Mutant of acylase and application of mutant in synthesis of sodium cocoyl glycinate

Abstract

The invention discloses a mutant of acylase and application of the mutant in synthesis of sodium cocoyl glycinate. The invention discloses a mutant of acylase, which is a mutant containing one, two, three or more substitution mutations of amino acid residues selected from the following sites on the basis of an amino acid sequence of the acylase as shown in SEQ ID NO.2: R209C, K218N, P244L, S249T, S249N, C271V, Q290K, M309T, C342A or L394I. The acylase mutant disclosed by the invention has the advantages that the activity of cocoyl glycine synthetase is obviously improved, and meanwhile, the mutant enzyme can tolerate cocinic acid and cocoyl glycine with relatively high concentration, so that the concentration of a reaction system is favorably improved, and the production cost is reduced.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 2025113575185, entitled "A mutant of acylase and its application in the synthesis of sodium cocoyl glycinate", filed on September 23, 2025. Technical Field

[0002] This invention belongs to the field of biotechnology, specifically relating to mutants of acylases, their encoding genes, and their application in the catalytic production of sodium cocoyl glycinate. Background Technology

[0003] Sodium Cocoyl Glycinate (SCG) is an amino acid surfactant whose chemical structure consists of coconut acid (a fatty acid) linked to glycine via an amide bond. Due to its mildness, low irritation, excellent foaming and cleaning power, SCG is widely used in high-end personal care products, such as baby washes, facial cleansers for sensitive skin, and shampoos. With the increasing consumer demand for mild, natural, and environmentally friendly products, the SCG market is rapidly expanding.

[0004] Currently, the industrial production of sodium cocoyl glycinate mainly employs chemical synthesis. This method typically uses cocoyl chloride or methyl cocoate as the acylation agent, reacting with glycine under alkaline conditions. Both methods have significant drawbacks. The acyl chloride method generates toxic chlorinating agents and highly corrosive hydrogen chloride gas, polluting the environment and causing severe equipment corrosion. The methyl cocoate method has low reaction yields, requires a catalyst, and produces methanol as a byproduct, necessitating additional separation and purification steps.

[0005] To overcome the shortcomings of traditional chemical synthesis, researchers began exploring the use of enzymatic methods to synthesize SCG. Enzymatic methods offer the following significant advantages: First, mild reaction conditions: enzymatic reactions typically occur at room temperature and pressure, in an aqueous phase or a water-organic mixture, resulting in low energy consumption and aligning better with the principles of green chemistry. Second, high selectivity and specificity: enzymes exhibit high substrate specificity and regioselectivity, effectively reducing side reactions and improving the yield and purity of the target product, SCG. Third, environmental friendliness: enzymes themselves and their metabolites are generally non-toxic and biodegradable, producing no harmful waste.

[0006] However, the currently reported bioenzymatic synthesis of SCG still faces many challenges. For example: (1) Substrate inhibition: Cocoolic acid and cocoyl glycine, as reaction substrates and products, inhibit many enzymes at high concentrations, affecting reaction efficiency. (2) Enzyme preparation cost: The production and purification costs of enzymes are high, increasing the overall production cost. (3) Low product yield: The reported yields of enzymatic SCG are generally low, making it difficult to meet the needs of industrial production.

[0007] Porcine kidney acyltransferase (pAcy) is a homodimeric zinc metalloenzyme. Its core function is the specific hydrolysis of N-acyl-L-amino acids. In 2002, Eiko Wada reported that porcine kidney acyltransferase can synthesize N-medium-chain and long-chain acyl amino acids from the corresponding L-amino acids and fatty acids (Eiko Wada, Masato Handa, Koreyoshi Imamura, et al. Enzymatic synthesis of -Acyl-L-amino acid in a glycerol-water system using acylase I from pig kidney. JAOCS, Vol.79, no.1 (2002).). In 2008, Holger A. Lindner reported that porcine kidney acylase possesses the hydrolytic ability of lauroyl amino acid surfactants (Holger A. Lindner, Alain Alary, Marsha Wike, and Triaian Sulea. Probing the acyl-binding pocket of aminoacylase-1. Biochemistry 2008, 47, 4266-4275.). However, its industrial production is still a long way off. Summary of the Invention

[0008] This invention aims to develop a mutant acylate for the highly efficient catalytic synthesis of sodium cocoyl glycinate. This enzyme can achieve efficient acylation of coconut oil acid and glycine, and has advantages such as mild reaction conditions, high yield, high product purity, and environmental friendliness. Sodium cocoyl glycinate prepared by this method can be widely used in various fields, especially in personal care products and the food industry, where its application potential is enormous.

