A horse-derived acylase mutant and its application in the synthesis of lauroylglycine

Abstract

This invention belongs to the field of biotechnology and discloses an equine-derived acylase mutant and its application in the synthesis of lauroylglycine. The equine-derived acylase mutant of this invention can efficiently and stably perform acylation reactions to generate lauroylglycine. Therefore, the equine-derived acylase mutant provided by this invention can be used as an enzyme in the production of amino acid surfactants and has wide applications in various fields, especially in personal care products and the food industry, where it has great potential.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a horse-derived acylase mutant and its application in the synthesis of lauroylglycine. Background Technology

[0002] Amino acid surfactants are not only widely available from biomass raw materials, but also have advantages such as low toxicity and side effects, mild performance, low irritation, and good foaming properties. They often have good surface activity, health and safety, and strong antibacterial ability, which has attracted much attention and led to their gradual application in many fields such as detergents, personal care, and the food industry.

[0003] N-acyl amino acid surfactants are the most common type of amino acid surfactants. Common examples include sodium lauroyl glycinate, sodium lauroyl sarcosinate, sodium lauroyl glutamate, potassium lauroyl glutamate, sodium cocoyl glutamate, sodium cocoyl glycinate, and potassium cocoyl glycinate. Their synthetic raw materials come from various acidic, basic, or neutral amino acids such as aspartic acid, glutamic acid, arginine, alanine, glycine, leucine, proline, serine, and protein hydrolysis products. Hydrophobic groups such as fatty acids or alkyl chlorides can be attached to the amino group, α-COOH group, or side chain group of the amino acid. If fatty acids or alkyl halides react with the amino group, corresponding N-acyl or N-alkyl amino acid derivatives are produced; if fatty amines or fatty alcohols condense with the carboxyl group, N-alkyl or O-alkyl ester amino acid derivatives are obtained. Different reaction methods yield different products; therefore, amino acid surfactants exhibit diversity in chemical structure and physicochemical and biological properties.

[0004] The synthesis methods of N-acyl amino acid surfactants are divided into direct and indirect methods. Indirect synthesis methods include acylation of fatty acid acyl chlorides, hydrolytic acylation of fatty acid nitriles, acylation of fatty acid anhydrides, and amide carbonylation reactions. Among these, the Schotten-Baumann condensation method, in which fatty acid acyl chlorides replace fatty acids in alkaline solutions, is currently the most widely used synthesis method in laboratories and industry. It has advantages such as relatively low equipment requirements, inexpensive and readily available raw materials, mild reaction conditions, and easy handling of byproducts. However, because it involves the use of phosgene to convert free fatty acids into the corresponding acyl chlorides during production, although this method produces high amide yields, it requires the use of stoichiometric amounts of harmful activating reagents and generates a large amount of waste, resulting in low atom economy. Direct synthesis methods from fatty acid raw materials include enzyme-catalyzed synthesis and dehydration condensation. Lipase-catalyzed synthesis is limited by low conversion rates, long reaction times, and expensive enzyme preparations; while dehydration condensation is limited by harsh reaction conditions, high equipment requirements, and high energy consumption, and its improvement is needed. Therefore, there is an urgent need to develop one or a series of novel synthetic schemes for N-acyl amino acid surfactants, providing a more convenient and efficient new route for laboratory / industrial production of N-acyl amino acid surfactants.

[0005] Aminoacylase I (ACY1) thus came into the public eye. ACY1 can specifically hydrolyze N-acyl-L-amino acids to generate L-amino acids without affecting their enantiomers, N-acyl-D-amino acids. Its physiological functions are believed to include rescuing post-metabolite amino acids and inhibiting cancer cell growth. Industrially, it is commonly used for amino acid resolution. Because it can also catalyze the formation of N-acyl amino acids in non-aqueous phases, it plays an important role in biosynthesis and metabolism. The mechanism for synthesizing lauroylglycine using it is as follows:

[0006]

[0007] However, despite the catalytic advantages of ACY1 for the above synthetic reactions, it is often limited by problems such as product hydrolysis, low enzyme catalytic efficiency, low enzyme stability leading to unsustainable reactions, or difficulty in recovery, resulting in low synthetic yields. Consequently, research on this type of reaction remains scarce. Currently, a major source of industrial ACY1 is extracted from pig kidneys. Although some studies have found that ACY1 from sheep, humans, and dogs has certain catalytic effects on acylation reactions, related research reports are still few, inevitably leading to the disadvantages of limited ACY1 sources and low abundance. Therefore, by performing BLAST on ACY1 sequences from humans and pigs and systematically checking sequence similarity, horse-derived ACY1 with high similarity was selected for research. The aim is to introduce mutation sites to give it certain catalytic efficiency, based on its acylation function from human and pig ACY1, thus providing a new direction for the ACY1 catalytic synthesis pathway of lauroylglycine. Summary of the Invention

[0008] This invention relates to a mutant with enhanced activity obtained by modifying the pAcy gene, which is derived from previously obtained equilibrase synthesis genes.

