Mutant enzyme and fermentation production method thereof and application in synthesizing SQ sugar
By performing site-directed mutation and transformation of natural enzymes to improve their activity and stability, a process for preparing SQ sugars was developed, which solved the problem of cumbersome and high cost in the existing technology, and achieved efficient, cheap and easy-to-scale preparation of SQ sugars.
Patent Information
- Application Number
- CN202411243312.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-05
- Publication Date
- 2025-03-07
- Estimated Expiration
- 2044-09-05
AI Technical Summary
The prior art is difficult to prepare SQ sugars on a large scale and inexpensive basis, and its isolation and purification process is cumbersome and expensive, and chemical synthesis is also very difficult.
By performing site-directed mutation and transformation of natural enzymes, the activity and stability of glucose sulfase, glucose thiooxidase and glucose kinase are significantly improved, and combined with fermentation production methods, a process for preparing SQ sugars was developed.
A short and efficient conversion route of SQ sugar is realized, and cheap D-glucose can be used as the starting material. Through multiple utilization of liquid enzymes or immobilized enzymes, green, cheap and easy-to-scale preparation of SQ sugar is achieved, and the yield is significantly improved.
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Figure CN119101671B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of enzyme engineering, and in particular relates to a mutant enzyme and a fermentation production method thereof and an application thereof in synthesizing SQ sugar. Background Art
[0002] SQ sugar (sulfoquinovose, also known as sulfoquinolone sugar, 6-deoxy-6-sulfoglucose) is a sulfonated monosaccharide commonly found in green vegetables such as spinach and cabbage. It is the polar head component of glycerol esters in the chloroplast membrane of all plants. It is one of the most abundant sulfur-containing organic compounds on the earth and an important part of the earth's sulfur cycle.
[0003] SQ sugar is easily absorbed by the intestines and converted into energy by intestinal bacteria. The importance of SQ sugar to the human body lies not only in the energy it provides, but also in the life-sustaining substances of beneficial intestinal bacteria such as Escherichia coli (E. coli). Eubacterium rectum and Bilophila vorticella can synergistically metabolize SQ sugar to produce acetate and hydrogen sulfide. SQ sugar can be made into SQ candy by mixing it with white sugar, starch, fruit, etc. in appropriate proportions.
[0004] Since the separation and purification process of SQ sugar is very cumbersome and expensive, and its chemical synthesis is also very difficult, it is of great significance to develop a scalable and inexpensive method for preparing SQ sugar for its large-scale application in the market. Summary of the invention
[0005] The object of the present invention is to provide a mutant enzyme of glucose sulfhydrylase (G6PHydra), a mutant enzyme of glucose thiolate oxidase (SQOxi) and a mutant enzyme of glucokinase (GluK), and a fermentation production method thereof and an application thereof in synthesizing SQ sugar.
[0006] The purpose of the present invention is achieved through the following technical solutions:
[0007] The mutant enzyme of glucose sulfhydrylase (G6PHydra), whose amino acid sequence is shown in SEQ.ID.NO.1, and whose encoding nucleotide sequence is shown in SEQ.ID.NO.2.
[0008] The glucose sulfhydrylase mutant enzyme of the present invention has a natural enzyme (WTG6PHydra, Uniprot ID: A0A6I4VAY7, EC 2.5.1.47) derived from Pantoea sp.Eser., and the substrate of the natural enzyme is L-serine-O-phosphate, but detection shows that the natural enzyme also has weak activity on glucose-6-phosphate. The present invention is subjected to a large number of rational designs and random mutations, and finally significantly improves the glucose sulfhydrylase activity of the enzyme, and the final mutation sites are: T72A, L89E, M91S, D93I, R99H, R123D, D145C, N146A, P207F.
[0009] The thioglucose oxidase (SQOxi) mutant enzyme, whose amino acid sequence is shown in SEQ.ID.NO.3, and whose encoding nucleotide sequence is shown in SEQ.ID.NO.4.
