 5.

[0044] Table 5: Double Digestion Verification System of Recombinant Plasmid

[0045] Reaction conditions: React at 37 °C for 1.5 h.

[0046] Individuals with correct sequencing are successfully constructed mutant expression vectors. Through the aforementioned steps, the present invention obtained a total of 4 mutant expression vectors: PdSIaseΔ13-pET-22b, PdSIaseΔ21-pET22b, PdSIaseΔ32-pET22b, PdSIaseΔ40-pET22b. The above recombinant expression vectors were respectively transformed into Escherichia coli BL21(DE3) competent cells by chemical transformation to obtain recombinant engineering strains PdSIaseΔ13-BL21, PdSIaseΔ21-BL21, PdSIaseΔ32-BL21 and PdSIaseΔ40-BL21.

[0047] (3) Expression and purification of PdSIase mutant enzyme The above recombinant engineering bacteria PdSIase-BL21, PdSIaseΔ13-BL21, PdSIaseΔ21-BL21, PdSIaseΔ32-BL21 and PdSIaseΔ40-BL21 were respectively cultured overnight at 37 °C and 200 rpm in LB medium to obtain seed solutions; the seed solutions were inoculated into LB medium at a volume ratio of 2% and cultured at 37 °C until the OD600 value reached 0.6 - 0.8, then the temperature was lowered to 16 °C, and IPTG with a final concentration of 1.0 mM was added to induce for 16 - 18 h to obtain fermentation broths.

[0048] The above 5 fermentation broths were respectively centrifuged at 4 °C and 8000 rpm for 20 min to collect the bacterial cells; after resuspending with solution A (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 20 mM imidazole, 2 mM DTT), lysozyme (final concentration of 200 μg / mL) and IPTG (final concentration of 1 mM) were added and placed on ice for 30 min, then sonicated on ice (sonication for 2 s, interval of 3 s, power 400 W), and finally centrifuged at low temperature and high speed (4 °C, 12000 r / min) to remove cell debris to obtain the supernatant.

[0049] The above 5 kinds of supernatants were respectively subjected to Ni affinity chromatography: 5 open columns were taken, and 1 mL of Ni-NTA resin (QIAGEN) was added to each; the resin was equilibrated with 20 mL of solution A, and then the supernatant was combined with 1 mL of resin and incubated at 4 °C for 40 - 60 min; the mixture was passed through the open column, and the resin bound with protein was retained; then the resin was rinsed with 20 mL of solution A; finally, the protein was eluted with 15 mL of solution B (20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 400 mM imidazole, 2 mM DTT). The eluted protein was replaced into 1×PBS buffer (pH 7.4) using an ultrafiltration tube to remove imidazole and other metal ions in the eluent, and 5 kinds of sucrose isomerase enzyme solutions were obtained and stored at 4 °C in a refrigerator for later use. The 5 enzyme solutions include: wild-type PdSIase (parent) and 4 mutants of PdSIase. The 5 mutants are: PdSIaseΔ13, PdSIaseΔ21, PdSIaseΔ32, and PdSIaseΔ40. Among them, the amino acid sequence and gene sequence of mutant PdSIaseΔ32 are shown in SEQ ID NO:3 and SEQ ID NO:4 respectively.

[0050] Determination of PdSIase enzyme activity and conversion rate: (1)Determination of relative enzyme activity of wild-type PdSIase and enzyme mutants Under standard reaction conditions (200 g / L sucrose, pure PdSIase enzyme solution, pH 7.0, reacting at 40 °C for 10 min), the relative enzyme activity of the mutants was based on the wild-type enzyme activity being 100%. Referring to the method for detecting the enzyme reaction system by HPLC described above, the relative enzyme activities of the mutants and the wild-type enzyme were measured. The results are as Figure 1 shown. The relative enzyme activities of mutant enzymes PdSIaseΔ13, PdSIaseΔ21, PdSIaseΔ32, and PdSIaseΔ40 were 136%, 106%, 229%, and 45% of the wild-type respectively ( Figure 1 shown). Among them, mutant PdSIaseΔ32 greatly improved the catalytic activity towards sucrose, with a specific enzyme activity reaching 1185.4 U / mg (the specific enzyme activity of the wild-type enzyme was 517.6 U / mg), which has great industrial application value.