[0009] This invention first provides a equine acylase mutant, which is based on the wild-type acylase with an amino acid sequence as shown in SEQ ID NO.1, and contains one, two, three, four, five, six or more amino acid residues selected from the following sites: T24Y, V35E, E40K, Q50G, A102C, T181N, S224G, S292A, Q160E, T14Y, K284L, V306E, S320A, K232D, A166S, A130S, T368H, L395S.

[0010] Specifically, mutants with amino acid residue mutations at the following two sites exist: E40K, A102C; E40K, T181N; E40K, S224G; E40K, S292A; A102C, T181N; A102C, S224G; A102C, S292A; T181N, S224G; T181N, S292A; S224G, S292A.

[0011] In a specific embodiment, there are mutants with amino acid residue mutations at the following three sites: E40K, T181N, S224G; E40K, T181N, S292A; A102C, T181N, S224G; A102C, T181N, S292A; T181N, S224G, S292A; E40K, T181N, S292A; E40K, T181N, S224G; S292A, E40K, T181N.

[0012] In a further embodiment, there are mutants with the following four amino acid residue mutations: E40K, T181N, S224G, S292A; E40K, T181N; S224G, S292A;

[0013] Or a mutant with mutations at the following five amino acid residue sites: E40K, T181N, S224G, S292A, T14Y.

[0014] Or a mutant with mutations in the following six amino acid residues: E40K, T181N, S224G, S292A, T14Y, K284L; E40K, T181N, S224G, S292A, T14Y, V306E; E40K, T181N, S224G, S292A, Q160E, K284L; E40K, T181N, S224G, S292A, Q160E, S320A;

[0015] Or a mutant with mutations at the following seven amino acid residue sites: E40K, T181N, S224G, S292A, T14Y, Q160E, S320A; E40K, T181N, S224G, S292A, Q160E, K232D, S320A; E40K, T181N, S224G, S292A, K232D, V 306E, S320A; E40K, T181N, S224G, S292A, Q160E, K284L, V306E; E40K, T181N, S224 G, S292A, Q160E, K284L, S320A; E40K, T181N, S224G, S292A, K284L, V306E, S320A;

[0016] Or a mutant with mutations at the following eight amino acid residue sites: E40K, T181N, S224G, S292A, T14Y, Q160E, K232D, K284L; E40K, T181N, S224G, S292A, T14Y, Q160E, K232D, V306E; E40K, T181N, S224G, S292A, T14Y, Q160E, K 284L, V306E; E40K, T181N, S224G, S292A, T14Y, Q160E, K284L, S320A; E40K, T181N, S224G, S 292A, Q160E, K284L, V306E, S320A; E40K, T181N, S224G, S292A, T14Y, Q160E, K284L, V306E,

[0017] Or a mutant with the following nine amino acid residue mutations: E40K, T181N, S224G, S292A, T14Y, Q160E, A166S, V306E, S320A; E40K, T181N, S224G, S292A, T14Y, Q160E, A166S, V306E, S320A;

[0018] Or a mutant with the following ten amino acid residue mutations: E40K, T181N, S224G, S292A, T14Y, Q160E, A166S, K284L, V306E, S320A.

[0019] Or a mutant with the following eleven amino acid residue mutations: E40K, T181N, S224G, S292A, T14Y, A130S, Q160E, A166S, K284L, V306E, S320A.

[0020] Alternatively, a mutant with mutations at the following twelve amino acid residue sites: E40K, T181N, S224G, S292A, T14Y, A130S, Q160E, A166S, K284L, V306E, S320A, T368H; E40K, T181N, S224G, S292A, T14Y, Q40G, A130S, Q160E, A166S, K284L. V306E, T368H; E40K, T181N, S224G, S292A, T14Y, V35E, Q160E, A166S, K284L, V306E, S320A, L395S; E40K, T181N, S224G, S292A, V35E, A130S, Q160E, A166S, K284L, V306E, T368H, L395S.