[0010] The thioglucose oxidase mutant enzyme of the invention has a natural enzyme (WTSQOxi, Uniprot ID: A6W7I7) derived from Kineococcus radiotolerans, which can oxidize a variety of sulfur-containing natural organic substances such as cysteine and ergothioneine, and also has weak oxidizing activity on thioglucose; after systematic enzyme modification, the activity and stability of the obtained mutant enzyme are significantly improved, and the specific mutation sites are: P20G, A21T, W43F, H60D, T62V, G63M, D73Q, L76I, G117S, and E118N.
[0011] The mutant enzyme of glucose kinase (GluK), whose amino acid sequence is shown in SEQ.ID.NO.5, and whose encoding nucleotide sequence is shown in SEQ.ID.NO.6.
[0012] The glucokinase mutant enzyme of the present invention, its natural enzyme (WTGluK, Uniprot ID: Q7WT42) is derived from Arthrobacter sp., and the natural enzyme can use ATP and polyphosphate as phosphate donors to phosphorylate glucose to glucose-6-phosphate. The mutant enzyme obtained after modification has significantly improved stability and activity, and its mutation sites are: R68S, H92K, E119R, N122Q, Y134T, G150V, L246M.
[0013] The fermentation production method of the mutant enzyme comprises the following steps:
[0014] The nucleotide sequence shown in SEQ.ID.NO.2, SEQ.ID.NO.4 or SEQ.ID.NO.6 was synthesized, subcloned into the pET28a plasmid through the NdeI / XhoI restriction site, and the plasmid was transferred into Escherichia coli cells for plate culture. Finally, a single clone was selected for liquid step-by-step amplification culture.
[0015] The step-by-step amplification culture comprises the following steps:
[0016] (1) First, transfer the single colony on the plate into 5 mL of LB culture medium containing 50 μM kanamycin for culture. When the cells grow to the logarithmic phase, inoculate them into 250 mL of LB culture medium containing the same antibiotic, and finally transfer them into a 5 L culture fermenter for culture. When the fermentation liquid OD 600 When the value reached 20, 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce protein expression at 25°C for at least 10 h, and then the wet cells were collected by centrifugation (4000 rpm, 15 min);
[0017] (2) The wet cells were first mixed with Tris-HCl buffer (50 mM, pH 8.0), and then the cells were disrupted by high pressure and centrifuged at high speed (16000 rpm, 45 min) to remove the cell wall, and finally a clear solution containing the mutant enzyme was obtained.
[0018] One or more of the above mutant enzymes can be used to synthesize SQ sugars.
[0019] A method for synthesizing SQ sugar comprises the following steps:
[0020] D-glucose-6-phosphate and / or its salt and sodium sulfide are added to the buffer, the pH value of the reaction system is adjusted to 7-9, glucose sulfhydrylase (G6PHydra) mutant enzyme and glucose thiophosphate oxidase (SQOxi) mutant enzyme are added to start the reaction, air is blown into the reaction process to maintain the above pH value, the reaction is completed after at least 3 hours, and SQ sugar is obtained after separation and purification;
[0021] The molar ratio of D-glucose-6-phosphate and / or its salt to sodium sulfide is 1:(1.5-3.0);
[0022] The activity unit ratio of the glucose sulfhydrylase (G6PHydra) mutant enzyme to the glucose thiosulfate oxidase (SQOxi) mutant enzyme is (1.5-3.0):1.
[0023] A method for synthesizing SQ sugar comprises the following steps:
[0024] D-glucose, sodium hexametaphosphate and sodium sulfide are added to the buffer, the pH value of the reaction system is adjusted to 7-9, and a glucose kinase (GluK) mutant enzyme, a glucose sulfhydrylase (G6PHydra) mutant enzyme and a glucose thiosulfate oxidase (SQOxi) mutant enzyme are added to start the reaction, and air is blown into the reaction process to maintain the above pH value. The reaction is completed after at least 6 hours, and SQ sugar is obtained after separation and purification;
[0025] The molar ratio of D-glucose, sodium hexametaphosphate and sodium sulfide is 1:(0.4-1.0):(1.2-3.0);
[0026] The activity unit ratio of the glucose kinase (GluK) mutant enzyme, the glucose sulfhydrylase (G6PHydra) mutant enzyme and the glucose thiophosphate oxidase (SQOxi) mutant enzyme is 1: (1.2-2.0): (0.8-1.5).