[0009] The first aspect of the invention provides a series of acylase mutants. The amino acid sequence of the wild-type porcine kidney acylase is shown in SEQ ID NO.2, and based thereon, the acylase mutants contain one, two, three or more mutants selected from the following sites for amino acid residue substitution mutations: S3T, P70A, L177C, G207V, R209C, K218N, P244L, S249T, S249N, C271V, Q290K, M309T, C342A or L394I.

[0010] Preferably, the acylase mutant contains a mutant with amino acid residue substitution mutations at the above two sites, and more specifically contains the following substitution mutations: L177C / C271V, R209C / L394I, G207V / C342A, L177C / P244L, G207V / M309T, L177C / S249N, R209C / C342A or P244L / L394I.

[0011] Preferably, the acylase mutant contains a mutant with amino acid residue mutations at the above three sites, and more specifically contains the following substitution mutations: L177C / C271V / G207V, L177C / C271V / R209C, L177C / C271V / P244L, L177C / C271V / L394I or L177C / C271V / M309T.

[0012] Preferably, the acylase mutant contains mutants with amino acid residue substitution mutations at four or more sites as described above, and more specifically contains the following substitution mutations: L177C / C271V / L394I / S3T, L177C / C271V / L394I / S3T / R209C or L177C / C271V / L394I / R209C / P70A.

[0013] A second aspect of the present invention provides the encoding nucleic acid of the above-described acylase mutant.

[0014] A third aspect of the invention provides a vector containing the above-mentioned acylase mutant encoding nucleic acid, such as an expression vector, and more specifically, a pET vector as a starting vector.

[0015] A fourth aspect of the invention provides genetically engineered bacteria containing the above-described acylase mutant encoding nucleic acid or the vector. For example, these are bacteria or fungi, specifically, the originating bacteria being *Escherichia coli*, *Bacillus subtilis*, or *Pichia pastoris*.

[0016] A fifth aspect of the invention provides the use of the porcine kidney acylase mutant, its encoded nucleic acid, vector, or genetically engineered bacteria in the biocatalytic synthesis of sodium cocoyl glycinate.

[0017] A sixth aspect of the present invention provides a method for preparing sodium cocoyl glycinate. The method comprises using the aforementioned acylase mutant as a biocatalyst, and using coconut oil acid and glycine as substrates to form a reaction system, thereby generating sodium cocoyl glycinate.

[0018] Specifically, the acylase mutant uses crude enzyme solution, pure enzyme, or wet cells obtained by fermentation culture of the genetically engineered bacteria as a biocatalyst. Preferably, the genetically engineered bacteria in wet cell form serve as a whole-cell catalyst.

[0019] More specifically, in the reaction system, the amount of wet bacterial cells is 5-30 g / L, the concentration of coconut oil acid is 10-300 g / L, and the concentration of saturated glycine solution is 60%-100%.

[0020] The reaction conditions were: temperature 35-65℃, pH 5.5-8.5, and stirring speed 100-300 rpm for biocatalytic reaction.

[0021] The wet bacterial cells are obtained by fermenting and culturing the genetically engineered bacteria, followed by centrifugation, discarding the supernatant, and collecting the precipitate.

