Acylase, encoding gene, engineered bacteria, and application in hydrolysis and synthesis of n-fatty acyl glutamic acid surfactants

Abstract

The application discloses an acylase, a coding gene, an engineering bacterium and application of the acylase in hydrolysis and synthesis of N-fatty acyl glutamic acid surfactant. The acylase is a recombinant acylase derived from pseudomonas, the nucleotide sequence of the acylase is shown in SEQ ID NO. 1, and the corresponding amino acid sequence is shown in SEQ ID NO. 2. The enzyme can catalyze hydrolysis of N-lauroyl glutamic acid sodium to generate lauric acid and glutamic acid sodium, and can also catalyze the reverse reaction, i.e. catalyzing synthesis of N-lauroyl glutamic acid sodium from lauric acid and glutamic acid sodium. The amino acid sequence of the enzyme is greatly different from reported acylases with N-fatty acyl amino acid as the main substrate (low homology), and is a new acylase with similar substrate catalytic performance. The application has potential application value in hydrolysis and synthesis of N-lauroyl glutamic acid sodium by using a biological enzyme method.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering, in particular to acylase from Pseudomonas, encoding gene and application in hydrolysis and synthesis of N-fatty acyl glutamic acid surfactants. BACKGROUND

[0002] N-fatty acyl amino acid surfactants are widely used in cosmetics, cleaning and care products. At present, the production of such surfactants mainly adopts chemical method, but the chemical method has the disadvantage of serious pollution. The use of biological enzyme method to synthesize such surfactants is a hot research at present. Enzymatic synthesis has the advantages of mild reaction conditions, low energy consumption and small pollution, which conforms to the development direction of green synthesis in the future. The most important thing in the research of enzymatic synthesis is the screening of biological catalysts. Generally known lipase preparations such as Novozyme435, Lipozyme lipase, Candida antarctica lipase B (CALB) are used to synthesize N-fatty acyl amino acid. However, these enzymes are limited, and the types of amino acid surfactants are various. They cannot be applied to all products, so it is urgent to screen enzymes with wide sources, different catalytic properties and strong substrate specificity.

[0003] Some researchers use acylase (or amide hydrolase, EC 3.5.1) to hydrolyze or synthesize N-acyl amino acids. Kazuhiro Nakanishi's team found three acylase genes in the genome of Streptomyces mobaraensis, namely Sm-PVA (Demin Zhang et al., 2007), Sm-ELA (Mayuko Koreishi, Ryoko Kawasaki et al., 2009) and Sm-AA (Mayuko Koreishi, Yasuyuki Nakatani et al., 2009). The gene sequences and catalytic functions of these three enzymes are different: Sm-PVA can catalyze the hydrolysis of various N-fatty acyl-L-amino acids, and can also catalyze the synthesis of amides from carboxylic acid methyl esters and amino compounds; Sm-ELA can catalyze the synthesis of Nε-lauroyl-L-lysine from lysine and lauric acid, which is an ε-lysine acylase; Sm-AA can catalyze the hydrolysis of N-mid-long short chain acyl amino acids. Yasuaki Takakura et al. (2019) reported an amino acylase gene from Burkholderia sp., which has the activity of hydrolyzing and synthesizing various N-acyl amino acids. Hu Laiyun et al. (2021) disclosed an invention patent "Recombinant amide hydrolase and its application" (CN 113481223A), which can catalyze the hydrolysis of N-lauroyl glycine and N-lauroyl glutamate sodium, and can also catalyze the synthesis of N-lauroyl glycine and N-lauroyl arginine, but the source of the enzyme (such as from which organism) is not mentioned.

