Mutant of horse-derived acylase with high pH stability and application of mutant

Abstract

The invention belongs to the technical field of biology, and relates to a mutant of horse-derived acylase with high pH stability, a coding gene and application of the mutant in catalytic generation of lauroyl glycine. The horse-derived acylase mutant sequence disclosed by the invention has the advantages of high enzyme activity stability in a wider pH environment, high synthesis efficiency of lauroyl glycine and very high production and application values.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and relates to a mutant of a horse-derived acyltransferase with high pH stability, a coding gene thereof, and an application thereof in catalyzing the production of lauroyl glycine. Background Art

[0002] Developing high-quality, multifunctional, and environmentally friendly surfactants has become the main direction of the surfactant industry, such as amino acid-based surfactants. Amino acid-based surfactants not only have a wide range of biomass raw material sources, low toxicity and side effects, mild performance, low irritation, and good biodegradability, but also their good emulsifying, wetting, solubilizing, dispersing, foaming and other properties have attracted much attention at present and are gradually applied to many fields such as washing, personal care, and food industry.

[0003] N-acyl amino acid-based surfactants are still the most important amino acid-based surfactants. At present, the synthesis of N-acyl amino acids has the disadvantages of high difficulty, low yield, and non-green synthesis process. The main reason is that the synthesis of commercially available N-acyl amino acids is mainly chemical method, and the most commonly used method is the Schotten-Baumann reaction, which is a reaction of fatty acyl chloride and amino acid in an organic solvent-water mixture as a reaction medium and a strong base as a catalyst. It is difficult to solve the problems of reaction by-products and environmental pollution.

[0004] Compared with the chemical method, the enzymatic synthesis of N-acyl amino acids has great advantages. The enzymatic synthesis is mainly through the amide reaction of fatty acids and their derivatives and amino acids catalyzed by biological enzymes, and has been widely studied and concerned due to its greenness and safety. Sodium lauroyl glycinate is an amino acid surfactant, which can be formed by a simple saponification reaction of lauroyl glycine. In previous studies, the horse-derived acyltransferase (Equus caballus aminoacylase-1, EcAcy) (GenBank: XP_001492888.2) discovered by our team has the synthesis activity of lauroyl glycine, but there is still a long way to go for its industrial production.

[0005] In the manufacturing process of lauroyl glycine using an enzymatic reaction, the following processes are generally carried out in sequence: a reaction process, in which at pH 7.5 - 8.0, under the catalytic action of acylating enzyme EcAcy, a reaction of synthesizing lauroyl glycine from lauric acid and glycine is carried out; a purification process, in which the pH of the above reaction solution is adjusted to 5.0 - 7.0 to achieve precipitation and separation of lauroyl glycine. In order to improve the efficiency of synthesizing lauroyl glycine compounds using equine acylating enzyme EcAcy, the inventors of this patent conducted in-depth research. The results showed that when the purification process is carried out within an acidic pH range and the reaction of synthesizing lauroyl glycine from lauric acid and glycine catalyzed by acylating enzyme EcAcy can proceed continuously, the catalytic efficiency of acylating enzyme EcAcy can be greatly improved and the process cost can be reduced. Summary of the Invention

[0006] The optimal pH of acylating enzyme EcAcy is 7.5 - 8.5, and the enzyme activity decreases significantly under conditions of pH below 7. Therefore, when using wild-type acylating enzyme EcAcy, the enzymatic reaction will not proceed to a satisfactory extent. Accordingly, the present invention provides, through research, a mutant of acylating enzyme with high pH stability, its encoding gene, and their application in catalyzing the formation of lauroyl glycine. More specifically, it provides an acylating enzyme mutant that still maintains good enzyme activity within the range of pH 5.5 - 8.5.

[0007] The first aspect of the present invention provides a series of acylating enzyme mutants. The amino acid sequence of its wild-type acylating enzyme is as shown in SEQ ID NO.2, and on this basis, the acylating enzyme mutant contains one, two, three or more mutants selected from the following site amino acid residue mutations:

[0008] (1) The 4th amino acid residue is mutated from lysine to glutamic acid or aspartic acid or asparagine;

[0009] (2) The 6th amino acid residue is mutated from arginine to glutamic acid or aspartic acid or asparagine;

[0010] (3) The 18th amino acid residue is mutated from glutamine to glutamic acid;

[0011] (4) The 50th amino acid residue is mutated from glutamine to glutamic acid;