[0027] In the above two methods of synthesizing SQ sugar:
[0028] Preferably, horseradish catalase is added at the same time as the mutant enzyme to remove hydrogen peroxide produced by the oxidation reaction; the activity unit ratio of the horseradish catalase to the mutant enzyme is 3:(10-20).
[0029] The buffer solution has a pH value of 7 to 9, preferably a Tris.HCl solution with a pH value of 8.0.
[0030] The separation and purification comprises the following steps:
[0031] After the reaction is completed, the pH value of the reaction solution is adjusted to below 1.0 to denature and precipitate various enzymes. The pH value of the solution is then adjusted back to 7.0, and solid impurities are removed by centrifugation. The supernatant is separated using D201 anion exchange resin to obtain the SQ crude product. Finally, the crude product is desalted using a reverse osmosis membrane, concentrated, and crystallized to obtain SQ sugar white granular solid.
[0032] The enzyme can be used after being immobilized;
[0033] The immobilization of the mutant enzyme comprises the following steps:
[0034] (1) adding ammonium sulfate solid to the clear solution containing the mutant enzyme gradually until the enzyme precipitates, then slowly dissolving it in a buffer solution with a pH value of 7 to 9, and finally desalting it through a G25 size exclusion chromatography column, and separating the mutant enzyme using a DEAE Seplite FF anion exchange column;
[0035] (2) Dissolve one or more mutant enzymes in a buffer solution with a pH value of 7 to 9 according to the activity unit ratio of GluK / G6PHydra / SQOxi mutant enzyme of 2:(2.5 to 4.0):(1.5 to 3.0), then add phenoxyacetic acid and epoxy resin to the buffer solution, stir at room temperature for at least 10 hours, filter out the immobilized enzyme, and finally wash with clean water and a buffer solution with a pH value of 7 to 9.
[0036] Compared with the prior art, the present invention has the following advantages and effects:
[0037] 1. Based on the natural enzyme, the present invention conducts site-directed mutagenesis tests to improve its substrate catalytic activity; at the same time, error-prone PCR high-throughput screening experiments are used to locate the remote sites to be replaced, thereby guiding the experiment to improve the activity and stability of the enzyme, and finally successfully integrating to obtain the mutant enzyme of GluK / G6PHydra / SQOxi with improved performance in all aspects.
[0038] 2. The present invention provides a fully enzymatic preparation process for SQ sugar, in which cheap bulk D-glucose (D-glucose) is first used as a starting material, and D-glucose-6-phosphate is generated under the catalysis of a glucose kinase (GluK) mutant enzyme, and then D-glucose-6-phosphate is converted into sulfoglucose by a sulfase (G6PHydra) mutant enzyme in the presence of sodium sulfide, and finally SQ sugar is obtained under the catalysis of an oxidase (SQOxi) mutant enzyme. This route is very short and has a high conversion efficiency. It can be achieved by a one-time conversion with a liquid enzyme, and it can also be achieved by multiple conversions using an immobilized enzyme. Therefore, the patented synthetic SQ sugar preparation process has many advantages such as being green, cheap, and easy to scale. BRIEF DESCRIPTION OF THE DRAWINGS
[0039] Figure 1 It is the SDS-PAGE gel detection diagram of the mutant enzyme of the present invention; wherein, lane 1: G6PHydra mutant enzyme; lane 2: GluK mutant enzyme; lane 3: SQOxi mutant enzyme.
[0040] Figure 2 It's SQ sugar 1 H-NMR spectrum; D2O as solvent, Varian 600MHz NMR. DETAILED DESCRIPTION
[0041] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.
[0042] Example 1: Transformation and fermentation production of mutant enzymes
[0043] Enzyme modification:
[0044] The basic idea of enzyme modification in this patent is to achieve enzyme amino acid site replacement by rationally / randomly replacing the gene template bases corresponding to the natural enzyme, thereby ultimately changing the performance of the enzyme.