[0022] More specifically, the wet bacterial cells are prepared as follows: Recombinant Escherichia coli containing the gene encoding a porcine kidney acylase mutant are inoculated into LB liquid medium containing 50 μg / ml kanamycin resistance, and cultured at 37°C and 200 rpm for 12 h. Then, 1% (v / v) inoculation is carried out into fresh LB liquid medium containing 50 μg / ml kanamycin resistance, and cultured at 37°C and 200 rpm until the bacterial cell OD reaches 100%. 600 When the concentration reaches 0.6-0.8, add IPTG to a final concentration of 0.1 mM, and induce culture at 20℃ and 200 rpm for 15 h; then centrifuge at 4℃ and 8000 rpm for 20 min, discard the supernatant, collect the precipitate, and obtain the wet bacterial cells.

[0023] This invention provides a series of porcine kidney acylase mutants that can be used to improve the efficiency of industrial production of sodium cocoyl glycinate. The provided acylase has the following characteristics: High catalytic efficiency: Through enzyme design and screening at different mutation sites, acylases with high catalytic activity were obtained, achieving a higher conversion rate for the synthesis of cocoyl glycinate. Environmental friendliness: This enzyme reaction can replace traditional chemical production, conforming to the principles of green chemistry and reducing environmental pollution. Industrial application potential: Preliminary performance evaluation tests have demonstrated that this acylase can significantly reduce costs and improve product quality in actual production, making it suitable for large-scale production. Detailed Implementation

[0024] The present invention will be described below through specific embodiments in order to better understand the present invention, but this does not constitute a limitation thereof.

[0025] Example 1: Establishment of genetically engineered bacteria

[0026] Based on the porcine kidney acyltransferase gene pAcy (GenBank: NP_999061.1) indexed by NCBI, whose nucleotide sequence is shown in SEQ ID NO.1, the porcine kidney acyltransferase gene was artificially synthesized. Using this gene as a template, the fragment was amplified by PCR (with NdeI and XhoI endonuclease fragments added to both sides of the fragment). The primers for PCR amplification of pAcy were: upstream primer 1: 5'-cggcagccatatgGCGAGCAAAGGCCGCGAAGG-3' (SEQ ID NO:3), and downstream primer 2: 5'-ggtggtggtggtgctcgagGCTTTCGCTCGGTAAGGCCGG-3' (SEQ ID NO:4).

[0027] The PCR system components (total volume 50 μL) were added as follows: Primestar 25 μL, ddH2O 20 μL, forward and reverse primers 1.5 μL each, and template 2 μL. The PCR amplification program was as follows: 98℃ pre-denaturation for 2 min; followed by cycling (98℃ denaturation for 10 s; 56℃ annealing for 15 s; 72℃ extension for 10 s), for a total of 25 cycles, with a final extension at 72℃ for 5 min. The amplified target gene fragment was approximately 1.3 kb in size.

[0028] PCR products were purified and recovered using a purification and recovery kit. The PCR products and the *E. coli* expression vector pET-28a(+) were double-digested with NdeI and XhoI, respectively, and the digested products were purified and recovered again. The target gene was ligated into the *E. coli* expression vector pET-28a(+) using T4 DNA ligase to obtain the recombinant plasmid pAcy-pET-28a(+). The ligated vector was then transformed into *E. coli* BL21(DE3) to establish the pAcy genetically engineered bacterium.

[0029] Example 2: Induced expression of porcine kidney acylase (pAcy)

[0030] The recombinant Escherichia coli BL21(DE3) / pAcy-pET-28a(+) obtained in Example 1 was inoculated into LB liquid medium containing 50 μg / ml kanamycin resistance and cultured at 37°C and 200 rpm for 12 h. Then, it was inoculated into fresh LB liquid medium containing 50 μg / ml kanamycin resistance at a 1% (v / v) inoculation rate and cultured at 37°C and 200 rpm until the bacterial cell OD reached 100%. 600The concentration of the culture medium was increased to 0.6-0.8, and IPTG was added to a final concentration of 0.1 mM. The mixture was then incubated at 20°C and 200 rpm for 15 h. After centrifugation at 4°C and 8000 rpm for 20 min, the supernatant was discarded, and the precipitate was collected to obtain wet cells of recombinant *E. coli* BL21(DE3) / pAcy-pET-28a(+) containing the recombinant plasmid. These cells can be used directly as a biocatalyst.