[0004] This invention will disclose a new acylase gene sequence and its function, and also disclose a screening method for the enzyme. This screening method takes advantage of the characteristics of microorganisms growing on specific nutrients. By using specific N-fatty acyl amino acid surfactants as the only carbon and nitrogen source, microorganisms that can specifically hydrolyze and utilize the substrate are first screened, and then the corresponding acylase gene is mined from the genome of the microorganism. This enzyme can catalyze the hydrolysis of specific N-fatty acyl amino acid surfactants, and under certain conditions, it can also catalyze the reverse reaction of hydrolysis, i.e. the synthesis of the surfactant. This method provides a good way for screening N-fatty acyl amino acid surfactant hydrolysis and synthesis enzyme catalysts with a wide range of sources, different properties and strong substrate specificity. SUMMARY

[0005] The present invention provides an acylase, a coding gene, a vector, an engineered bacterium and its application in N-fatty acyl amino acid surfactants. The enzyme can catalyze the hydrolysis of N-lauroyl glutamate sodium to produce lauric acid and glutamate sodium, and can also catalyze the reverse reaction, i.e. catalyze the synthesis of N-lauroyl glutamate sodium from lauric acid and glutamate sodium.

[0006] The present application provides an acylase, the amino acid sequence of which is shown in SEQ ID NO. 2, and specifically as follows:

[0007] MSQHPTIERLAASTEDFARIRRDLHQHPELGFEEARTSAIVAGYLREWGYEVHEGIGGTGVVGVLRQGNSARSIGIRADMDALPIDEASGVPYASQHAGRMHACGHDGHTAILLCAARDLAEQRLFDGTLNLIFQPAEETLGGAVAMMDDGLFERFPCDAVYALHNAPGLPVGCFLTREGALTASSDRVSIRLTGVGGHGAMPHLTKDPIVAAAELVLALQSIVARNVPSTEVGVVTVGMLKAGEAANVIPDHADLRLSVRATRPDVRELLKRRIGEITRGVAAVHGMELQYEYEELVPVLVNTPEETRLAREVLTELVGPQRLLSEIPSGFLGSEDFAWMLERRPGCYIALGNGNSGPSGCMVHNPGYDFNDAAIPFGAALWVRLVETFLGTGARA

[0008] or also includes any amino acid sequence with homology of more than 80% to the amino acid sequence shown in SEQ ID NO. 2 and with the same function.

[0009] Due to the particularity of the amino acid sequence, any polypeptide fragment or variant thereof, such as conservative variant, bioactive fragment or derivative, containing the amino acid sequence shown in SEQ ID NO. 2, as long as the polypeptide fragment or polypeptide variant has homology of more than 80% to the aforementioned amino acid sequence, all belong to the protection scope of the present application. Specifically, the changes can include deletion, insertion or substitution of amino acids in the amino acid sequence; wherein, for conservative changes of the variant, the substituted amino acid has similar structure or chemical property to the original amino acid, such as substitution of leucine for isoleucine, and the variant can also have non-conservative changes, such as substitution of tryptophan for glycine.

[0010] The present application provides a coding gene of the acylase, the nucleotide sequence of the coding gene is shown in SEQ ID NO. 1, and specifically as follows:

[0011]

[0012] The application provides a vector constructed from the acylase-encoding gene.

[0013] The application provides a genetically engineered bacterium obtained by transformation of the vector.

[0014] Further, the vector (specifically, Acy2-pET28a(+)) constructed from the acylase-encoding gene, and the genetically engineered bacterium (specifically, Acy2-pET-28a(+)-E.coli BL21 Gold(DE3)) obtained by transformation of the vector.

[0015] The application provides an application of the acylase in catalyzing hydrolysis of N-fatty acyl amino acid surfactant.

[0016] The N-fatty acyl amino acid surfactant comprises at least one of N-decanoyl glutamic acid sodium, N-lauroyl glutamic acid sodium, N-myristoyl glutamic acid sodium and N-palmitoyl glutamic acid sodium, and is preferably N-lauroyl glutamic acid sodium.

[0017] The crude enzyme solution obtained by crushing the cell bodies of the engineered bacterium containing the acylase-encoding gene and obtained through fermentation culture is used as a catalyst, and the N-fatty acyl amino acid surfactant is used as a substrate to perform a hydrolysis reaction.