[0021] The present invention provides the encoding gene of the acylase mutant.

[0022] The present invention also provides a recombinant expression vector containing the aforementioned coding gene, specifically in the pET vector framework.

[0023] The present invention provides genetically engineered bacteria containing the aforementioned coding gene or the aforementioned recombinant expression vector, such as Escherichia coli.

[0024] The present invention further provides the application of the acylase mutant, or the nucleic acid encoded thereon, in the preparation of lauroylglycine, specifically in the catalytic conversion of lauric acid to lauroylglycine.

[0025] This invention also provides a method for preparing lauroylglycine, which includes using the genetically engineered bacteria as a whole-cell catalyst and lauric acid and glycine as substrates to synthesize lauroylglycine;

[0026] The whole-cell catalyst is obtained by the following steps: centrifuging the fermentation culture, discarding the supernatant, collecting the precipitate, and thus obtaining wet cells.

[0027] The reaction conditions are as follows: the amount of wet bacterial cells is 5-30 g / L, the concentration of lauric acid is 1.44-72 g / L, and the concentration of 60%-100% saturated glycine solution is used. The reaction conditions are: temperature 35-65℃, pH 5.5-8.5, and stirring speed 100-300 rpm for biocatalytic reaction to obtain lauroylglycine.

[0028] This invention provides a equine-derived acylase mutant with significantly enhanced catalytic activity compared to the wild-type acylase. Using wet bacterial cells obtained through fermentation culture of recombinant bacteria containing the mutant encoding gene of the acylase as a biocatalyst, and lauric acid and glycine as substrates, lauroylglycine surfactants can be synthesized, thus greatly improving production efficiency and possessing high production application value. Detailed Implementation

[0029] The embodiments are described in more detail below, but the present invention is not limited to any of the embodiments described below. It should be noted that the so-called mutation site in the following embodiments refers to the position in the amino acid sequence that differs from that of the equine mutant acylase containing the amino acid sequence with sequence number 1.

[0030] Comparative Example 1

[0031] Based on the horse acylase sequence obtained from previous experiments, the gene pAcy was synthesized. Its amino acid sequence is shown in Sequence 1, and its nucleic acid sequence of the cDNA coding region is shown in Sequence 2.

[0032] After artificially synthesizing the wild-type acylase cDNA coding region, this gene was used as a template, and primer pair sequences 3 and 4 were used to amplify the fragment by PCR, introducing NdeI and XhoI endonuclease sites on both sides of the fragment. PCR reaction system: Takara Max DNA Polymerase 25 μL, ddH2O 20 μL, primer 3 (Sequence 3) 1.5 μL, primer 4 (Sequence 4) 1.5 μL, template 2 (Sequence 2) 2 μL. PCR reaction program: 98℃ pre-denaturation for 2 min, followed by 25 cycles of 98℃ denaturation for 10 s, 58℃ annealing for 15 s, and 72℃ extension for 10 s, with a final extension at 72℃ for 5 min.

[0033] Analysis of the PCR products using 1% agarose gel electrophoresis confirmed the presence of an amplification product of approximately 1.2 kb.

[0034] Only a DNA fragment of about 1.2kb was excised from the agarose gel. The DNA in the gel was purified and recovered using the Tiangen ordinary agarose gel DNA recovery kit. The purified product was double-digested with restriction enzymes NdeI and XhoI. After confirming that the band size was about 1.2kb by 1% agarose gel electrophoresis, the DNA was purified and recovered again to obtain the gene digested fragment.

[0035] Similarly, the E. coli expression vector pET-28a(+) was digested using restriction enzymes NdeⅠ and XhoⅠ, and a DNA fragment of about 5.3 kb was excised from the agarose gel by agarose gel electrophoresis. After purification and recovery, the vector digested fragment was obtained.

[0036] The approximately 1.2 kb and 5.3 kb restriction fragments obtained above were ligated using T4 DNA ligase and then transformed into *E. coli* BL21(DE3) to obtain transformant (1). A plasmid was prepared from the bacterial cells using the Tiangen plasmid miniprep kit to obtain the recombinant plasmid pET-28a(+)-Acy. DNA sequencing confirmed that the coding region nucleotide sequence of this plasmid was identical to sequence 2. Transformant (1) produced acylase (1), a mutant acylase derived from horse.