[0012] (5) The 69th amino acid residue is mutated from asparagine to aspartic acid or glutamic acid;

[0013] (6) The 71st amino acid residue is mutated from arginine to glutamic acid;

[0014] (7) The 79th amino acid residue is mutated from serine to glutamic acid;

[0015] (8) The 112th amino acid residue is mutated from glutamine to glutamic acid;

[0016] (9) The 165th amino acid residue is changed from glutamine to glutamate;

[0017] (10) The 235th amino acid residue is changed from glutamine to glutamate;

[0018] (11) The 240th amino acid residue is changed from asparagine to aspartic acid;

[0019] (12) The 243rd amino acid residue is changed from glutamine to glutamate;

[0020] (13) The 284th amino acid residue is changed from lysine to glutamate;

[0021] (14) The 295th amino acid residue is changed from glutamine to glutamate;

[0022] (15) The 335th amino acid residue is changed from asparagine to aspartic acid;

[0023] (16) The 367th amino acid residue is changed from lysine to glutamate or aspartic acid;

[0024] (17) The 393rd amino acid residue is changed from glutamine to glutamate or aspartic acid;

[0025] Preferably, the acylating enzyme mutant contains mutants with amino acid residue mutations at the above two sites, more specifically, it contains the following mutations:

[0026] K4E / Q243E, R6E / Q243E, N69E / Q243E, R71E / Q243E, Q165E / Q243E, Q235E / Q243E, K284E / Q243E, N335D / Q243E, R367D / Q243E, Q393E / Q243E, Q393D / Q243E.

[0027] Preferably, the acylating enzyme mutant contains mutants with amino acid residue mutations at the above three sites, more specifically, it contains:

[0028] K4E / N335D / Q243E, R71E / N335D / Q243E, K284E / N335D / Q243E, R367D / N335D / Q243E, Q393E / N335D / Q243E.

[0029] Preferably, the acylating enzyme mutant contains mutants with amino acid residue mutations at the above four or more sites, more specifically, it contains:

[0030] K4E / R367D / N335D / Q243E, R71E / R367D / N335D / Q243E, K284E / Q393E / R367D / N335D / Q243E.

[0031] Under reaction conditions such as pH 5.5 - 8.5, the acylase mutants containing the above mutations have more stable enzyme activity for lauroyl glycine synthesis than the wild type.

[0032] The second aspect of the present invention provides the coding nucleic acid of the above acylase mutant.

[0033] The third aspect of the present invention provides a vector containing the coding nucleic acid of the above acylase mutant, such as an expression vector, more specifically, the starting vector is a pET vector.

[0034] The fourth aspect of the present invention provides a genetically engineered bacterium containing the coding nucleic acid of the above acylase mutant or the said vector. For example, it is a bacterium or a fungus, specifically, the starting bacterium is Escherichia coli or Bacillus subtilis or Pichia pastoris.

[0035] The fifth aspect of the present invention provides the application of the said kidney acylase mutant, its coding nucleic acid, vector or genetically engineered bacterium in the biocatalytic synthesis of octanoyl surfactants.

[0036] The sixth aspect of the present invention provides a method for preparing lauroyl glycine. The preparation method includes using the above acylase mutant as a biocatalyst, and using lauric acid and glycine as substrates to form a reaction system, and reacting to generate lauroyl glycine compounds.

[0037] Specifically, the wet cells or crude enzyme solution or pure enzyme obtained by fermenting and culturing the said genetically engineered bacterium containing the acylase mutant is used as the biocatalyst. Preferably, the said genetically engineered bacterium in the form of wet cells is used as a whole cell catalyst.

[0038] More specifically, in the said reaction system, the dosage of the wet cells is 5 - 50 g / L, the concentration of lauric acid is 2 - 200 g / L, and 20% - 100% saturated glycine solution.

[0039] The reaction conditions are: carrying out the biocatalytic reaction under the conditions of temperature 35 - 65°C, pH 5.5 - 8.5, and stirring speed 100 - 300 rpm.

[0040] The said wet cells are obtained by centrifuging the fermented and cultured genetically engineered bacterium, discarding the supernatant, and collecting the precipitate.

