Mutant enzyme of alkylglucosamide condensing enzyme, fermentation production method thereof and application in synthesizing alkylglucosamide

Abstract

The present application discloses mutant enzymes of alkyl glucamide condensing enzymes, fermentation production methods thereof, and applications in the synthesis of alkyl glucamides. The amino acid sequences of mutant enzymes 1 (CARTrucA) and 2 (CARTrucAT) of the alkyl glucamide condensing enzymes are shown in SEQ.ID.NO.1 and SEQ.ID.NO.3, respectively. The method for synthesizing alkyl glucamides comprises the following steps: adding a solvent, meglumine, magnesium chloride hexahydrate, and adenosine triphosphate disodium salt to a buffer, adding mutant enzymes 1 and / or 2 of the alkyl glucamide condensing enzymes, and finally dripping an alcohol solution of fatty acids into the system to initiate the reaction. The preparation method of the present invention utilizes bulk, inexpensive fatty acids and meglumine as starting materials, and directly performs condensation preparation using mutant enzymes of the alkyl glucamide condensing enzymes. The reaction conditions are mild and the conversion rate is high.

Description

Technical Field

[0001] The invention belongs to the field of enzyme engineering, and in particular relates to a mutant enzyme of alkylglucosamide condensing enzyme, a fermentation production method thereof, and application of the mutant enzyme in synthesizing alkylglucosamide. Background Art

[0002] Alkyl glucamide, also known as N-alkanoyl-N-methylglucose, abbreviated as MEGA, is a non-ionic surfactant. The raw materials used are all from renewable resources. Its biodegradability can reach 98%. It has mild performance and is highly environmentally and biologically safe. Through testing and comparison, MEGA's wetting power, emulsifying power and foaming performance are all better than those of alkyl polyglycosides under the same conditions. Therefore, MEGA is an excellent green surfactant.

[0003] The current MEGA production process is primarily a chemical synthesis process, typically involving glucose, an alkylamine, hydrogen, and a methyl ester in the presence of a chemical catalyst. The first step is the hydrogenation-reductive amination of glucose and methylamine under Raney-Ni catalysis to produce the glucose methylamine intermediate. The second step is the condensation of glucose methylamine with an alkyl acid using sodium methoxide as a catalyst.

[0004] From the above preparation methods, although MEGA products themselves are very green, their production process is not green and environmentally friendly; at the same time, the price is relatively high. Therefore, developing a low-cost green production process is conducive to fully leveraging the green and environmental advantages of MEGA surfactants. Summary of the Invention

[0005] The present invention aims to provide a mutant enzyme of alkylglucosamide condensing enzyme, a fermentation production method thereof and an application of the mutant enzyme in synthesizing alkylglucosamide.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The mutant enzyme 1 of alkylglucosamide condensase (CARTrucA), its amino acid sequence is shown in SEQ.ID.NO.1, and its encoding nucleotide sequence is shown in SEQ.ID.NO.2.

[0008] The mutant enzyme 1 of the alkylglucosamide condensase, its wild type (Uniprot ID: B2HN69, EC1.2.1.-) is derived from Mycobacterium marinum, the first 1-646aa is truncated, and then the following mutations are performed: T77L, R80P, S233M, P266I, D592Q, T598D.

[0009] The mutant enzyme 2 of alkylglucosamide condensase (CARTrucAT), its amino acid sequence is shown in SEQ.ID.NO.3, and its encoding nucleotide sequence is shown in SEQ.ID.NO.4.

[0010] The mutant enzyme 2 of the alkylglucosamide condensase, its wild type (Uniprot ID: B2HN69, EC1.2.1.-) is derived from Mycobacterium marinum, the first 1-728aa is truncated, and the following site mutations are performed: T77L, R80P, S233M, P266I, D592Q, L665S, N693V.