[0037] The recombinant *E. coli* BL21(DE3) / pAcy-pET-28a(+) obtained above 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 at 1% (v / v) into fresh LB liquid medium containing 50 μg / ml kanamycin resistance and cultured at 37°C and 200 rpm until the bacterial OD600 reached 0.6-0.8. IPTG was added to a final concentration of 0.1 mM, and the culture was induced 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 bacterial cells containing the recombinant plasmid expressing *E. coli* BL21(DE3) / pAcy-pET-28a(+). These bacterial cells can be directly used as a biocatalyst.

[0038] The wet bacterial cells obtained above were used to determine the synthetic activity of lauroylglycine. The synthetic reaction system (10 mL) consisted of 360 mg lauric acid, 10 mL saturated glycine solution, and 0.2 g wet bacterial cells. The reaction was allowed to proceed for 14 h.

[0039] Reaction conditions: temperature 35-65℃, pH 5.5-8.5.

[0040] 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: 15 min.

[0041] Comparative Example 2

[0042] Candidate modification sites are introduced through sequence conservation analysis and random mutation. Since the mutation risk of low-conservation sites is unknown, it is not possible to stack sites one by one. Therefore, it is necessary to first screen for single-point transformation rates, eliminate low-transformation-rate sites, retain high-transformation-rate advantageous mutation sites, and then combine and stack them, introducing new mutation sites during the stacking process, and conducting multiple rounds of evolutionary screening.

[0043] After artificially synthesizing the recombinant plasmid pET-28a(+)-Acy, this plasmid was used as a template, and primer pair sequence 5 was used to amplify the fragment by PCR to introduce the mutation site required for transformant (2). PCR reaction system: Takara Max DNA Polymerase 25 μL, ddH2O 20 μL, primer F 1.5 μL, primer R 1.5 μL, pET-28a(+)-Acy template 2 μL. PCR reaction program: 98℃ pre-denaturation for 2 min, followed by 25 cycles of 98℃ denaturation for 10 s, 58℃ annealing for 15 s, and 72℃ extension for 40 s, with a final extension at 72℃ for 5 min.

[0044] Analysis of the PCR products using 1% agarose gel electrophoresis confirmed the presence of an amplification product of approximately 6.5 kb.

[0045] Only about 6.5kb of DNA was excised from the agarose gel. The DNA in the gel was purified and recovered using the Tiangen General Agarose Gel DNA Recovery Kit.

[0046] Escherichia coli BL21(DE3) was transformed to obtain transformant (2). Transformant (2) produced mutant (2) as a dominant mutant acylase derived from horses.

[0047] Transformant (2) produces acylase (2) with mutations based on Comparative Example 1 as described in the table, and its culture and activity assay are the same as those of Comparative Example 1.

[0048] [Example 1] Obtaining mutant acylase (3)

[0049] To obtain transformant (3) expressing a mutant acyltransferase with the amino acid residue substitutions shown in the table, suitable primers were designed, and plasmids were obtained by operating in the same manner as in Comparative Example 2, resulting in transformant (3). Transformant (3) produced acyltransferase (3) with the mutations described in the table.

[0050] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 1.

[0051] [Example 2] Obtaining mutant acylase (4)

[0052] To obtain transformant (4) expressing a mutant acyltransferase with the amino acid residue substitutions shown in the table, suitable primers were designed, and plasmids were obtained by operating in the same manner as in Comparative Example 2, resulting in transformant (4). Transformant (4) produced acyltransferase (4) with the mutations described in the table.

[0053] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 1.

[0054] [Example 3] Obtaining mutant acylase (5)

[0055] To obtain transformant (5) expressing a mutant acyltransferase with the amino acid residue substitutions shown in the table, suitable primers were designed, and plasmids were obtained by operating in the same manner as in Comparative Example 2, resulting in transformant (5). Transformant (5) produced acyltransferase (5) with the mutations described in the table.

[0056] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 1.

[0057] [Example 4] Obtaining mutant acylase (6)

[0058] To obtain a transformant (6) expressing a mutant acyltransferase with the amino acid residue substitutions shown in the table, suitable primers were designed, and plasmids were obtained by operating in the same manner as in Comparative Example 2, resulting in transformant (6). Transformant (6) produced acyltransferase (6) with the mutations described in the table.

[0059] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 1.