[0051] (2)Determination of isomaltulose conversion rate of wild-type PdSIase and enzyme mutants Using 200 g / L sucrose as the substrate, an appropriate amount of wild-type and mutant enzyme solutions of PdSIase were added, and the reaction was carried out at pH 7.0 and 40 °C until the sucrose was completely consumed. Referring to the method for detecting the enzyme reaction system by HPLC described above, the isomaltulose conversion rates of the mutants and the wild-type enzyme were measured. The results are asFigure 2 As shown, the isomaltulose conversion rates of the wild-type PdSIase enzyme and the mutant enzymes PdSIaseΔ13, PdSIaseΔ21, PdSIaseΔ32, and PdSIaseΔ40 were 91.0%, 91.4%, 91.6%, 98.9%, and 91.2% respectively. Among them, the mutant PdSIaseΔ32 greatly improved the conversion rate of sucrose to isomaltulose, indicating that the mutant enzyme PdSIaseΔ32 obtained in the present invention has high industrial application value.

[0052] The amino acid and nucleotide sequences used in this example are as follows: SEQ ID NO:1: MFLNGFKTVIALTMASSFYLAASPLTKPSTPIAATNIQKSADFPIWWKQAVFYQIYPRSFKDSNGDGIGDIPGIIEKLDYLKMLGVDAIWINPHYESPNTDNGYDISDYRKIMKEYGSMADFDRLVAEMNKRGMRLMIDIVINHTSDRHRWFVQSRSGKDNPYRDYYFWRDGKQGQAPNNYPSFFGGSAWQLDKQTDQYYLHYFAPQQPDLNWDNPKVRAELYDILRFWLDKGVSGLRFDTVATFSKIPGFPDLSKAQLKNFAEAYTEGPNIHKYIHEMNRQVLSKYNVATAGEIFGVPVSAMPDYFDRRREELNIAFTFDLIRLDRYPDQRWRRKPWTLSQFRQVISQTDRAAGEFGWNAFFLDNHDNPRQVSHFGDDSPQWRERSAKALATLLLTQRATPFIFQGAELGMTNYPFKNIEEFDDIEVKGFWNDYVASGKVNAAEFLQEVRMTSRDNSRTPMQWNDSVNAGFTQGKPWFHLNPNYKQINAAREVNKPDSVFSYYRQLINLRHQIPALTSGEYRDLDPQNNQVYAYTRILDNEKYLVVVNFKPEQLHYALPDNLTIASSLLENVHQPSLQENASTLTLAPWQAGIYKLN. SEQ ID NO:2:

[0053] SEQ ID NO:3: MAATNIQKSADFPIWWKQAVFYQIYPRSFKDSNGDGIGDIPGIIEKLDYLKMLGVDAIWINPHYESPNTDNGYDISDYRKIMKEYGSMADFDRLVAEMNKRGMRLMIDIVINHTSDRHRWFVQSRSGKDNPYRDYYFWRDGKQGQAPNNYPSFFGGSAWQLDKQTDQYYLHYFAPQQPDLNWDNPKVRAELYDILRFWLDKGVSGLRFDTVATFSKIPGFPDLSKAQLKNFAEAYTEGPNIHKYIHEMNRQVLSKYNVATAGEIFGVPVSAMPDYFDRRREELNIAFTFDLIRLDRYPDQRWRRKPWTLSQFRQVISQTDRAAGEFGWNAFFLDNHDNPRQVSHFGDDSPQWRERSAKALATLLLTQRATPFIFQGAELGMTNYPFKNIEEFDDIEVKGFWNDYVASGKVNAAEFLQEVRMTSRDNSRTPMQWNDSVNAGFTQGKPWFHLNPNYKQINAAREVNKPDSVFSYYRQLINLRHQIPALTSGEYRDLDPQNNQVYAYTRILDNEKYLVVVNFKPEQLHYALPDNLTIASSLLENVHQPSLQENASTLTLAPWQAGIYKLN。

[0054] SEQ ID NO:4:

[0055] Example 2: Construction of Recombinant Engineering Bacteria of Sucrose Isomerase (1)Construction of the Skeleton of Sucrose Isomerase Expression Vector According to the amino acid sequences of the solubility-enhancing proteins SUMO tag and SSO tag (shown as SEQ ID NO:5 and SEQ ID NO:7 respectively), the nucleic acid sequences shown as SEQ ID NO:6 and SEQ ID NO:8 were obtained after codon optimization. The nucleic acid sequences were synthesized by total gene synthesis, and two restriction enzyme sites, Nde I and BamH I, were introduced at the 5' end and 3' end of the above gene nucleotide sequences respectively. The synthesized gene was ligated between the restriction enzyme sites Nde I and BamH I of the Escherichia coli expression vector pET22b to obtain the expression vectors SUMO-pET22b and SSO-pET22b respectively, so as to optimize and screen the solubility-enhancing tags and the gene positions of the solubility-enhancing tags.