[0041] More specifically, the wet bacteria were prepared as follows: the recombinant Escherichia coli containing the gene encoding the mutant of horseradish acylase was inoculated into a LB liquid medium containing 50 μg / ml kanamycin resistance, and cultured at 37°C, 200 rpm for 12 h, and then inoculated into a fresh LB liquid medium containing 50 μg / ml kanamycin resistance at a 1% (v / v) inoculation amount, and cultured at 37°C, 200 rpm until the bacteria OD 600 When the pH value reaches 0.6-0.8, add IPTG with a final concentration of 0.1 mM, and induce and culture at 20°C, 200 rpm for 15 hours; then centrifuge at 4°C, 8000 rpm for 20 minutes, discard the supernatant, collect the precipitate, and obtain the wet bacteria.

[0042] The present invention provides an acylase mutant for catalyzing the synthesis of lauroylglycine from lauric acid and glycine, and the pH stability of the acylase is significantly improved compared with that of the wild-type acylase. More specifically, the enzyme activity is still well maintained in the range of pH 5.5 to 8.5. The acylase mutant provided by the present invention has high lauroylglycine synthase activity, a high yield of lauroylglycine compounds, reduced reaction costs, and has high production application value. DETAILED DESCRIPTION

[0043] The present invention is described below through specific implementation modes in order to better understand the present invention, but it does not constitute a limitation of the present invention.

[0044] Example 1. Obtaining wild-type acylase (1) from horse

[0045] According to the wild-type horse-derived acylase EcAcy (GenBank: XP_001492888.2) included in NCBI, the amino acid sequence of which is shown in SEQ ID NO.2, the wild-type plant-derived acylase gene was artificially synthesized, and the gene was used as a template to extend the fragment by PCR amplification (with NdeI and XhoI endonuclease fragments on both sides of the fragment). The nucleotide sequence is shown in SEQ ID NO.1.

[0046] The PCR product was purified and recovered by a purification and recovery kit, and the PCR product and the E. coli expression vector pET-28a(+) were double-digested with NdeI and XhoI, respectively, and the digested product was purified and recovered again. Under the action of T4 DNA ligase, the target gene was connected to the E. coli expression vector pET-28a(+) to obtain the EcAcy recombinant expression plasmid. It was transferred into E. coli BL21(DE3) to establish the EcAcy genetic engineering bacteria.

[0047] The above genetically engineered bacteria were inoculated into LB liquid medium containing 50 μg / ml kanamycin resistance, cultured at 37 °C and 200 rpm for 12 h, and then inoculated into fresh LB liquid medium containing 50 μg / ml kanamycin resistance at an inoculation amount of 1% (v / v), cultured at 37 °C and 200 rpm until the OD600 of the bacteria reached 0.6 - 0.8. IPTG with a final concentration of 0.1 mM was added, and the culture was induced at 20 °C and 200 rpm for 15 h. After that, the mixture was centrifuged at 4 °C and 8000 rpm for 20 min, the supernatant was discarded, and the precipitate was collected to obtain wet bacteria expressing wild-type EcAcy, that is, wild-type acyltransferase (1).

[0048] Example 2. Comparison of enzyme activity and pH stability

[0049] The wet bacteria obtained in Example 2 were used for the synthesis of octanoyl alanine at different pH values. Synthesis reaction system (10 mL): 360 mg of lauric acid, 10 ml of saturated glycine solution, 0.2 g of wet bacteria, and the pH values were 6.5 and 8.0 respectively. The reaction was carried out at 37 °C for 24 h. After the reaction, the reaction solution was analyzed by HPLC to obtain the synthase activity of lauric acid glycine. The reaction and analysis were carried out more than 3 times for each reaction system, and the data deviation caused by operations and the like was corrected.

[0050] Analysis conditions: Chromatographic column: C18, 4.6 mm × 250 mm, 5 μm; Detection wavelength: ultraviolet (UV) = 200 nm; Mobile phase: 85% acetonitrile and 15% 0.05% trifluoroacetic acid aqueous solution; Column temperature: 30 °C; Flow rate: 1 mL / min; Detection time: 15 min.

[0051] The ratio of the synthase activity of octanoyl alanine at pH 6.5 to that at pH 8.0 was taken as the basis for pH stability.

[0052] Example 3. Obtaining acyltransferase mutants with single mutation sites

[0053] Based on the optimization of the surface charge of horse-derived acyltransferase, it was found that the 4th, 6th, 18th, 50th, 69th, 71st, 79th, 112th, 165th, 235th, 240th, 243rd, 284th, 295th, 335th, 367th, 393rd and other sites of horse-derived acyltransferase may be related to pH stability. An attempt was made to mutate the horse-derived acyltransferase gene to obtain mutants, and their enzyme activity (pH 8.0) and pH stability were verified.