[0060] [Example 5] Obtaining mutant acylase (7)

[0061] To obtain transformants (7) expressing mutant acyltransferases with the amino acid residue substitutions shown in the table, suitable primers were designed, and plasmids were obtained by the same procedure as in Comparative Example 2, resulting in transformants (7). Transformants (7) produced acyltransferases (7) with the mutations described in the table.

[0062] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 1.

[0063] Table 1

[0064]

[0065] For superimposed point mutations, a relative conversion efficiency of >1.5 compared to the wild type is defined as a significant increase, 1.2–1.5 as an increase, 1–1.2 as a slight increase, 0.8–1 as a slight decrease, 0.5–0.8 as a decrease, and <0.5 as a drastic decrease. Table 1 shows that the mutation at amino acid position 24 of Comparative Example 2, resulting in a Y amino acid mutation, drastically decreased the conversion efficiency, demonstrating that not all low-conservation sites can be used for conversion efficiency enhancement. This site is detrimental to evolution and was therefore eliminated. Mutations T181N and S224G showed increases, while S292A, E40K, and A102C showed slight increases; these mutations can be considered advantageous sites for conversion efficiency enhancement.

[0066] [Example 6] Obtaining mutant acylase (8)

[0067] To obtain a transformant (8) expressing a mutant acylase with amino acid residue substitutions as shown in the table, the vector obtained by plasmid extraction from transformant (1) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain transformant (8). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 2.

[0068] [Example 7] Obtaining mutant acylase (9)

[0069] To obtain a transformant (9) expressing a mutant acylase with amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from transformant (1) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain transformant (9). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 2.

[0070] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 2.

[0071] [Example 8] Obtaining mutant acylase (10)

[0072] To obtain a transformant (10) expressing a mutant acylase with amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from transformant (1) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain transformant (10). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 2.

[0073] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 2.

[0074] [Example 9] Obtaining mutant acylase (11)

[0075] To obtain a transformant (11) expressing a mutant acylase with the amino acid residue substitutions shown in the table, a vector obtained by plasmid extraction from transformant (1) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain transformant (11). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 2.

[0076] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 2.

[0077] [Example 10] Obtaining mutant acylase (11)

[0078] To obtain a transformant (11) expressing a mutant acylase with the amino acid residue substitutions shown in the table, a vector obtained by plasmid extraction from transformant (1) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain transformant (11). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 2.

[0079] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 2.

[0080] [Example 11] Obtaining mutant acylase (12)

[0081] To obtain a transformant (12) expressing a mutant acylase with amino acid residue substitutions as shown in the table, the vector obtained by plasmid extraction from transformant (1) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain transformant (12). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 2.

[0082] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 2.

[0083] [Example 11] Obtaining mutant acylase (13)

[0084] To obtain a transformant (13) expressing a mutant acylase with amino acid residue substitutions as shown in the table, the vector obtained by plasmid extraction from transformant (1) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain transformant (13). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 2.

[0085] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 2.

[0086] [Example 12] Obtaining mutant acylase (14)

[0087] To obtain a transformant (14) expressing a mutant acylase with amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from transformant (1) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain transformant (14). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 2.

[0088] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 2.

[0089] [Example 13] Obtaining mutant acylase (15)

[0090] To obtain a transformant (15) expressing a mutant acylase with amino acid residue substitutions as shown in the table, the vector obtained by plasmid extraction from transformant (1) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain transformant (15). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 2.

[0091] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 2.

[0092] [Example 14] Obtaining mutant acylase (16)

[0093] To obtain a transformant (16) expressing a mutant acylase with amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from transformant (1) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain transformant (16). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 2.

[0094] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 2.

[0095] [Example 15] Obtaining mutant acylase (17)

[0096] To obtain a transformant (17) expressing a mutant acylase with amino acid residue substitutions as shown in the table, the vector obtained by plasmid extraction from transformant (1) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain transformant (17). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 2.

[0097] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 2.

[0098] Table 2

[0099]

[0100]

[0101] Table 2 shows that mutations at sites 40 and 102 have a slight improvement, while the combination of sites 181, 224 and 292 has a greater effect on improving the transformation rate. The combination of sites 181 and 224 has a significant improvement. These mutations can be considered as advantageous sites for improving the transformation rate.

[0102] [Example 16] Obtaining mutant acylase (18)

[0103] To obtain a transformant (18) expressing a mutant acylase with amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (5) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain the transformant (18). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 3.