[0018] Further, the crude enzyme solution is used as a catalyst, lauric acid and sodium glutamate are used as substrates, a mixed solution of pH 7.0 phosphate buffer and butyl acetate is used as a reaction medium (butyl acetate: buffer = 80:20, v / v) to form a reaction system, and the synthesis reaction is performed under the conditions of 40-60 DEG C and 200-300 rpm, and a mixed solution containing N-lauroyl glutamate sodium is obtained through the reaction.

[0019] The application provides an application of the acylase in catalyzing synthesis of N-lauroyl glutamate sodium from lauric acid and sodium glutamate.

[0020] The crude enzyme solution obtained by crushing the cell bodies of the engineered bacterium containing the acylase-encoding gene and obtained through fermentation culture is used as a catalyst, and lauric acid and sodium glutamate are used as substrates to perform a synthesis reaction, and a mixed solution containing N-lauroyl glutamate sodium is obtained through the reaction.

[0021] Further, the crude enzyme solution is used as a catalyst, N-lauroyl glutamate sodium is used as a substrate, and a 50 mM Tris-HCl buffer solution with pH 8.0 is used as a reaction medium to form a reaction system, and the hydrolysis reaction is performed under the conditions of 35-40 DEG C and 150-200 r / min, and the reaction product is lauric acid and sodium glutamate.

[0022] In this invention, wet bacterial cells obtained by fermentation culture of engineered bacteria containing acylase encoding genes are suspended in buffer solution, homogenized under high pressure, crushed, centrifuged, and the supernatant is taken as crude enzyme solution.

[0023] Furthermore, the catalyst (crude enzyme solution) was prepared as follows: The engineered bacterium Acy2-pET-28a(+)-E. coli BL21 Gold(DE3) containing the recombinant acylase encoding gene Acy2 was inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C and 180 r / min until the bacterial culture reached OD200. 600 The concentration of the enzyme solution is approximately 0.5-0.8. IPTG is added to a final concentration of 0.6 mmol / L, and the culture is induced at 25°C and 180 r / min for 10 h. The bacterial culture is then centrifuged at 4°C and 8000 r / min for 10 min, and the supernatant is discarded. The precipitated cells, i.e., the wet bacterial cells, are resuspended in 100 mmol / L pH 8.0 Tris-HCl buffer, with a bacterial concentration of 1.5-3 g / L (preferably 1.5 g / L). The cells are homogenized under high pressure at 4°C, centrifuged at 12000 r / min for 20 min, and the supernatant is collected to obtain the crude enzyme solution.

[0024] Using DNAMAN software, the amino acid sequences of acylase Acy2 (amino acid sequence shown in SEQ ID NO.2) and the five previously reported acylases mentioned in the "Background Art" section (all with N-ester acyl amino acids as their main substrates) were compared. The comparison results are shown below. Figure 1 An evolutionary tree was drawn based on the comparison results; see below. Figure 2 .

[0025] Depend on Figure 1 and 2 It is known that acylase Acy2 has very low amino acid sequence homology with the five previously reported acylases that act on N-fatty acyl amino acids. Its highest homology is with enzyme Sm-AA, but even that is only 15% (while enzyme E-CN11 (patent CN113481223A) and enzyme E-Burk (Yasuaki Takakura et al., 2019) share as much as 80% amino acid sequence homology). This indicates that acylase Acy2 is a novel acylase protein that uses N-fatty acyl amino acids as substrates.