[0045] In the practice of this patent, we firstly conducted a homologous multiple sequence alignment on the WTGluK / WTG6PHydra / WTSQOxi enzyme to preliminarily determine the overall analysis of its family amino acid conserved sites, and then used the commercial software Discovery Studio to perform homologous modeling and ligand docking on the WTGluK / WTG6PHydra / WTSQOxi enzyme, and finally determined the substrate binding site; under the guidance of the above two aspects, we conducted a site-directed mutagenesis test to improve its substrate catalytic activity; at the same time, we used error-prone PCR high-throughput screening experiments to locate the remote sites to be replaced, thereby guiding the experiment to improve the activity and stability of the enzyme. Finally, through the integration of the above aspects, we successfully obtained a mutant enzyme of GluK / G6PHydra / SQOxi with improved performance in all aspects.
[0046] The mutant enzyme of glucose sulfhydrylase (G6PHydra), whose amino acid sequence is shown in SEQ.ID.NO.1, and whose encoding nucleotide sequence is shown in SEQ.ID.NO.2.
[0047] The glucose sulfhydrylase mutant enzyme of the present invention has a natural enzyme (WTG6PHydra, Uniprot ID: A0A6I4VAY7, EC 2.5.1.47) derived from Pantoea sp.Eser., and the substrate of the natural enzyme is L-serine-O-phosphate, but detection shows that the natural enzyme also has weak activity on glucose-6-phosphate. The present invention is subjected to a large number of rational designs and random mutations, and finally significantly improves the glucose sulfhydrylase activity of the enzyme, and the final mutation sites are: T72A, L89E, M91S, D93I, R99H, R123D, D145C, N146A, P207F.
[0048] The thioglucose oxidase (SQOxi) mutant enzyme, whose amino acid sequence is shown in SEQ.ID.NO.3, and whose encoding nucleotide sequence is shown in SEQ.ID.NO.4.
[0049] The thioglucose oxidase mutant enzyme of the invention has a natural enzyme (WTSQOxi, Uniprot ID: A6W7I7) derived from Kineococcus radiotolerans, which can oxidize a variety of sulfur-containing natural organic substances such as cysteine and ergothioneine, and also has weak oxidizing activity on thioglucose; after systematic enzyme modification, the activity and stability of the obtained mutant enzyme are significantly improved, and the specific mutation sites are: P20G, A21T, W43F, H60D, T62V, G63M, D73Q, L76I, G117S, and E118N.
[0050] The mutant enzyme of glucose kinase (GluK), whose amino acid sequence is shown in SEQ.ID.NO.5, and whose encoding nucleotide sequence is shown in SEQ.ID.NO.6.
[0051] The glucokinase mutant enzyme of the present invention, its natural enzyme (WTGluK, Uniprot ID: Q7WT42) is derived from Arthrobacter sp., and the natural enzyme can use ATP and polyphosphate as phosphate donors to phosphorylate glucose to glucose-6-phosphate. The mutant enzyme obtained after modification has significantly improved stability and activity, and its mutation sites are: R68S, H92K, E119R, N122Q, Y134T, G150V, L246M.
[0052] Fermentation production of the above mutant enzyme:
[0053] The nucleotide sequence shown in SEQ.ID.NO.2, SEQ.ID.NO.4 or SEQ.ID.NO.6 was synthesized, subcloned into the pET28a plasmid through the NdeI / XhoI restriction site, and the plasmid was transferred into E.coli (BL21) cells for plate culture. Finally, a single clone was selected for liquid step-by-step amplification culture.
[0054] The following is the basic process of cell amplification culture:
[0055] (1) Transfer a single colony on the plate into 5 mL of LB culture medium containing 50 μM kanamycin (37°C) for culture. When the cells grow to the logarithmic phase, inoculate them into 250 mL of LB culture medium containing the same antibiotic, and finally transfer them into a 5 L culture fermenter for culture. When the fermentation liquid OD 600 When the value reached 20, 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce protein expression at 25°C for 10 h, and then 40-60 g of wet cells were collected by centrifugation (4000 rpm, 15 min).