[0104] The synthesis of lauroyl glycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 3.

[0105] [Example 17] Obtaining mutant acylase (19)

[0106] To obtain a transformant (19) expressing a mutant acylase with amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (5) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain the transformant (19). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 3.

[0107] The synthesis of lauroyl glycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 3.

[0108] [Example 18] Obtaining mutant acylase (20)

[0109] To obtain a transformant (20) expressing a mutant acylase with amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (5) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain the transformant (20). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 3.

[0110] The synthesis of lauroyl glycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 3.

[0111] [Example 19] Obtaining mutant acylase (21)

[0112] To obtain a transformant (21) expressing a mutant acylase with amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (5) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain the transformant (21). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 3.

[0113] The synthesis of lauroyl glycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 3.

[0114] [Example 20] Obtaining mutant acylase (22)

[0115] To obtain a transformant (22) expressing a mutant acylase with the amino acid residue substitutions shown in the table, a vector obtained by plasmid extraction from the transformant (5) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain the transformant (22). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 3.

[0116] The synthesis of lauroyl glycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 3.

[0117] Table 3

[0118]

[0119]

[0120] For superimposed point mutations, a relative value of >1.5 for the synthetic transformation rate relative to the parent is defined as a significant improvement, 1.2–1.5 as an improvement, and 1–1.2 as a slight improvement. Table 3 shows that the combination of sites 40, 181, and 292 results in a significant improvement. The combination of site 224 with sites 181 and 292, and sites 40 and 181, also contributes to the improvement in transformation rate. Therefore, these four sites are further superimposed.

[0121] [Example 21] Obtaining mutant acylase (23)

[0122] To obtain a transformant (23) expressing a mutant acylase with amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (19) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain the transformant (23). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 4.

[0123] The synthesis of lauroyl glycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 4.

[0124] Table 4

[0125]

[0126] As can be seen from Table 4, the four-site combination of 40, 181, 224, and 292 has a higher conversion rate than the three-site combination, and can be used as the parent material for a new round of evolutionary superposition to screen sites.

[0127] [Example 22] Obtaining mutant acylase (24)

[0128] To obtain a transformant (24) expressing a mutant acylase with the amino acid residue substitutions shown in the table, a vector obtained by plasmid extraction from the transformant (23) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain the transformant (24). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 5.

[0129] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 5.

[0130] [Example 23] Obtaining mutant acylase (25)

[0131] To obtain a transformant (25) expressing a mutant acylase with the amino acid residue substitutions shown in the table, a vector obtained by plasmid extraction from the transformant (23) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain the transformant (25). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 5.

[0132] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 5.

[0133] [Example 24] Obtaining mutant acylase (26)

[0134] To obtain a transformant (26) expressing a mutant acylase with amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (23) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain the transformant (26). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 5.

[0135] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 5.

[0136] [Example 25] Obtaining mutant acylase (27)

[0137] To obtain a transformant (27) expressing a mutant acylase with amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (23) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain the transformant (27). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 5.

[0138] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 5.

[0139] [Example 26] Obtaining mutant acylase (28)

[0140] To obtain a transformant (28) expressing a mutant acylase with amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (23) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain the transformant (28). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 5.

[0141] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 5.

[0142] Table 5

[0143]

[0144] As can be seen from Table 5, the introduction of sites 160 and 320 has improved the transformation rate of the previous four-site combination to a certain extent. These mutations can be regarded as advantageous sites for improving the transformation rate.

[0145] [Example 27] Obtaining mutant acylase (29)

[0146] To obtain a transformant (29) expressing a mutant acylase with the amino acid residue substitutions shown in the table, a vector obtained by plasmid extraction from the transformant (23) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain the transformant (29). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 6.

[0147] The synthesis of lauroyl glycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 6.

[0148] [Example 28] Obtaining mutant acylase (30)

[0149] To obtain a transformant (30) expressing a mutant acylase with the amino acid residue substitutions shown in the table, a vector obtained by plasmid extraction from the transformant (23) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain the transformant (30). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 6.

[0150] The synthesis of lauroyl glycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 6.

[0151] [Example 29] Obtaining mutant acylase (31)

[0152] To obtain a transformant (31) expressing a mutant acylase with the amino acid residue substitutions shown in the table, a vector obtained by plasmid extraction from the transformant (23) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain the transformant (31). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 6.