[0011] The fermentation production method of a mutant enzyme of alkylglucosamide condensing enzyme comprises the following steps:

[0012] (1) Synthesize the nucleotide sequence shown in SEQ.ID.NO.2 or SEQ.ID.NO.4, then subclone into the pET28a plasmid through the NdeI / XhoI restriction sites, and transform the plasmid into Escherichia coli cells for plate culture. Finally, select a single clone for liquid amplification culture;

[0013] (2) Transfer the single colony on the plate into LB culture medium containing at least 50 μM kanamycin for culture. When the cells grow to the logarithmic phase, inoculate them into LB culture medium containing the same antibiotic, and finally transfer them to a fermenter for culture. When the OD value of the fermentation liquid reaches 20, add 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) to induce protein expression for at least 8 hours, and then collect the wet cells by centrifugation.

[0014] (3) The wet cells were mixed with the buffer solution, and then the cells were disrupted by high pressure and centrifuged at high speed (16000 rpm, 45 min) to remove the cell walls, and finally the enzyme-containing clear solution was obtained;

[0015] The pH value of the buffer solution in step (3) is 7.0 to 9.0, preferably 50 mM Tris-HCl buffer solution.

[0016] The mutant enzymes 1 and / or 2 of the alkylglucamide condensase can be used to synthesize alkylglucamide.

[0017] A method for synthesizing alkyl glucamide comprises the following steps:

[0018] A solvent, meglumine, magnesium chloride hexahydrate, and adenosine triphosphate disodium salt are added to a buffer solution, the pH of the reaction system is adjusted to 7.0-9.0, and mutant enzymes 1 and / or 2 of alkyl glucosamide condensing enzyme are added. Finally, an alcohol solution of a fatty acid is added dropwise to the system to initiate the reaction. The addition process is maintained for more than 2 hours. After the addition is completed, stirring is continued at room temperature for more than 10 hours to complete the reaction. The pH of the reaction system is maintained at 7.0-9.0 during the addition process and the reaction process.

[0019] After the reaction is completed and alkyl glucose amide is generated, the alkyl glucose amide solid is obtained by separation and purification.

[0020] The molar ratio of meglumine to fatty acid is (1.2-2.5):1;

[0021] In the method, pyrophosphohydrolase BtPPase can be added;

[0022] In the method, polyphosphate kinase can be added to achieve ATP cycle regeneration in the reaction system;

[0023] In the method, the enzyme can be immobilized and the multiple enzyme system can be recycled multiple times, thereby further reducing the production price and improving the convenience of the production process;

[0024] The pH value of the buffer solution is 7.0 to 9.0; the buffer solution is preferably a Tris-HCl solution with a pH of 8.0;

[0025] The solvent is DMSO (dimethyl sulfoxide), isopropanol or acetonitrile;

[0026] The fatty acid has 8 to 10 carbon atoms;

[0027] The alcohol solution of the fatty acid, the solvent of which is preferably isopropanol;

[0028] The enzyme immobilization comprises the following steps:

[0029] The mutant enzyme, polyphosphate kinase, and BtPPase were dissolved in a buffer solution at an activity unit ratio of (1.8-2.5):(1.5-2.8):1, followed by the addition of phenoxyacetic acid and epoxy resin. The mixture was stirred at room temperature for at least 5 hours, and the immobilized enzyme was filtered out. The mixture was then washed three times with water and three times with buffer solution, and dried for later use.

[0030] In the method, the ratio of enzyme to epoxy resin is (5-20) U:1 g;

[0031] The pH value of the buffer solution is 8.0, preferably 50 mM potassium phosphate solution or 25 mM phosphate buffer;

[0032] The epoxy resin is preferably LX-1000EP epoxy resin.

[0033] The separation and purification comprises the following steps:

[0034] The reaction solution is separated and removed from phosphoric acid-containing impurities by anion exchange resin, and then the product is purified and collected by non-polar resin. Finally, the product is desalted by a reverse osmosis membrane and concentrated to obtain alkyl glucose amide solid.