[0031] Example 3: Construction and high-throughput screening of pAcy mutant libraries

[0032] Using plasmid pAcy-pET-28a(+) from Example 1 as a template, error-prone PCR was performed. Error-prone PCR was conducted with the help of upstream primer 1 and downstream primer 2. The PCR reaction system (total volume 50 μL) consisted of: 5 μL of 10×Taq DNA polymerase Buffer, 4 μL of 10 mM dNTP mixture (2.5 mM each of dATP, dCTP, dGTP, and dTTP), 0.8 μL of 10 mM dCTP, 0.8 μL of 10 mM dTTP, 1 μL each of primer 1 and primer 2 (both at 10 μM), 2 μL of plasmid template, 1 μL of R-Taq enzyme, and Mg... 2+ (25mM) 4μL, Mn 2+ (0.5mM) 4μL, deionized water to make up to 50μL. PCR amplification program: 95℃ pre-denaturation for 5min; then cycling (95℃ denaturation for 30s; 56℃ annealing for 20s; 72℃ extension for 1min), for a total of 25 cycles, with a final extension at 72℃ for 10min. After purifying the error-prone PCR product, it was homologously recombinated with the vector fragment obtained in Example 1. The homologous recombination product was finally transformed into E. coli BL21(DE3) competent cells and cultured overnight at 37℃ to obtain the pAcy mutant library.

[0033] From the obtained pAcy mutant library, 2000 single clones were selected and inoculated into 96-well plates. Each single clone contained 500 μL of LB liquid medium and was cultured at 37°C and 220 rpm for 5 h. Then, IPTG was added to a final concentration of 0.1 mM, and the plates were cultured at 20°C and 200 rpm for 15 h. After centrifugation at 5000 rpm for 5 min, the supernatant was discarded, and the precipitate was collected to obtain 2000 mutant wet cells.

[0034] The above wet bacterial cells were used for the biosynthesis of cocoyl glycine. The final concentration composition and catalytic conditions of the catalytic system (200 μL) were as follows: 50 mg of coconut oil acid and 200 μL of saturated glycine solution were added to the wet bacterial cells, pH 6.0. The reaction conditions were: temperature 37℃, 800 rpm, and reaction time 24 h. After the reaction was completed, the mixture was shaken to homogenize, and the reaction solution was analyzed by HPLC to obtain the cocoyl glycine synthase activity.

[0035] Analytical conditions: Column: C18, 4.6 mm × 250 mm, 5 μm; Detection wavelength: UV = 200 nm; Mobile phase: 85% acetonitrile and 15% 0.05% trifluoroacetic acid aqueous solution; Column temperature: 30 ℃; Flow rate: 1 mL / min; Detection time: 30 min.

[0036] By comparing the relative values ​​of enzyme activity between mutants and wild-type, a total of 14 superior mutants were screened, and the results are shown in Table 1.

[0037] Table 1. Dominant Mutants

[0038]

[0039] As shown in Table 1, individual mutations of S3T, P70A, L177C, G207V, R209C, K218N, P244L, S249T, S249N, C271V, Q290K, M309T, C342A, and L394I can increase the activity of cocoyl glycine synthase in porcine kidney acyltransferase.

[0040] Example 4: Obtaining acylase mutants with double mutation sites

[0041] Based on the enzyme activity results of single mutants, mutants with higher enzyme activity were selected for verification of the superposition of enzyme activity at two mutation sites.

[0042] Based on specific primer design and homologous recombination, dual-site acyltransferase mutants were obtained as listed in Table 2. These acyltransferase mutants were evaluated according to the enzyme activity assay method described in Example 3, and the results of the relative enzyme activities of the dual-site acyltransferase mutants compared to the wild type are shown in Table 2.

[0043] Table 2

[0044]

[0045] As shown in Table 2, the prepared two-site mutants all exhibited further improvements compared to the wild type. This indicates that selecting two mutations from amino acid residues such as L177C, C271V, R209C, L394I, G207V, C342V, P244L, M309T, and S249N can also enhance the cocoyl glycine synthase activity of porcine kidney acyltransferase.