[0026] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0027] The acylase gene Acy2 provided by the application is cloned and expressed in Escherichia coli, and the expressed recombinant enzyme has high activity in hydrolyzing N-lauroyl glutamate sodium. The recombinant enzyme is used to catalyze the hydrolysis of N-lauroyl glutamate sodium, the concentration of which is 1 g / L, and the reaction time is 10 min, and the conversion rate of the substrate is 78.1%. The recombinant enzyme can also be used to catalyze the synthesis of N-lauroyl glutamate, and the concentrations of lauric acid and sodium glutamate, two kinds of substrates, are 20 mM and 100 mM respectively, and the reaction time is 5 h, and the conversion rate of lauric acid is 13.4%. The amino acid sequence of the enzyme Acy2 is quite different from the reported acylases which mainly use N-fatty acyl amino acids as substrates (low homology), and is a new acylase with similar substrate catalytic performance. The application has potential application value in the hydrolysis and synthesis of N-lauroyl glutamate sodium by using biological enzyme method. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Comparison of the amino acid sequences of the acylase Acy2 and the reported five acylases (the main substrates are all N-fatty acyl amino acids);

[0029] Figure 2 Evolution tree established by the comparison results of the amino acid sequences of Figure 1

[0030] Figure 3 Strain Pseudomonas sp. 131 strain morphology under a microscope;

[0031] Figure 4 16S rDNA phylogenetic tree of the strain Pseudomonas sp. 131;

[0032] Figure 5 Agarose electrophoresis map of the DNA fragment of the acylase gene Acy2 amplified by PCR; lanes 1 and 2: target genes;

[0033] Figure 6 PCR verification of the recombinant plasmid; M: Marker; lane 1: Acy2-pET-28a(+) recombinant plasmid; lane 2: gene Acy2 amplified fragment; lane 3: pET-28a(+) empty plasmid;

[0034] Figure 7 SDS-PAGE map of the expression product of the recombinant bacteria Acy2-pET-28a(+)-E. coli BL21 Gold (DE3); M: Marker; lane 1: with IPTG induction; lane 2: without IPTG induction; lane 3: enzyme Acy2;

[0035] Figure 8 ​HPLC profile of N-lauroyl glutamic acid hydrolysis catalyzed by acylase crude enzyme solution; A reaction 0 min, B reaction 10 min. DETAILED DESCRIPTION

[0036] The application will be further described in the following with specific examples, but the protection scope of the application is not limited to this.

[0037] The experimental methods in the following examples are all the routine methods, unless otherwise specified; the reagents and biological materials, unless otherwise specified, can be obtained from commercial channels.

[0038] For better illustrating the purpose, technical scheme and advantages of the present application, the present application will be further described in the following with the attached drawings and specific examples.

[0039] The recombinant acylase in the present application is derived from Pseudomonas sp. 131, which is screened by Changsha Puji Biological Technology Co., Ltd. in the environment.

[0040] Example 1: Screening and identification of strains

[0041] 1. Screening of strains

[0042] Primary screening: solid medium was prepared containing substrate N-lauroyl glutamate sodium, and the plate was poured. Acylase can hydrolyze the substrate into lauric acid and glutamic acid, and the microorganism containing the enzyme grows by taking them as carbon source and nitrogen source, respectively. This method can be used to quickly screen effective strains. The specific operation is as follows: 100 μL of bacteria liquid from the environment was mixed with 900 μL of physiological saline (sterilized, the same below) to carry out dilution by 10 times, and 10 -4 , 10 -5 , 10 -6 , 10 -7 Bacteria liquid with different dilution degrees was coated on the solid medium containing N-lauroyl glutamate sodium, and after 2-4 days of culture at 30°C, the growth on the plate was observed.

[0043] Solid medium (g / L): K2HPO4 2.0, KH2PO4 0.4, NaCl 0.1, MgSO4·7H2O 0.2, CaCl2 0.025, NH4NO3 0.5, N-lauroyl glutamate sodium 1.0, agar 20, sterilized at 115°C for 30 min.

[0044] Secondary screening: the well-grown strains from the primary screening medium were inoculated into the liquid medium, and cultured at 30°C, 200 r / min on a shaker for 24 h. The sample was analyzed by high performance liquid chromatography, and the strains with good N-lauroyl glutamate sodium hydrolysis effect were selected and stored at -80°C.