[0153] The synthesis of lauroyl glycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 6.

[0154] [Example 30] Obtaining mutant acylase (32)

[0155] To obtain a transformant (32) expressing a mutant acylase with the amino acid residue substitutions shown in the table, a vector obtained by plasmid extraction from the transformant (23) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain the transformant (32). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 6.

[0156] The synthesis of lauroyl glycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 6.

[0157] [Example 31] Obtaining mutant acylase (33)

[0158] To obtain a transformant (33) expressing a mutant acylase with amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from transformant (23) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain transformant (33). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 6.

[0159] The synthesis of lauroyl glycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 6.

[0160] [Example 32] Obtaining mutant acylase (34)

[0161] To obtain a transformant (34) expressing a mutant acylase with amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (23) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain the transformant (34). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 6.

[0162] The synthesis of lauroyl glycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 6.

[0163] Table 6

[0164]

[0165]

[0166] As shown in Table 6, the introduction of sites 160, 232, 284, 306, and 320 all helped to improve the transformation rate. These mutations can be considered as advantageous sites for improving the transformation rate.

[0167] [Example 33] Obtaining mutant acylase (35)

[0168] To obtain a transformant (35) expressing a mutant acylase with amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (24) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain the transformant (35). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 7.

[0169] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 7.

[0170] [Example 34] Obtaining mutant acylase (36)

[0171] To obtain a transformant (36) expressing a mutant acylase with amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (24) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain the transformant (36). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 7.

[0172] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 7.

[0173] [Example 35] Obtaining mutant acylase (37)

[0174] To obtain a transformant (37) expressing a mutant acylase with amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (24) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain the transformant (37). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 7.

[0175] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 7.

[0176] [Example 36] Obtaining mutant acylase (38)

[0177] To obtain a transformant (38) expressing a mutant acylase with amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (24) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain the transformant (38). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 7.

[0178] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 7.

[0179] [Example 37] Obtaining mutant acylase (39)

[0180] To obtain a transformant (39) expressing a mutant acylase with amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (24) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain the transformant (39). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 7.

[0181] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 7.

[0182] Table 7

[0183]

[0184]

[0185] As shown in Table 7, the introduction of 14 sites helps to improve the transformation rate. The combination of 160, 232, 284, 306 and 320 sites can also improve the transformation rate. These mutation combinations can be regarded as advantageous solutions for improving the transformation rate.

[0186] [Example 38] Obtaining mutant acylase (40)

[0187] To obtain a transformant (40) expressing a mutant acylase with the amino acid residue substitutions shown in the table, a vector obtained by plasmid extraction from the transformant (38) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain the transformant (40). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 8.

[0188] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 8.

[0189] [Example 39] Obtaining mutant acylase (41)

[0190] To obtain a transformant (41) expressing a mutant acylase with amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (38) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain the transformant (41). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 8.

[0191] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 8.

[0192] Table 8

[0193]

[0194] Table 8 shows that the substitution at site 166 slightly improved the conversion rate, and site 306 also helped improve the conversion rate. Based on the eight-site combination, alternative or superimposed advantageous sites can still be used to enhance enzyme activity. These mutant combinations can be considered advantageous strategies for improving conversion rate. [Example 40] Obtaining mutant acylase (42)

[0195] To obtain a transformant (42) expressing a mutant acylase with amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (40) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain the transformant (42). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 9.

[0196] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 9.

[0197] Table 9

[0198]

[0199] As can be seen from Table 9, when the advantageous sites obtained from the above evolution are superimposed, the conversion rate of the ten-site combination mutation is improved compared with the nine-site combination.

[0200] [Example 41] Obtaining mutant acylase (43)

[0201] To obtain a transformant (43) expressing a mutant acylase with the amino acid residue substitutions shown in the table, a vector obtained by plasmid extraction from the transformant (42) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain the transformant (43). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 10.

[0202] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 10.

[0203] Table 10

[0204]

[0205]

[0206] As shown in Table 10, the introduction of 130 sites helps to improve the transformation rate, resulting in mutants with eleven combinations.

[0207] [Example 41] Obtaining mutant acylase (43)

[0208] To obtain a transformant (43) expressing a mutant acylase with amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (42) was used as the cloning parent. Suitable primers were designed, and plasmids were obtained using the same procedure as in Comparative Example 2 to obtain the transformant (43). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 11.

[0209] The synthesis of lauroylglycine was assessed according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 11.