[0035] The present invention has the following advantages and effects compared to the prior art:

[0036] 1. The present invention obtains two mutant enzymes (CARTrucA & CARTrucAT) by mutating a wild enzyme in Mycobacterium marinum, which can effectively dock a variety of saturated fatty acids to the amino group of meglumine (N-Methyl-D-Glucamine), thereby effectively generating MEGA surfactant.

[0037] 2. The present invention provides an enzymatic preparation route for alkyl glucosamide. The entire preparation utilizes bulk, inexpensive fatty acids and meglumine as starting materials, and utilizes a mutant enzyme of alkyl glucosamide condensing enzyme to directly carry out condensation preparation. The reaction conditions are mild and the conversion rate is high. At the same time, by further optimizing the catalytic process, such as coupling polyphosphate kinase (BaPPK) to achieve ATP recycling in the reaction system, by adding pyrophosphate hydrolase BtPPase to hydrolyze the dipolyphosphate generated in the reaction system into monophosphate, thereby further improving the conversion rate of the reaction, or by using an immobilized enzyme to recycle the enzyme system multiple times, thereby further reducing the production price and improving the production process. Therefore, the preparation process of alkyl glucosamide of this patent has many production advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The SDS-PAGE gel detection images of the prepared enzymes are as follows: 1) CARTrucAT, 2) CARTrucA, 3) BaPPK, and 4) BtPPase.

[0039] Figure 2 、 Figure 3 、 Figure 4 Alkyl glucamide (MEGA-8, MEGA-9, MEGA-10) 1 H-NMR spectrum. DETAILED DESCRIPTION

[0040] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0041] Related information about the enzymes used in this patent:

[0042] Polyphosphate kinase BaPPK (Uniprot ID: A0A2U8PYE2): Derived from the rhizobium Bradyrhizobium amphicarpaeae, this enzyme can efficiently recycle adenosine monophosphate (AMP) to adenosine triphosphate (ATP) using polyphosphate;

[0043] Pyrophosphate hydrolase BtPPase (Uniprot ID: Q8A5V9): Derived from Bacteroides thetaiotaomicron, this enzyme can selectively hydrolyze dipolyphosphate PPi into monophosphate Pi.

[0044] Example 1: Modification of wild-type alkylglucosamide condensase

[0045] Alkylglucosamide condensase (CARTruc) (ID: B2HN69, EC 1.2.1.-): derived from Mycobacterium marinum (Uniprot), the enzyme has a total length of 1174 amino acids;

[0046] The natural enzyme (WTCARTrucA) was obtained by cutting the first 1-646aa; then the following mutations were made: T77L, R80P, S233M, P266I, D592Q, T598D to obtain mutant enzyme 1 (CARTrucA);

[0047] Alternatively, the natural enzyme (WTCARTrucAT) can be obtained by cutting off the first 1-728aa and performing mutations at the following sites: T77L, R80P, S233M, P266I, D592Q, L665S, and N693V, ultimately obtaining mutant enzyme 2 (CARTrucAT).

[0048] Enzyme modification:

[0049] The basic idea is to achieve enzyme amino acid site replacement by rationally / randomly replacing the gene template bases corresponding to the modified enzyme, thereby changing the performance of the enzyme.

[0050] First, a multiple sequence alignment was performed on more than 50 homologous proteins of the B2HN69 enzyme that have similar functions but large sequence differences. The overall sequence similarity of the selected homologous proteins was between 40 and 65%. Then, the generally conserved regions of the sequence (1-728aa) were analyzed and amino acid sites were selected for mutation testing. At the same time, the known crystal structure of B2HN69 (PDB: 5MSO) and its ligand cavity were analyzed using DiscoveryStudio commercial software. After locating the enzyme-substrate binding site, single-point and multi-point mutation experiments were performed. On the other hand, error-prone PCR technology can be used to randomly construct a CARTruc mutant library with wider coverage, so that more remote related sites related to the performance to be modified can be located. The thermal stability and quantitative activity of the enzyme need to be characterized throughout the entire analysis and testing process. The above three methods can be used alone or in combination, and ultimately, the mutants CARTrucA and CARTrucAT with improved performance in multiple aspects were successfully prepared.