[0056] To verify the expression of the enzyme, a small amount of cells was first mixed with Tris-HCl buffer (50 mM, pH 8.0), and then the cells were broken by freeze-thaw method. After high-speed centrifugation, the supernatant was run on SDS-PAGE protein gel (sodium dodecyl sulfate-polyacrylamide gel) to determine the soluble expression of the protein ( Figure 1 );
[0057] (2) After confirming that the remaining cells are correct, mix them with the buffer solution (10 g of wet cells are mixed with about 200 mL of the above buffer solution), and then the cells are crushed by high pressure and centrifuged at high speed (16000 rpm, 45 min) to remove the cell walls. The resulting enzyme-containing clear liquid can be used directly in subsequent use (the liquid enzyme activity is between 320 and 500 U / mL, where U is the amount of enzyme required to convert 1 μmol of substrate in one minute at room temperature) or further purified and immobilized for use (for solid enzyme reactions).
[0058] LB medium consists of: 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium hydrogen phosphate, 1% dipotassium hydrogen phosphate and 5% glycerol.
[0059] Example 2
[0060] Using D-glucose-6-phosphate as raw material, SQ sugar was prepared by two-step conversion of G6PHydra mutant enzyme and SQOxi mutant enzyme.
[0061]
[0062] 28.2 g of glucose hexaphosphate monosodium salt (100 mM) and 11.7 g of sodium sulfide (150 mM) were added to 1 L of 50 mM pH 8.0 tris(hydroxymethylaminomethane) hydrochloric acid (Tris.HCl) solution, and then the pH value of the solution was adjusted back to 8.0; before starting the reaction, 3000 U of G6PHydra mutant enzyme and 2000 U of SQOxi mutant enzyme were added to start the reaction, and air was uniformly blown into the reaction solution during the reaction to maintain the pH between 7.0 and 9.0. After 4 hours, the reaction was basically completed. A dilute HCl aqueous solution was added to adjust the pH value of the reaction solution to 1.0 to denature and precipitate the G6PHydra mutant enzyme and the SQOxi mutant enzyme to stop the reaction, and then the pH value of the solution was adjusted back to 7.0 and centrifuged at 12,000 rpm for 15 minutes to remove solid impurities. The supernatant was separated by D201 anion exchange resin to obtain the SQ crude product ( Figure 2 ), and finally the crude product was desalted by reverse osmosis membrane, concentrated and crystallized (ethanol:H2O=1:1, V:V) to obtain 10 g of white granular solid (41% yield).
[0063] Example 3
[0064] Using D-glucose-6-phosphate as raw material, SQ sugar was prepared by three-step conversion of G6PHydra mutant enzyme, SQOxi mutant enzyme and Catalase.
[0065]
[0066] Similar to Example 2, horseradish catalase (Catalase) was added to the reaction solution to remove hydrogen peroxide generated by the oxidation reaction.
[0067] 28.2 g of glucose hexaphosphate monosodium salt (100 mM) and 11.7 g of sodium sulfide (150 mM) were added to 1 L of 50 mM pH 8.0 tris(hydroxymethylaminomethane) hydrochloric acid (Tris.HCl) solution, and then the pH value of the solution was adjusted back to 8.0; before starting the reaction, 3000 U of G6PHydra mutant enzyme, 2000 U of SQOxi mutant enzyme and 1000 U of Catalase enzyme were added to start the reaction. During the reaction, air was uniformly blown into the reaction solution and the pH was maintained between 7.0 and 9.0. The reaction was basically completed after 3 hours. A dilute HCl aqueous solution was added to adjust the pH value of the reaction solution to 1.0 to denature and precipitate the G6PHydra mutant enzyme, SQOxi mutant enzyme and Catalase enzyme to stop the reaction. The pH value of the solution was then adjusted back to 7.0 and centrifuged to remove solid impurities. The supernatant was separated using D201 anion exchange resin to obtain a crude SQ product. Finally, the crude product was desalted using a reverse osmosis membrane and then concentrated and crystallized (ethanol:H2O=1:1, V:V) to obtain 21.7 grams of white granular solid (89% yield).