[0046] Example 5: Obtaining acylase mutants with three mutation sites

[0047] Based on the enzyme activity results of the acylase mutant with two mutation sites described in Example 4, an acylase mutant with three mutation sites was constructed, and the above acylase mutant was evaluated according to the enzyme activity detection method described in Example 3. The results of the relative activity values ​​of the mutants of acylase (16) are shown in Table 3.

[0048] Table 3 Information on acylase mutants

[0049]

[0050] As shown in Table 3, the three point mutations of the above acylase mutants can also further enhance the cocoyl glycine synthase activity of porcine kidney acylase.

[0051] Example 6: Obtaining acylase mutants with multiple mutation sites

[0052] Based on the pH stability results of the three-point mutation acylase mutant described in Example 5, acylase mutants with more mutation sites were constructed, and the above acylase mutants were evaluated according to the enzyme activity detection method described in Example 3. The results of their relative enzyme activity with respect to acylase (27) are shown in Table 4.

[0053] Table 4 Information on acylase mutants

[0054]

[0055] As shown in Table 4, based on L177C / C271V / L394I, further addition of one or two point mutations from amino acid residues such as S3T, R209C, and P70A can further enhance the cocoyl glycine synthase activity of porcine kidney acyltransferase.

Claims

1. An acylating enzyme mutant, characterized in that, which is a mutant having only substitution mutation of amino acid residue selected from the following sites based on the amino acid sequence of acylase as shown in SEQ ID NO. 2: R209C, K218N, P244L, S249T, S249N, C271V, Q290K, M309T, C342A or L394I; or a mutant having only substitution mutation of amino acid residue of two sites as follows: R209C / L394I, G207V / C342A, G207V / M309T, R209C / C342A or P244L / L394I.

2. Nucleic acid encoding the mutant acylase according to claim 1.

3. Expression vector containing the nucleic acid encoding according to claim 2.

4. The expression vector of claim 3, wherein, which is pET vector.

5. Genetically engineered bacteria containing the nucleic acid encoding according to claim 2.

6. The genetically engineered bacteria as described in claim 5, characterized in that, which is Escherichia coli or Bacillus subtilis or Pichia pastoris.

7. Use of the mutant acylase according to claim 1, nucleic acid encoding thereof, vector according to claim 3 or 4, or genetically engineered bacteria according to claim 5 or 6 in the biocatalytic synthesis of sodium cocoyl glycinate.

8. A process for the preparation of sodium cocoyl glycinate, characterized in that, The mutant acylase according to claim 1 is used as the biocatalyst, and a reaction system is formed with coconut oil and glycine as substrates, and sodium cocoyl glycinate is produced in the reaction; Preferably, the mutant acylase is wet bacteria or crude enzyme solution or pure enzyme obtained by fermentation culture of the genetically engineered bacteria according to claim 6, which is used as the biocatalyst.

9. The production method according to claim 8, wherein In the reaction system, the amount of the wet bacteria is 5-30 g / L, the concentration of coconut oil is 10-300 g / L, and the saturated glycine solution is 60%-100%; The reaction conditions are as follows: temperature 35-65℃, pH 5.5-8.5, and stirring speed 100-300 rpm. The wet bacteria are obtained by centrifugation of the genetically engineered bacteria after fermentation culture, discarding the supernatant, and collecting the precipitate.

10. The preparation method according to claim 9, characterized in that, The wet bacteria are prepared as follows: the recombinant E. coli containing the gene encoding the mutant of pig kidney enzyme is inoculated into LB liquid medium containing 50 μg / ml kanamycin resistance, and cultured at 37°C, 200 rpm for 12 h, then inoculated into fresh LB liquid medium containing 50 μg / ml kanamycin resistance at a volume percentage of 1%, and cultured at 37°C, 200 rpm until the OD 600 of the bacteria is 0.6-0.8, 0.1 mM IPTG is added, and the culture is induced at 20°C, 200 rpm for 15 h; then the culture is centrifuged at 4°C, 8000 rpm for 20 min, the supernatant is discarded, and the precipitate is collected, thus obtaining the wet bacteria.

Images