[0051] Example 2: Fermentation production method of mutant enzyme of alkylglucosamide condensing enzyme

[0052] (1) The nucleotide sequence shown in SEQ.ID.NO.2 or SEQ.ID.NO.4 was synthesized, and then subcloned into the pET28a plasmid through the NdeI / XhoI restriction site. The plasmid was transferred into E. coli (BL21) (Qingke Biotechnology) cells for plate culture, and finally a single clone was selected for liquid step-by-step amplification culture.

[0053] (2) First, transfer the 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 OD value of the fermentation liquid reaches 20, add 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 25°C to induce protein expression for 8 hours, and then centrifuge (4000 rpm, 15 min) to collect 25-35 g of wet cells.

[0054] (3) 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; the results are as follows Figure 1 shown.

[0055] After confirming the correct amount of cells, the remaining cells are first mixed with buffer (10 grams of wet cells are mixed with about 200 mL of buffer), and then the cells are crushed by high pressure and centrifuged at high speed (16000 rpm, 45 min) to remove the cell walls. The enzyme-containing clear liquid obtained can be used directly in subsequent use (the liquid enzyme activity is 150-300 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).

[0056] LB medium consists of: 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium hydrogen phosphate, 1% dipotassium hydrogen phosphate and 5% glycerol.

[0057] Mixed immobilization of enzymes:

[0058] Solid ammonium sulfate was gradually added to crude enzyme solutions of alkylglucosamide condensing enzyme (CARTruc), polyphosphate kinase (BaPPK), and pyrophosphohydrolase (BtPPase) until the enzymes precipitated (30%-60%, w / v ammonium sulfate / buffer). The enzyme solution was then collected by centrifugation (10,000 rpm, 12 minutes) and slowly dissolved in 25 mM Tris buffer (pH 8.0). Finally, the solution was desalted using a G25 size exclusion chromatography column (purchased from Sigma) and separated using a DEAE Seplite FF (Xi'an Lanxiao Company) anion exchange column to obtain the purified liquid enzymes CARTruc, BaPPK, and BtPPase. This enzyme solution was then used directly for subsequent enzyme immobilization. For the mixed immobilization of CARTruc / BaPPK / BtPPase, the purified enzymes CARTruc, BaPPK, and BtPPase were immobilized using LX-1000EP epoxy resin (Xi'an Lanxiao Company) at a ratio of 2:2:1 activity units. The basic immobilization method is as follows: 6000 U of the enzyme mixture, mixed according to the above activity unit ratio, is dissolved in 2 L of 50 mM potassium phosphate solution, pH 8.0. 40 mM phenoxyacetic acid and 600 g of LX-1000EP epoxy resin are then added to the buffer. After stirring at room temperature for 5 hours, the immobilized enzyme is filtered out and washed three times with water and three times with 25 mM phosphate buffer, pH 8.0, before being dried at low temperature for use. The immobilized CARTruc / BaPPK / BtPPase enzyme mixture exhibits 77-89% of the activity of the corresponding liquid enzyme.

[0059] Example 3: Preparation of alkyl glucamide (MEGA) using fatty acids (C8, C9, C10) and meglumine as raw materials and liquid enzyme (CARTrucA)

[0060]

[0061] To 1L of 100mM Tris-HCl (pH 8.0) solution, add 20mL of DMSO, 23.4g of meglumine (120mM), 4.2g of magnesium chloride hexahydrate (20mM), and 60.8g of adenosine triphosphate disodium salt (110mM). The pH of the reaction is then adjusted back to 8.0, and 2000U of ARTrucA and 1000U of crude BtPPase enzyme are added. Finally, 50mL of a 2mol / L fatty acid alcohol solution (isopropanol) is slowly and uniformly added to the solution to initiate the reaction. The entire addition process is maintained for 2 hours, during which the reaction system pH is maintained between 7.0 and 9.0. After the addition is complete, stirring is continued at room temperature for 10 hours to allow the reaction to complete.