[0210] Table 11

[0211]

[0212]

[0213] Table 11 shows that introducing V35E, Q50G, T368H, and L395S sites using different combinations of substitution methods helps improve the transformation rate, resulting in mutants with twelve site combinations, proving that there is still room for improvement in this mutation site superposition scheme.

Claims

1. A equine-derived acylate mutant, characterized in that, It is a mutant based on the wild-type acylase with the amino acid sequence shown in SEQ ID NO.1, which contains mutations of amino acid residues selected from the following sites: (1) T181N; (2) T181N, S224G; (3) T181N, S292A; (4) E40K, T181N, S224G; (5) E40K, T181N, S292A; (6) A102C, T181N, S224G; (7) A102C, T181N, S292A; (8) T181N, S224G, S292A; (9) E40K, T181N, S292A; (10) E40K, T181N, S224G, S292A; (11) E40K, T181N, S224G, S292A, T14Y, K284L; (12) E40K, T181N, S224G, S292A, T14Y, V306E; (13) E40K, T181N, S224G, S292A, Q160E, K284L; (14) E40K, T181N, S224G, S292A, Q160E, S320A; (15) E40K, T181N, S224G, S292A, T14Y, Q160E, S320A; (16) E40K, T181N, S224G, S292A, Q160E, K232D, S320A; (17) E40K, T181N, S224G, S292A, K232D, V306E, S320A; (18) E40K, T181N, S224G, S292A, Q160E, K284L, V306E; (19) E40K, T181N, S224G, S292A, Q160E, K284L, S320A; (20) E40K, T181N, S224G, S292A, K284L, V306E, S320A; (21) E40K, T181N, S224G, S292A, T14Y, Q160E, K232D, K284L; (22) E40K, T181N, S224G, S292A, T14Y, Q160E, K232D, V306E; (23) E40K, T181N, S224G, S292A, T14Y, Q160E, K284L, V306E; (24) E40K, T181N, S224G, S292A, T14Y, Q160E, K284L, S320A; (25) E40K, T181N, S224G, S292A, Q160E, K284L, V306E, S320A; (26) E40K, T181N, S224G, S292A, T14Y, Q160E, A166S, V306E, S320A; (27) E40K, T181N, S224G, S292A, T14Y, Q160E, A166S, K284L, V306E, S320A; (28) E40K, T181N, S224G, S292A, T14Y, A130S, Q160E, A166S, K284L, V306E, S320A; (29) E40K, T181N, S224G, S292A, T14Y, A130S, Q160E, A166S, K284L, V306E, S320A, T368H; (30) E40K, T181N, S224G, S292A, T14Y, Q40G, A130S, Q160E, A166S, K284L, V306E, T368H; (31) E40K, T181N, S224G, S292A, T14Y, V35E, Q160E, A166S, K284L, V306E, S320A, L395S; (32) E40K, T181N, S224G, S292A, V35E, A130S, Q160E, A166S, K284L, V306E, T368H, L395S.

2. The encoding gene of the acylase mutant as described in claim 1.

3. A recombinant expression vector containing the encoding gene as described in claim 2.

4. The recombinant expression vector as described in claim 3, characterized in that, It is a pET carrier.

5. Genetically engineered bacteria containing the coding gene as described in claim 2, or the recombinant expression vector as described in claim 3 or 4.

6. The genetically engineered bacteria as described in claim 5, characterized in that, It is Escherichia coli.

7. The use of the acylase mutant as described in claim 1, or its encoded nucleic acid, in the preparation of lauroylglycine, characterized in that, It is used in the catalytic conversion of lauric acid to lauroylglycine.

8. A method for preparing lauroyl glycine, characterized in that, This includes using the genetically engineered bacteria as described in claim 5 or 6 as a whole-cell catalyst and lauric acid and glycine as substrates to synthesize lauroylglycine.

9. The preparation method according to claim 8, characterized in that, The whole-cell catalyst is obtained by the following steps: centrifuging the fermented bacterial culture, discarding the supernatant, collecting the precipitate, and obtaining wet bacterial cells, which are the whole-cell catalyst.

10. The preparation method according to claim 8 or 9, characterized in that, The reaction conditions include: the amount of wet bacterial cells is 5-30 g / L, the concentration of lauric acid is 1.44-72 g / L, and a 60%-100% saturated glycine solution is used; 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 to obtain lauroylglycine.

Images