[0045] Liquid medium (g / L): NaCl 0.5, MgSO4·7H2O 1.0, K2HPO4 1.0, NH4NO3 1.0, yeast powder 5.0, glycerol 5.0, CaCl2 0.03, N-lauroyl glutamic acid sodium 1.0, sterilized at 115℃ for 30 min.

[0046] 2. Identification of the strain

[0047] Morphological identification: the screened strain was inoculated into liquid medium and cultured at 30℃ for 24 h, then the bacterial liquid was diluted to an appropriate multiple and spread on solid medium, which was then cultured at 30℃ for 48 h. The size and morphology of the strain were observed under an optical microscope, and a single colony was subjected to Gram staining (see Fig. 1). Figure 3 The strain was sent to a biological company for 16S rDNA identification, and a phylogenetic tree of the strain was made based on the identification (see Fig. 2), Figure 4 Figure 4 which showed that it belonged to Pseudomonas sp. and was named Pseudomonas sp. 131.

[0048] Example 2: Amplification of acylase gene and construction of recombinant engineering strain

[0049] The strain was sent to a biological company for second-generation genome sequencing, and an acylase Acy2 gene was mined from the annotated genome, which was cloned into E. coli for expression.

[0050] 1. Extraction of genomic DNA of Pseudomonas sp. 131

[0051] The strain was inoculated into liquid medium and cultured at 30℃ and 200 r / min on a shaker for 24 h to generate seed liquid. The seed liquid was transferred to liquid medium at a volume concentration of 6% inoculation amount, and cultured at 30℃ and 200 r / min on a shaker for 24 h to obtain bacterial cells. The genomic DNA of the target strain was extracted according to the instructions of the DNA extraction kit and the steps.

[0052] 2. Amplification of acylase gene Acy2 and construction of recombinant engineering strain

[0053] The genomic DNA of Pseudomonas sp. 131 was used as a template, and specific primers were designed for amplification of the target gene. The designed primers were as follows:

[0054] Upstream primer: 5'-CGCGGATCCGAATTCGAGATGAGTCAGCACCCCACG-3' (see SEQ ID NO. 3), BamH I

[0055] ​Downstream primer: 5'-CGCAAGCTTGTCACACCACCACCACCACCACCACTGCTCTGGCTC-3' (see SEQ ID NO. 4), Hind III.

[0056] PCR system (50 μL):

[0057] Table 1 PCR system

[0058]

[0059] PCR reaction parameters:

[0060]

[0061] After the PCR reaction, the PCR product purification kit was used to clean the target gene PCR product: according to the operation steps in the kit instructions, the PCR product was recovered by centrifugation, and the nucleic acid product was subjected to agarose gel electrophoresis to detect the target band, and the results are shown in Figure 5 As can be seen from the figure, there is a clear band of 1000-1500 bp, and there is no non-specific band, which is consistent with the expected size. The target fragment was recovered, and the target fragment and pET-28a(+) were double-digested by restriction endonuclease BamH I and Hind III, respectively. The enzyme-digested target fragment (nucleotide sequence is shown in SEQ ID NO. 1) and pET-28a(+) were connected by high-efficiency ligation liquid, and the ligation product was transformed into E. coli BL21 Gold (DE3) host bacteria to obtain recombinant E. coli Acy2-pET-28a(+)-E. coli BL21 Gold (DE3).

[0062] The recombinant bacteria were plated on LB plates containing 50 μg / mL kanamycin and incubated at 37°C in an incubator overnight. Several single colonies on the plates were randomly picked and cultured in LB liquid medium (containing 50 μg / mL kanamycin) overnight. The recombinant plasmid was extracted using a small amount of plasmid DNA kit, double-digested, and subjected to plasmid PCR verification (see Figure 6 ), and the results were consistent with the expected. The recombinant plasmid was sent for sequencing, and the sequencing results showed that the recombinant E. coli was successfully constructed.