[0062] The resulting reaction solution was first treated with D201 anion exchange resin (Tianjin Yunkai Resin Technology Co., Ltd.) in a 0-1N (NH4)2CO3 aqueous solution gradient to retain various phosphoric acid-containing impurities in the solution. The product MEGA was able to elute quickly due to its weak polarity. The crude product was then purified and collected using D101 non-polar resin (Xi'an Lanxiao Technology New Materials Co., Ltd.) by rinsing with a 10% ethanol aqueous solution. Finally, the product was desalted using a reverse osmosis membrane and concentrated to obtain 20-24 grams of alkyl glucose amide solid (all white solids with a yield of 63-74%).

[0063] Example 4: Preparation of alkyl glucamide (MEGA) using fatty acids (C8, C9, C10) and meglumine as raw materials, liquid enzyme (CARTrucAT) and ATP cycle regeneration enzyme (BaPPK)

[0064]

[0065] To 1 L of 100 mM Tris-HCl (pH 8.0) solution, add 20 mL of DMSO, 48.7 g of meglumine (250 mM), 4.2 g of magnesium chloride hexahydrate (20 mM), 5.6 g of adenosine triphosphate disodium salt (10 mM), and 41.5 g of sodium hexametaphosphate (68 mM). The pH of the reaction is then adjusted back to 8.0, and 3000 U of CARTrucAT, 2000 U of BaPPK, and 1000 U of crude BtPPase enzyme are added. Finally, 50 mL of a 2 mol / L fatty acid alcohol solution (isopropanol) is slowly and uniformly added dropwise to initiate the reaction. This addition is continued over 2 hours, during which the pH of the reaction system is maintained between 7.0 and 9.0. After the addition is complete, the reaction is stirred at room temperature for 6 hours to complete.

[0066] Using D201 anion exchange resin (Tianjin Yunkai Resin Technology Co., Ltd.), various phosphoric acid-containing impurities in the solution were retained in a gradient of 0-1N (NH4)2CO3 aqueous solution. The product MEGA was able to elute quickly due to its weak polarity; the crude product was then purified and collected using D101 non-polar resin (Xi'an Lanxiao Technology New Materials Co., Ltd.) by rinsing with 10% ethanol aqueous solution. Finally, the product was desalted using a reverse osmosis membrane and concentrated to obtain 23-26 grams of alkyl glucamide solid (all white solids with a yield of 66-81%).

[0067] Example 5: Preparation of alkyl glucamide (MEGA) using fatty acids (C8, C9, C10) and meglumine as raw materials, liquid enzyme (CARTrucA) and ATP cycle regeneration enzyme (BaPPK)

[0068]

[0069] Similar to Example 4, except that the CARTrucAT enzyme was replaced by the CARTrucA enzyme.

[0070] To 1 L of 100 mM Tris-HCl (pH 8.0) solution, add 20 mL of DMSO, 29.3 g of meglumine (150 mM), 4.2 g of magnesium chloride hexahydrate (20 mM), 5.6 g of adenosine triphosphate disodium salt (10 mM), and 41.5 g of sodium hexametaphosphate (68 mM). The pH of the reaction is then adjusted back to 8.0, and 2000 U of CARTrucAT, 2000 U of BaPPK, and 1000 U of crude BtPPase enzyme are added. Finally, 50 mL of a 2 mol / L fatty acid alcohol solution (isopropanol) is slowly and uniformly added dropwise to initiate the reaction. This addition is continued over 2 hours, during which the pH of the reaction system is maintained between 7.0 and 9.0. After the addition is complete, the reaction is stirred at room temperature for 5 hours to complete.