[0068] Example 4
[0069] Using D-glucose as raw material, SQ sugar was prepared by four-step conversion of GluK mutant enzyme, G6PHydra mutant enzyme, SQOxi mutant enzyme and Catalase.
[0070]
[0071] 18 g of D-glucose (100 mM), 18.3 g of sodium hexametaphosphate (30 mM), and 11.7 g of sodium sulfide (150 mM) were added to 1 L of 50 mM pH 7.5 tris(hydroxymethylaminomethane) hydrochloric acid (Tris.HCl) solution, and then the pH value of the solution was adjusted back to 7.5; before starting the reaction, 3000 U of GluK mutant enzyme, 4000 U of G6PHydra mutant enzyme, 3000 U of SQOxi mutant enzyme, and 1500 U of Catalase were added to start the reaction. During the reaction, air was uniformly blown into the reaction solution and the pH was maintained between 7.0 and 8.0. The reaction was completed after 6 hours. A dilute HCl aqueous solution was added to adjust the pH value of the reaction solution to 1.0 to denature and precipitate the GluK mutant enzyme, G6PHydra mutant enzyme, SQOxi mutant enzyme, and Catalase enzyme to stop the reaction. The pH value of the solution was then adjusted back to 7.0 and centrifuged to remove solid impurities. The supernatant was removed by D201 anion exchange resin to remove phosphate impurities in the solution and obtain a crude SQ product. Finally, the crude product was desalted by a reverse osmosis membrane and concentrated and crystallized (ethanol:H2O=1:1, V:V) to obtain 17.6 grams of white granular solid (72% yield).
[0072] Example 5
[0073] Using glucose as raw material, immobilized mixed enzymes are used to convert SQ sugar in one step.
[0074]
[0075] Reaction Example 4 is similar, but uses an immobilized enzyme for the reaction, which can be recycled after the reaction is completed.
[0076] 18 g of D-glucose (100 mM), 18.3 g of sodium hexametaphosphate (30 mM), and 11.7 g of sodium sulfide (150 mM) were added to 1 L of 50 mM pH 8.0 tris(hydroxymethyl)aminomethane hydrochloric acid (Tris.HCl) solution, and then the pH value of the solution was adjusted back to 8.0; before starting the reaction, 7000 U of immobilized mixed enzyme and 1000 U of Catalase were added to start the reaction, and air was uniformly blown into the reaction solution during the reaction to maintain the pH between 7.0 and 9.0. The reaction was completed after 10 hours; then the immobilized mixed enzyme was collected by filtration (the immobilized mixed enzyme retained 72% of its initial activity after being used 8 times). The filtered clear liquid was used to remove phosphate impurities in the solution using D201 anion exchange resin to obtain a crude SQ product, and finally the crude product was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol: H2O = 1:1, V:V) to obtain 19.7 g of white granular solid (81% yield).
[0077] Mixed immobilization of enzymes:
[0078] Solid ammonium sulfate was gradually added to the clear solution containing the mutant enzymes of glucokinase (GluK), glucose sulfhydrylase (G6PHydra), and thioglucose oxidase (SQOxi) collected in Example 1 until the enzyme precipitated (40%-60%, w / v ammonium sulfate / buffer). The enzyme solid was then collected by centrifugation (10000rpm, 12min), and slowly dissolved in 25mM Tris buffer of pH 8.0, and finally desalted by G25 size exclusion chromatography column (purchased from Sigma) and separated by DEAESeplite FF (Xi'an Lanxiao Company) anion exchange column to obtain the initially purified liquid enzymes GluK mutant enzyme, G6PHydra mutant enzyme, and SQOxi mutant enzyme, and the enzyme solution can be directly used for subsequent enzyme fixation.