[0063] Example 3: Expression of acylase and obtaining of crude enzyme solution

[0064] A single colony of recombinant E. coli Acy2-pET-28a (+) -E. coli BL21 Gold (DE3) preserved on a plate in Example 2 was inoculated into 50 ml of LB medium containing 50 μg / mL kanamycin and incubated at 37°C and 180 r / min for 12 h. The culture was inoculated into 50 ml of liquid LB medium (containing 50 μg / mL kanamycin) at a volume concentration of 1%. The culture was incubated at 37°C and 180 r / min for 3 h, and then IPTG was added to a final concentration of 0.6 mmol / L. The culture was induced at 25°C and 180 r / min for 10 h. The intracellular liquid of the uninduced and induced recombinant bacteria was subjected to SDS-PAGE electrophoresis analysis, respectively (the supernatant was obtained by centrifugation of the wet bacteria after high-pressure homogenization and disruption of the fermentation broth). Figure 7 As shown in Figure 1, compared with lane 2 (uninduced group), there was an overexpressed protein band between 40-55 KDa in lane 1 (induced group), indicating that the recombinant E. coli expressed the target protein after induction by IPTG, and the size was about 42 kDa, which was consistent with the expectation.

[0065] The engineered bacteria containing the acylase-encoding gene were inoculated into LB liquid medium containing 50 μg / mL kanamycin and incubated at 37°C and 180 r / min until the OD 600 of the bacterial liquid was 0.5-0.8. Then, IPTG was added to a final concentration of 0.6 mmol / L, and the culture was induced at 25°C and 180 r / min for 10 h. The bacterial liquid was centrifuged at 8000 r / min at 4°C for 10 min, and the supernatant was discarded. The precipitated cells (wet bacteria) were suspended in 100 mmol / L Tris-HCl buffer (pH 8.0) to a bacterial concentration of 1.5 g / L. The bacteria were disrupted by high-pressure homogenization at 4°C, and the supernatant was obtained by centrifugation at 12000 r / min for 20 min.

[0066] Example 4: Hydrolysis of N-lauroyl glutamate sodium and other N-fatty acyl amino acid surfactants by acylase

[0067] N-lauroyl glutamate sodium 30 mg was used as the substrate, 50 mM Tris-HCl buffer (pH 8.0) was used as the reaction medium, and 50 μL of the crude enzyme solution prepared in Example 3 was added to a total reaction system of 30 mL. The reaction was carried out in a shaking flask at 37°C and 180 r / min. During the reaction, samples were taken at 0 min and 10 min, respectively. The samples were centrifuged at 8000 rpm for 5 min, and 100 μL of the supernatant was mixed with 900 μL of mobile phase for dilution. The mixture was filtered through a 0.22 μm organic membrane, and the concentration changes of the substrate and product glutamic acid in the reaction solution were detected by HPLC (as shown in Figure 2). Figure 8The conversion of the substrate N-lauroyl glutamate sodium (retention time 14.2 min) was 78.1% in the first 10 min and the average hydrolysis rate was 4.69 g / L / h (i.e. 13.4 mmol / L / h).

[0068] High performance liquid chromatography detection conditions (light scattering low temperature evaporation detector): Column: 125 x 2.1 mm (4.6 μm) C18 (Altech, France); mobile phase A: methanol / water / trifluoroacetic acid (TFA, 60 / 40 / 0.1, v / v / v); mobile phase B: methanol / TFA (100 / 0.07, v / v); flow rate: 0.5 mL / min; column temperature: 25°C; injection volume: 10 μL.

[0069] Gradient elution: starting with methanol / water / TFA (60 / 40 / 0.1 v / v / v), after 6 min a linear elution gradient was applied to reach methanol / water / TFA (95 / 5 / 0.1 v / v / v). The methanol concentration was maintained for 13 min, then decreased to the initial methanol / water / TFA (60 / 40 / 0.1 v / v / v) in 1 min, and maintained until the end of the run (35 min).