[0054] Using the recombinant plasmid EcAcy recombinant expression plasmid constructed in Example 1 as a template, whole plasmid PCR amplification was performed. After the PCR reaction was completed, 1 μL of DpnI was added and digested at 37 °C for 30 min. Then, it was transformed into competent cells of Escherichia coli BL21(DE3) to obtain the corresponding mutants. The corresponding wet cells were obtained according to the method described in Example 1. Then, the above acylase mutants were evaluated according to the method described in Example 2. The results of the relative enzyme activity (pH 8.0) and relative pH stability compared to the wild type are shown in Table 1.

[0055] Table 1 Enzyme activity and pH stability data of acylase mutants with single mutation sites

[0056]

[0057]

[0058] As can be seen from the results shown in Table 1, single mutations of K4E, K4D, K4N, R6N, R6D, R6E, Q18E, Q50E, N69D, N69E, R71E, S79E, Q112E, Q165E, Q235E, N240D, Q243E, K284E, Q295E, N335D, R367E, R367D, Q393E, Q393D can not only improve the enzyme activity (pH 8.0) of acylase, but also improve its pH stability.

[0059] Example 4 Obtaining of acylase mutants with double mutation sites

[0060] According to the enzyme activity and pH stability data of acylase mutants with single mutation sites shown in Table 1, the mutant (19) with relatively high enzyme activity and pH stability was used as the evolutionary parent of acylase mutants with double mutation sites. Site-directed mutagenesis was performed according to the method described in Example 3 to obtain acylase mutants with double mutation sites as shown in Table 2. The corresponding wet cells were obtained according to the method described in Example 1. Then, the above acylase mutants were evaluated according to the method described in Example 2. The results of the relative enzyme activity (pH 8.0) and relative pH stability compared to mutant (19) are shown in Table 2.

[0061] Table 2 Enzyme activity and pH stability data of acylase mutants with double mutation sites

[0062]

[0063]

[0064] As can be seen from the results shown in Table 2, the double-site mutations of K4E / Q243E, R6E / Q243E, N69E / Q243E, R71E / Q243E, Q165E / Q243E, Q235E / Q243E, K284E / Q243E, N335D / Q243E, R367D / Q243E, Q393E / Q243E, and Q393D / Q243E can not only improve the enzyme activity (pH 8.0) of the acylase compared with the single-site mutation Q243E, but also improve its pH stability.

[0065] Example 5. Obtaining of Acylase Mutants with Three Mutation Sites

[0066] Based on the enzyme activity and pH stability data of the acylase mutants with double mutation sites shown in Table 2, the mutant (34) with relatively high enzyme activity and pH stability was used as the evolutionary parent of the acylase mutants with three mutation sites. Site-directed mutagenesis was carried out by the method shown in Example 3 to obtain the acylase mutants with three mutation sites shown in Table 3. The corresponding wet cells were obtained according to the method described in Example 1. Then, the above acylase mutants were evaluated according to the method described in Example 2. The relative values of the enzyme activity (pH 8.0) and the relative values of pH stability relative to the mutant (34) are shown in Table 3.

[0067] Table 3. Enzyme Activity and pH Stability Data of Acylase Mutants with Three Mutation Sites

[0068]

[0069]

[0070] As can be seen from the results shown in Table 3, the triple-site mutations of K4E / N335D / Q243E, R71E / N335D / Q243E, K284E / N335D / Q243E, R367D / N335D / Q243E, and Q393E / N335D / Q243E can not only further improve the enzyme activity (pH 8.0) of the acylase compared with the double-site mutation N335D / Q243E, but also improve its pH stability.

[0071] Example 6. Obtaining of Acylase Mutants with More than Three Mutation Sites

[0072] According to the enzyme activity and pH stability data of the acylase mutants at the three mutation sites shown in Table 3, the mutant (41) with relatively high enzyme activity and pH stability was used as the evolutionary parent of the acylase mutants with more than three mutation sites. Site-directed mutagenesis was carried out by the method shown in Example 3 to obtain the acylase mutants with multiple mutation sites shown in Table 4. The corresponding wet cells were obtained according to the method described in Example 1. Then, the above acylase mutants were evaluated according to the method described in Example 2. The relative values of the enzyme activity (pH 8.0) and the relative values of pH stability relative to the mutant (41) are shown in Table 4.

