A Streptomyces-derived acylase and acylase mutant and use thereof

Abstract

The invention discloses an acylase and an acylase mutant derived from Streptomyces and applications thereof. The acylase mutant derived from Streptomyces has high enzyme activity in synthesizing octanoyl amino acid compounds, and the preparation method of the octanoyl amino acid surfactant provided by the invention has high substrate conversion rate and high product yield, thus providing a solid foundation for the industrial production of the octanoyl amino acid surfactant prepared by enzyme catalysis.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to an acylase Sam-aa derived from Streptomyces and an acylase mutant and application of the acylase in catalyzing the synthesis of octanoyl amino acid compounds. Background Art

[0002] Acyl amino acid surfactants have excellent surface activity, wettability, emulsification, solubilization and foaming properties, and are widely used in daily chemicals, textiles, oil extraction, lubrication and other fields. In addition, N-acyl amino acid surfactants can be used as drug sustained-release agents, wound dressings and other materials in the biomedical field due to their advantages such as good biodegradability, low toxicity and low irritation. In recent years, with the promotion of the amino acid personal care product market, the demand for N-acyl amino acid surfactant products has continued to increase. Therefore, the development of environmentally friendly and efficient N-acyl amino acid surfactant synthesis routes has become a research hotspot.

[0003] The current industrial production of N-acyl amino acid surfactants mostly adopts chemical production, and its general process is as follows:

[0004]

[0005] The chemical synthesis of N-amino acid surfactants involves a large number of toxic and harmful substances, and the process is complex and energy-intensive, which brings about serious environmental pollution and other problems. Therefore, in recent years, the enzyme catalysis method has gradually become a new direction for the synthesis of amino acid surfactants due to its low energy consumption and environmental friendliness. The process of enzymatic synthesis of amino acid surfactants is as follows:

[0006]

[0007] The process is to generate N-acyl amino acids from fatty acids and amino acids by enzyme catalysis in one step, and then the N-acyl amino acids are saponified in one step to synthesize N-acyl amino acid type surfactants. The process is simple, has no by-products, is green and environmentally friendly, and has high safety, so it has attracted widespread attention.

[0008] 2020, Mochamed Chafik Bourkaib found from S. ambofaciens ATCC23877The acylase in aqueous media has the ability to catalyze the production of N-acyl amino acids (Mochamed Chafik Bourkaib, Stephane Delaunay, Xavier Framboisier, Laurence Hotel, Bertrand Aigle, Catherine Humeau, Yann Guiavarch, Isabelle Chevalot. N-acylation of L-amino acids in aqueous media: Evaluation of the catalytic performance of Streptomyces ambofaciens aminoacylases. Enzyme and microbial technology 137(2020)109536.). However, its enzyme activity is low and it is difficult to be used directly in industrial production. At the same time, they also confirmed that the acylase Sam-AA is a wild Streptomyces succinate. ATCC23877 The main reason for possessing N-α-acylation activity.

[0009] Streptomyces succinate ATCC23877 The strain grows slowly and the culture time is too long. ATCC23877 The N-α-acylation activity of the crude extract is low and insufficient for industrial application. Therefore, how to provide a method for large-scale production of acylase with simple operation and high production efficiency has become an urgent problem to be solved. Summary of the invention

[0010] In order to overcome the above shortcomings, the present invention provides a genetically engineered bacterium of an acylase Sam-aa derived from Streptomyces. Further, the present invention provides a mutant of the acylase, which has significantly improved enzyme activity in synthesizing octanoyl amino acid compounds and has high production and application value.

[0011] The invention provides an acylase and an acylase mutant derived from Streptomyces. The amino acid sequence of the acylase and the mutant is shown in SEQ ID NO.2 (NCBI No. WP_053128363.1), and the amino acid sequence of the acylase and the mutant is shown in SEQ ID NO.2 (NCBI No. WP_053128363.1), and the mutations of one or more of the following sites are present on the acylase: A156S, V158G, V183I, V183L, F186T, F186Y, F186K, R195I, A330E, Y379V, L381M, L381I, G384A, G384K, T385G, A412M, A412P, and F415Y.