[0070] The same method was used to determine the hydrolytic activity of the acylase on several other common N-ester acyl amino acid surfactants, and the results are shown in Table 2.

[0071] Table 2 Hydrolytic activity of the acylase on common N-ester acyl amino acid surfactants

[0072] Substrate Relative activity (%) * ]] N-decanoyl glutamic acid sodium salt 88.3 N-lauroyl glutamic acid sodium salt 100 N-myristoyl glutamic acid sodium salt 93.6 N-palmitoyl glutamic acid sodium salt 82.7 N-lauroyl alanine sodium salt N-decanoyl glutamic acid sodium salt N-lauroyl glutamic acid sodium salt N-myristoyl glutamic acid sodium salt N-palmitoyl glutamic acid sodium salt N-lauroyl alanine sodium salt 3.6

[0073] * The hydrolysis rate of N-lauroyl glutamate sodium (in mol / L / h) under the reaction conditions (37°C, pH 8.0) was taken as 100% activity.

[0074] As shown in Table 2, the acylase has relatively high hydrolytic activity on N-fatty acyl glutamate sodium of C10-C16, and the activity on C12 (lauroyl) is the highest. However, the hydrolytic activity of the acylase on N-lauroyl alanine sodium is very low, and the possible reason is that glutamic acid has a γ-carboxyl group, and the binding center of the enzyme and the substrate can have an amino acid residue that specifically binds with the γ-carboxyl group, but alanine does not have a second carboxyl group and thus cannot bind with the amino acid residue at the corresponding position of the center. This result shows that the screening method of the present application can screen a microbial enzyme with relatively high substrate specificity.

[0075] Example 5: Synthesis of N-lauroyl glutamate sodium catalyzed by recombinant enzyme

[0076] ​The crude enzyme solution prepared in Example 3, 50 μL, was added to a mixture of butyl acetate and phosphate buffer (100 mmol / L, pH 7.0) (butyl acetate: buffer = 80:20, v / v), sodium glutamate 100 mM, lauric acid 20 mM, and the total reaction system was 30 mL. The reaction was carried out at 40°C in a 200 rpm shaker for 5 h. The concentration of the product, sodium N-lauroyl glutamate, was 0.938 g / L (2.68 mM) as determined by the HPLC method in Example 4, and the average synthesis rate was 0.536 mmol / L / h. The conversion rate of lauric acid was 13.4%.

Claims

1. Use of an acylating enzyme in the catalysis of the hydrolysis of N-fatty acyl amino acid surfactants, characterized in that, The acylase amino acid sequence is shown as SEQ ID NO. 2; The N-fatty acyl amino acid surfactant is at least one of N-decanoyl glutamic acid sodium, N-lauroyl glutamic acid sodium, N-myristoyl glutamic acid sodium, and N-palmitoyl glutamic acid sodium.

2. Use according to claim 1, wherein The N-fatty acyl amino acid surfactant is N-lauroyl glutamic acid sodium.

3. The use according to claim 1, wherein The crude enzyme solution obtained by crushing the wet bacterial cells obtained by fermenting the engineering bacteria containing acylase-encoding genes is used as a catalyst, and the N-fatty acyl amino acid surfactant is used as a substrate to perform a hydrolysis reaction.

4. The use of the acylase in claim 1 in catalyzing the synthesis of N-lauroyl glutamic acid sodium from lauric acid and sodium glutamate.

5. The use according to claim 4, wherein the compound is ###0002### The crude enzyme solution obtained by crushing the wet bacterial cells obtained by fermenting the engineering bacteria containing acylase-encoding genes is used as a catalyst, and lauric acid and sodium glutamate are used as substrates to perform a synthesis reaction, and N-lauroyl glutamic acid sodium is obtained.

6. Use according to claim 3 or 5, wherein: The wet bacterial cells obtained by fermenting the engineering bacteria containing acylase-encoding genes are suspended with a buffer, crushed by high-pressure homogenization, and centrifuged, and the supernatant is the crude enzyme solution.

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