[0056] (2)Construction of Recombinant Engineering Bacteria of Sucrose Isomerase Using the PdSIaseΔ32-pET22b recombinant plasmid as a template, the upstream and downstream primers PdSIaseΔ32-F2 and PdSIaseΔ32-R2 (Table 6) were designed. Restriction enzyme sites BamH I and Hind III were introduced through the primers for PCR amplification. The total volume of the reaction system was 50 μL, and the components of the reaction system were referred to Table 1. The PCR reaction procedure was: 98 °C, 1 min (pre-denaturation); 98 °C, 15 s (denaturation); 68 °C, 15 s (annealing); 72 °C, 20 s (extension); cycle 30 times; 72 °C, 5 min (final extension); 4 °C, ∞ (storage).

[0057] Table 6: Primer Sequences (the following primer sequences are shown as SEQ ID NO:14 - 15 in sequence)

[0058] After the PCR amplification reaction was completed, the PCR product was double-digested and the target gene fragment was recovered. At the same time, the SUMO-pET22b and SSO-pET22b vector fragments were double-digested respectively. The total volume of the double-digestion system of the target gene and the vector was 50 μL, and the components of the reaction system were referred to Table 1. The reaction conditions were: reaction at 37 °C for 1.5 h.

[0059] After the double-digestion reaction was completed, the target gene fragment, SUMO-pET22b, and SSO-pET22b vector fragments were recovered and ligated using Ligation Solution I. The total volume of the ligation system of the target gene and the vector was 8 μL, and the components of the reaction system were referred to Table 3. The reaction conditions were: overnight reaction at 16 °C.

[0060] After the ligation reaction of the target gene and the vector, the ligation product was transferred into competent Escherichia coli JM109 cells by chemical transformation, and the transformants were screened on an ampicillin-resistant plate. The transformants were picked and the recombinant plasmids were extracted. After verification by double digestion, the plasmids were sequenced. The total volume of the double digestion verification system for the recombinant plasmid was 10 μL. The components of the reaction system are shown in Table 4, and the reaction conditions were: reaction at 37 °C for 1.5 h.

[0061] After correct sequencing, the recombinant expression vectors of sucrose isomerase SUMO-PdSIaseΔ32-pET22b and SSO-PdSIaseΔ32-pET22b were obtained respectively. The above recombinant expression vectors were transferred into competent Escherichia coli BL21(DE3) cells by chemical transformation to obtain the recombinant engineering strains of sucrose isomerase SUMO-PdSIaseΔ32-BL21 and SSO-PdSIaseΔ32-BL21.

[0062] (3)Flask fermentation of recombinant engineering bacteria of sucrose isomerase The above four recombinant engineering bacteria of sucrose isomerase PdSIase-BL21, PdSIaseΔ32-BL21, SUMO-PdSIaseΔ32-BL21 and SSO-PdSIaseΔ32-BL21 were respectively inoculated into LB medium and cultured overnight at 37 °C and 200 rpm on a shaker to obtain seed solutions. The seed solutions were inoculated into fresh LB medium at a volume ratio of 2% and cultured at 37 °C until the OD600 value reached 0.6 - 0.8, then the temperature was lowered to 16 °C, and IPTG with a final concentration of 1.0 mM was added to induce for 16 - 18 h to obtain fermentation broths. The fermentation broths were centrifuged at 6000 rpm for 10 min to collect the bacterial cells. After washing the bacterial cells twice with pure water, the bacterial cells were resuspended with pure water (bacterial concentration was 250 g / L), and sonicated on ice (sonication for 2 s, interval 3 s, power 400 W). After cell disruption, crude enzyme solutions PdSIase, PdSIaseΔ32, SUMO-PdSIaseΔ32 and SSO-PdSIaseΔ32 were obtained respectively. The enzyme activities of the obtained crude enzyme solutions were detected. The sucrose isomerase activities of the four recombinant engineering bacteria of sucrose isomerase are shown in Table 7. Among them, the enzyme activity of the recombinant engineering strain SUMO-PdSIaseΔ32-BL21 was the highest, reaching 40.5 U / mg wet cells.

[0063] Table 7: Enzyme activities of recombinant engineering bacteria of sucrose isomerase

[0064] The amino acid and nucleotide sequences used in this example are as follows: SEQ ID NO:5: MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGS。

[0065] SEQ ID NO:6: ATGTCGGACTCAGAAGTCAATCAAGAAGCTAAGCCAGAGGTCAAGCCAGAAGTCAAGCCTGAGACTCACATCAATTTAAAGGTGTCCGATGGATCTTCAGAGATCTTCTTCAAGATCAAAAAGACCACTCCTTTAAGAAGGCTGATGGAAGCGTTCGCTAAAAGACAGGGTAAGGAAATGGACTCCTTAAGATTCTTGTACGACGGTATTAGAATCCAAGCTGATCAGACCCCTGAAGATTTGGACATGGAGGATAACGATATTATTGAGGCTCACAGAGAACAGATTGGTGGATCC。