[0071] The reaction solution was then treated with D201 anion exchange resin (Tianjin Yunkai Resin Technology Co., Ltd.) in a 0-1N (NH4)2CO3 aqueous solution gradient to retain various phosphoric acid-containing impurities in the solution. The product MEGA was able to flow out quickly due to its weak polarity; the crude product was then purified and collected using D101 non-polar resin (Xi'an Lanxiao Technology New Materials Co., Ltd.) by rinsing with 10% ethanol aqueous solution. Finally, the product was desalted using a reverse osmosis membrane and concentrated to obtain 25-27 grams of alkyl glucose amide solid (all white solids with a yield of 71-84%).

[0072] Example 6: Preparation of alkyl glucamide (MEGA) by immobilized mixed enzyme using fatty acids (C8, C9, C10) and meglumine as raw materials

[0073]

[0074] In the tris(hydroxymethyl)aminomethane hydrochloric acid (Tris.HCl) solution of 1L 100mM pH 8.0, add 20mL DMSO solvent, 39 grams of meglumine (200mM), 4.2 grams of magnesium chloride hexahydrate (20mM), 5.6 grams of adenosine triphosphate disodium salt (10mM), 41.5 grams of sodium hexametaphosphate (68mM), then the pH value of reaction is adjusted back to 8.0, and add 6000U immobilized mixed enzyme (prepared by embodiment 2).Finally, in the solution, slowly drip the fatty acid alcohol solution (isopropanol solvent) of 50mL 2mol / L to start the reaction, and the whole instillation process maintained 2 hours. During this period, utilize acid and alkali to maintain the reaction system pH 7.0~9.0 before, continue to maintain stirring at room temperature after being added dropwise to complete the reaction in 12 hours and promptly complete.Filter the reaction solution subsequently and reclaim the immobilized mixed enzyme (enzyme activity retains 87% after this immobilized enzyme is used 6 times).

[0075] The reaction solution was treated similarly to the other embodiments. The reaction solution was treated with D201 anion exchange resin (Tianjin Yunkai Resin Technology Co., Ltd.) in a 0-1N (NH4)2CO3 aqueous solution gradient to retain various phosphoric acid-containing impurities in the solution. The product MEGA was able to flow out quickly due to its weak polarity. The crude product was then purified and collected using D101 non-polar resin (Xi'an Lanxiao Technology New Materials Co., Ltd.) by washing with 10% ethanol aqueous solution. Finally, the product was desalted using a reverse osmosis membrane and concentrated to obtain 27-29 grams of alkyl glucose amide solid (all white solids with a yield of 77-90%).

[0076] Comparative Example: Using fatty acids (C8, C9, C10) and meglumine as raw materials, liquid enzyme (WTCARTrucA) was used to prepare alkyl glucamide (MEGA)

[0077]

[0078] Similar to the above Example 3, but the mutant enzyme CARTrucA was replaced by the natural enzyme WTCARTrucA;

[0079] To 1L of 100mM Tris-HCl (pH 8.0) solution, add 20mL of DMSO, 23.4g of meglumine (120mM), 4.2g of magnesium chloride hexahydrate (20mM), and 60.8g of adenosine triphosphate disodium salt (110mM). The pH of the reaction is then adjusted back to 8.0, and 3000U of WtCARTrucA and 2000U of crude BtPPase enzyme are added. Finally, 50mL of a 2mol / L fatty acid alcohol solution (isopropanol) is slowly and uniformly added to the solution to initiate the reaction. The entire addition process is maintained over 2 hours, during which the reaction system pH is maintained between 7.0 and 9.0. After the addition is complete, stirring is continued at room temperature for 12 hours to allow the reaction to complete.

[0080] After the reaction, the pH of the solution was adjusted to 1.0 and stirred to remove protein precipitates. The pH was then adjusted back to pH 7.0, and the solution was treated with D201 anion exchange resin (Tianjin Yunkai Resin Technology Co., Ltd.) in a 0-1N (NH4)2CO3 aqueous solution gradient to retain various phosphoric acid-containing impurities in the solution. The product MEGA, due to its weak polarity, was able to flow out quickly. The crude product was then purified and collected using D101 non-polar resin (Xi'an Lanxiao Technology New Materials Co., Ltd.) by rinsing with 10% ethanol aqueous solution. Finally, the product was desalted using a reverse osmosis membrane and concentrated to obtain 3.8-4.9 grams of alkyl glucose amide solid (all gray solids with a yield of 11-15%).