[0079] In the mixed fixation of GluK / G6PHydra / SQOxi mutant enzymes, the initially purified enzymes were mixed and fixed using LX-1000EP epoxy resin (Xi'an Lanxiao Company) at an activity unit ratio of 2:3:2.
[0080] The basic method of fixation is: 7000U mixed enzyme mixed according to the above activity unit ratio is dissolved in 3L 50mM pH8.0 potassium phosphate solution, then 40mM phenoxyacetic acid and 800g LX-1000EP epoxy resin are added to the buffer solution, and the immobilized enzyme is filtered out after stirring at room temperature for 10 hours, and finally washed three times with clean water and 25mM pH 8.0 phosphate buffer, and then dried at low temperature for use; the GluK / G6PHydra / SQOxi mutant enzyme immobilized mixed enzyme has 78-85% of the activity of the corresponding liquid enzyme.
[0081] Comparative Example
[0082] Using D-glucose as raw material, SQ sugar was prepared by four-step conversion using natural enzymes (WTGluK, WTG6PHydra, WTSQOxi, Catalase)
[0083] The same as the above Example 4, but the mutant enzyme was replaced by the natural enzyme, and finally 1.7 grams of gray solid was obtained (7% yield).
[0084] The performance comparison of the mutant enzyme involved in the present invention and its corresponding natural enzyme is as follows:
[0085] Table 1: Performance comparison of the mutant enzyme of the present invention and its natural enzyme
[0086]
[0087] It can be seen from Table 1 that, compared with the natural enzyme, the mutant enzyme obtained after modification has obvious improvements in activity, stability and expression level.
[0088] From the comparison between Example 4 and the comparative example, it can be seen that the yield of SQ sugar prepared by using the mutant enzyme is significantly improved.
[0089] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.
Claims
1. A glucose sulfhydrylase mutant enzyme, characterized in that The amino acid sequence is shown in SEQ.ID.NO.
1.
2. A glucose thiophosphate oxidase mutant enzyme, characterized in that The amino acid sequence is shown in SEQ.ID.NO.
3.
3. A glucose kinase mutant enzyme characterized in that The amino acid sequence is shown in SEQ.ID.NO.
5.
4. The fermentation production method of the mutant enzyme according to any one of claims 1 to 3, characterized in that The following steps are involved: The nucleotide sequence shown in SEQ.ID.NO.2, SEQ.ID.NO.4 or SEQ.ID.NO.6 was synthesized, subcloned into the pET28a plasmid through the NdeI / XhoI restriction site, and the plasmid was transferred into Escherichia coli cells for plate culture. Finally, a single clone was selected for liquid step-by-step amplification culture.
5. Use of the mutant enzyme according to any one of claims 1 to 3 in the synthesis of SQ sugar.
6. A method for synthesizing SQ sugar, characterized in that The following steps are involved: D-glucose-6-phosphate and / or its salt, and sodium sulfide are added to the buffer, the pH value of the reaction system is adjusted to 7-9, the glucose sulfhydrylase mutant enzyme according to claim 1 and the thioglucose oxidase mutant enzyme according to claim 2 are added to start the reaction, air is blown into the reaction process to maintain the above pH value, the reaction is completed after at least 3 hours, and SQ sugar is obtained after separation and purification.
7. A method for synthesizing SQ sugar, characterized in that The following steps are involved: D-glucose, sodium hexametaphosphate and sodium sulfide are added to the buffer, the pH value of the reaction system is adjusted to 7-9, the glucose kinase mutant enzyme according to claim 3, the glucose sulfhydrylase mutant enzyme according to claim 1 and the glucosinolate oxidase mutant enzyme according to claim 2 are added to start the reaction, air is blown into the reaction process to maintain the above pH value, the reaction is completed after at least 6 hours, and SQ sugar is obtained after separation and purification.
8. The method according to claim 6 or 7, characterized in that: Horseradish catalase was added at the same time as the mutant enzyme.
9. The method according to claim 6 or 7, characterized in that: The pH value of the buffer solution is 7-9.
10. The method according to claim 6 or 7, characterized in that: The enzyme is used after being immobilized.