[0066] SEQ ID NO:7: MATVKFKYKGEEKEVDISKIKKVWRVGKMISFTYDEGGGKTGRGAVSEKDAPKELLQMLEKQKK。

[0067] SEQ ID NO:8: ATGGCAACAGTAAAGTTCAAGTACAAGGGAGAAGAGAAGGAAGTAGATATAAGTAAGATAAAGAAGGTATGGAGAGTAGGCAAAATGATAAGTTTCACCTATGATGAGGGTGGAGGAAAGACTGGTAGAGGAGCTGTAAGCGAGAAAGACGCTCCAAAAGAACTACTACAAATGTTAGAGAAGCAAAAGAAG。

[0068] Example 3: Preparation of Isomaltulose In a 1 L reaction system, using 800 g / L sucrose as the substrate, 1% (v / v) PdSIase or 0.2% (v / v) SUMO-PdSIaseΔ32 crude sucrose isomerase solution was added respectively, and the reaction was carried out in a water bath shaker at 40 °C. The reaction process was monitored by high performance liquid chromatography until the sucrose was completely consumed. The experimental results are shown in Table 8, indicating that the conversion rate of the mutant SUMO-PdSIaseΔ32 crude enzyme solution for catalyzing sucrose to isomaltulose reached 98.9%.

[0069] Table 8: Production and conversion rate of isomaltulose

[0070] In summary, based on the rational design of the protein structure of the highly active sucrose isomerase (PdSIase) from Pantoea dispersa ( Pantoea dispersa UQ68J), the mutant PdSIase-Δ32 with both significantly improved catalytic activity and isomaltulose conversion rate was screened. In this invention, the gene encoding the above sucrose isomerase mutant was fused with the SUMO gene of the ubiquitin-like protein modification molecule, and an efficient sucrose isomerase engineering strain was constructed using Escherichia coli BL21 ( Escherichia coli B21) as the chassis strain. After shake flask fermentation for 16 - 18 h, the sucrose isomerase activity of the recombinant engineering strain could reach 40.5 U / mg wet cells. Using the method of this invention to produce isomaltulose by transformation for 4 h, 800 g / L sucrose in the reaction system could be converted into 791.2 g / L isomaltulose, and the isomaltulose yield was as high as 98.9%. The mutant in this invention is more suitable for industrial production than the natural sucrose isomerase, and has great application prospects and industrial value.

Claims

1. A sucrose isomerase mutant, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

3.

2. A gene encoding the sucrose isomerase mutant according to claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID NO:

4.

3. A recombinant plasmid, characterized in that: The recombinant plasmid carries the gene with the nucleotide sequence as claimed in claim 2.

4. The recombinant plasmid according to claim 3, characterized in that The recombinant plasmid uses any one of pET series vectors, pMA5 vectors, pPICZαA vectors, pPIC9k vectors and pPIC3.5k vectors as an expression vector.

5. A high-yield isomaltulose engineering bacterium, characterized in that: It carries the gene according to claim 2 or the recombinant plasmid according to any one of claims 3-4.

6. The high-yield isomaltulose engineering bacteria according to claim 5, characterized in that The host cell of the high-yield isomaltulose engineering bacteria is Escherichia coli ( Escherichia coli )、Bacillus subtilis( Bacillus subtilis ) and yeast ( Saccharomyces ) 7. The high-yield isomaltulose engineering bacteria according to claim 5 or 6, characterized in that: The high-yield isomaltulose engineering bacteria also carry a solubilizing protein SUMO tag or SSO tag, and the amino acid sequences thereof are shown in SEQ ID NO: 5 and SEQ ID NO: 7, respectively.

8. A method for constructing a high-yield isomaltulose engineering bacterium as claimed in claim 5 or 6, characterized in that: The method comprises the following steps: connecting the gene according to claim 2 to an expression vector to obtain a recombinant plasmid, and transferring the recombinant plasmid into a host cell to obtain the high-yield isomaltulose engineering bacteria.

9. The construction method according to claim 8, characterized in that: The expression vector is also connected to a gene encoding a solubilizing protein SUMO tag or a SSO tag, and the nucleotide sequence thereof is shown in SEQ ID NO: 6 or SEQ ID NO: 8, respectively.

10. Use of the sucrose isomerase mutant according to claim 1, the gene according to claim 2, the recombinant vector according to any one of claims 3-4, or the high-yield isomaltulose engineering bacteria according to any one of claims 5-6 in preparing sucrose isomerase or converting isomaltulose.

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