[0081] The solid alkyl glucose amide obtained in each embodiment and comparative example was subjected to nuclear magnetic resonance hydrogen spectrum, D2O was used as solvent, Varian 600 MHz nuclear magnetic resonance, and the results were as follows: Figures 2 to 4 As shown, it is proved that the product is alkylglucamide.

[0082] Table 1: Performance comparison of the mutant enzyme of the present invention and its wild-type enzyme

[0083]

[0084] Note: The expression levels of the enzymes were determined after fermentation according to the method of Example 2.

[0085] It can be seen from the results in Table 1, Examples and Comparative Examples that, compared with the corresponding wild-type enzyme, the activity, thermal stability and expression level of the mutant enzyme of the present invention are significantly improved; and the yield is greatly increased when used to synthesize alkyl glucamide.

[0086] 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 considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A mutant enzyme 1 of alkylglucosamide condensase, characterized in that: The amino acid sequence is shown in SEQ.ID.NO.1, and the encoding nucleotide sequence is shown in SEQ.ID.NO.

2.

2. A mutant enzyme 2 of alkylglucosamide condensase, characterized in that: The amino acid sequence is shown in SEQ.ID.NO.3, and the encoding nucleotide sequence is shown in SEQ.ID.NO.

4.

3. A fermentation method for producing an alkylglucosamide condensing enzyme mutant, characterized in that The following steps are involved: (1) Synthesize the nucleotide sequence shown in SEQ.ID.NO.2 or SEQ.ID.NO.4, then subclone into the pET28a plasmid through the NdeI / XhoI restriction sites, and transform the plasmid into Escherichia coli cells for plate culture. Finally, select a single clone for liquid amplification culture; (2) Transfer the single colony on the plate into LB culture medium containing at least 50 μM kanamycin for culture. When the cells grow to the logarithmic phase, inoculate them into LB culture medium containing the same antibiotic, and finally transfer them to a fermenter for culture. When the OD value of the fermentation liquid reaches 20, add 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) to induce protein expression for at least 8 hours, and then collect the wet cells by centrifugation. (3) The wet cells are mixed with the buffer solution, and then the cells are broken by high pressure and centrifuged at high speed to remove the cell walls, and finally the enzyme-containing clear solution is obtained.

4. Use of the alkylglucosamide condensing enzyme mutant 1 according to claim 1 and / or the alkylglucosamide condensing enzyme mutant 2 according to claim 2 in the synthesis of alkylglucosamide.

5. A method for synthesizing alkyl glucamide, characterized in that The following steps are involved: Add a solvent, meglumine, magnesium chloride hexahydrate, and adenosine triphosphate disodium salt to a buffer solution, adjust the pH value of the reaction system to 7.0-9.0, and add the alkyl glucosamide condensing enzyme mutant 1 described in claim 1 and / or the alkyl glucosamide condensing enzyme mutant 2 described in claim 2. Finally, add a fatty acid alcohol solution dropwise to the system to initiate the reaction, and maintain the addition process for more than 2 hours. After the addition is completed, continue to stir at room temperature for more than 10 hours to complete the reaction. Maintain the pH value of the reaction system at 7.0-9.0 during the addition process and the reaction process. The fatty acid has 8 to 10 carbon atoms.

6. The method according to claim 5, characterized in that: The molar ratio of meglumine to fatty acid is (1.2-2.5):

1.

7. The method according to claim 5, characterized in that: When the alkylglucose amide condensing enzyme mutant is added, pyrophosphohydrolase BtPPase is added.

8. The method according to claim 5, characterized in that: Polyphosphate kinase was added simultaneously with the addition of the alkylglucosamide condensase mutant enzyme.

9. The method according to any one of claims 5 to 8, characterized in that: The enzyme was immobilized for use.

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