[0073] Table 4. Enzyme activity and pH stability data of acylase mutants with four mutation sites

[0074]

[0075] As can be seen from the results shown in Table 4, multi-site mutations such as K4E / R367D / N335D / Q243E, R71E / R367D / N335D / Q243E, and K284E / Q393E / R367D / N335D / Q243E can not only improve the enzyme activity (pH 8.0) of acylase, but also improve its pH stability.

Claims

1. An acylase mutant, which is a mutant containing one, two, three or more amino acid residue mutations selected from the following positions based on the amino acid sequence of the wild-type acylase as shown in SEQ ID NO.2: K4E, K4D, K4N, R6N, R6D, R6E, Q18E, Q50E, N69D, N69E, R71E, S79E, Q112E, Q16 5E, Q235E, N240D, Q243E, K284E, Q295E, N335D, R367E, R367D, Q393E, Q393D.

2. The acylase mutant according to claim 1, characterized in that The mutant containing two mutations of amino acid residues at the above positions, more specifically, contains the following mutations: K4E / Q243E, R6E / Q243E, N69E / Q243E, R71E / Q243E, Q165E / Q243E, Q235E / Q24 3E, K284E / Q243E, N335D / Q243E, R367D / Q243E, Q393E / Q243E, Q393D / Q243E.

3. The acylase mutant according to claim 1, characterized in that The mutants containing three mutations of amino acid residues at the above positions more specifically contain: K4E / N335D / Q243E, R71E / N335D / Q243E, K284E / N335D / Q243E, R367D / N335D / Q243E, Q393E / N335D / Q243E.

4. The acylase mutant according to claim 1, characterized in that The mutants containing four or more amino acid residue mutations at the above positions more specifically contain: K4E / R367D / N335D / Q243E, R71E / R367D / N335D / Q243E, K284E / Q393E / R367D / N335D / Q243E.

5. A nucleic acid encoding the acylase mutant according to any one of claims 1 to 4.

6. A vector containing the encoding nucleic acid according to any one of claims 1 to 4, such as an expression vector, more specifically a pET vector.

7. A genetically engineered bacterium containing the nucleic acid encoding the acylase mutant according to any one of claims 1 to 4, or the vector according to claim 6, such as bacteria or fungi, specifically, the starting bacterium is Escherichia coli, Bacillus subtilis or Pichia pastoris.

8. Use of the horse acylase mutant according to any one of claims 1 to 4, the encoding nucleic acid thereof, the vector according to claim 6, or the genetically engineered bacteria according to claim 7 in biocatalytic synthesis of lauroylglycine.

9. A method for preparing lauroyl glycine, characterized in that: The method comprises using the acylase mutant according to any one of claims 1 to 4 as a biocatalyst, and using lauric acid and glycine as substrates to form a reaction system to generate a lauroylglycine compound.

10. The preparation method according to claim 9, characterized in that: The acylase mutant is a wet cell, crude enzyme solution or pure enzyme obtained by fermentation culture of the genetically engineered bacteria as a biocatalyst; preferably, the genetically engineered bacteria is used as a whole cell catalyst in the form of wet cells; More specifically, in the reaction system, the wet cell dosage is 5-50 g / L, the lauric acid concentration is 2-200 g / L, and the saturated glycine solution is 20%-100%; The reaction conditions are: the biocatalytic reaction is carried out at a temperature of 35-65°C, a pH of 5.5-8.5, and a stirring speed of 100-300 rpm; The wet bacteria are obtained by fermenting and culturing the genetically engineered bacteria, centrifuging, discarding the supernatant, and collecting the precipitate; More specifically, the wet bacteria were prepared as follows: the recombinant Escherichia coli containing the gene encoding the mutant of horseradish acylase was inoculated into a LB liquid medium containing 50 μg / ml kanamycin resistance, and cultured at 37°C, 200 rpm for 12 h, and then inoculated into a fresh LB liquid medium containing 50 μg / ml kanamycin resistance at a 1% (v / v) inoculation amount, and cultured at 37°C, 200 rpm until the bacteria OD 600 When the pH value reaches 0.6-0.8, add IPTG with a final concentration of 0.1 mM, and induce and culture at 20°C, 200 rpm for 15 hours; then centrifuge at 4°C, 8000 rpm for 20 minutes, discard the supernatant, collect the precipitate, and obtain the wet bacteria.

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