[0012] Furthermore, based on the amino acid sequence as shown in SEQ ID NO.2, there are mutations in two, three, four or more of the following sites: A156S, V158G, V183I, V183L, F186T, F186Y, F186K, R195I, A330E, Y379V, L381M, L381I, G384A, G384K, T385G, A412M, A412P, and F415Y.

[0013] Preferably, the following mutations exist based on the amino acid sequence as shown in SEQ ID NO.2:

[0014] A156S / V158G, A156S / V183I, V158G / Y379V, V183L / A412M, F186T / R195I, F186K / A412P, R195I / F415Y, A330E / T385G, L381M / G384K;

[0015] A156S / V158G / V183I, A156S / V158G / F186K, A156S / V158G / Y379V, A156S / V158G / L381I, A156S / V158G / A412P.

[0016] The present invention also provides a nucleic acid encoding the acylase or a mutant thereof.

[0017] The present invention provides a recombinant expression vector containing the nucleic acid, specifically using the pDD57 vector as the framework.

[0018] The present invention also provides a genetically engineered bacterium containing the nucleic acid or the recombinant expression vector.

[0019] Preferably, it is Rhodococcus.

[0020] The present invention provides application of the acylase or its mutant, or its encoding nucleic acid in preparing octanoyl amino acid compounds.

[0021] The present invention also provides a method for preparing an octanoyl amino acid compound, which comprises using the genetically engineered bacteria as a whole-cell catalyst, octanoic acid and amino acids as substrates, and synthesizing the octanoyl amino acid compound.

[0022] Furthermore, the method specifically comprises the following steps: centrifuging the fermented bacterial liquid, discarding the supernatant, and collecting the precipitate to obtain wet bacterial cells;

[0023] Preferably, the amino acids include glycine, arginine, glutamic acid, lysine, and alanine;

[0024] More specifically, the reaction system is composed of 5-30 g / L wet bacterial cell dosage, 1.44-72 g / L caprylic acid concentration, and 60%-100% saturated amino acid solution;

[0025] The reaction conditions are as follows: 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 an octanoyl amino acid compound.

[0026] The method for preparing the octanoyl amino acid surfactant provided by the invention has high substrate conversion rate and high product yield, and provides a solid foundation for the industrial production of preparing the octanoyl amino acid surfactant by enzyme catalysis. DETAILED DESCRIPTION

[0027] The present invention is further described below through specific examples in order to better understand the present invention, but does not constitute a limitation of the present invention.

[0028] The product detection method used in the embodiment is as follows:

[0029] The product prepared by the present invention was detected by high performance liquid chromatography, chromatographic column: C18, 4.6 mm × 250 mm, 5 μm, detection wavelength: ultraviolet (UV) = 200 nm; mobile phase: 50% acetonitrile and 50% 0.05% trifluoroacetic acid aqueous solution; column temperature: 30 ° C; flow rate: 1 mL / min; detection time: 15 min.

[0030] The primers used in the examples are as follows:

[0031] .

[0032] Example 1: Acquisition of Sam-aa expression strain

[0033] The Sam-aa gene fragment was synthesized by Nanjing GenScript Biotechnology Co., Ltd. and recombined into the PUC57 vector. Its nucleotide sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO:2. The fragment was amplified by PCR using the gene as a template. The primers for PCR amplification of Sam-aa are: upstream primer 1 is: 5'-attaagaaggagatatacatATGAGTGATTCAGGAACAGCT-3',

[0034] Downstream primer 2: 5'-aagctggatccagctcgaatTCAGTGATGATGATGATGATG-3'

[0035] The amount of each component added to the PCR system (total volume 50 μL): PrimerStar 25 μL, ddH2O 20 μL, upstream and downstream primers 1.5 μL each, template 2 uL. The PCR amplification program is: 98℃ pre-denaturation for 2 min; enter the cycle (98℃ denaturation for 10 s; 56℃ annealing for 15 s; 72℃ extension for 30 s), a total of 25 cycles, 72℃ final extension for 5 min. The amplified target gene fragment size is about 1.3 kb.

[0036] Obtaining the vector fragment: Use the upstream primer 3: 5'-CATCATCATCATCATCACTGAattcgagctggatccagctt-3' and the downstream primer 4: 5'-atgtatatctccttcttaattaagcatgc-3' to amplify the PDD57 plasmid. The PCR system and reaction conditions are as described above.

[0037] The PCR product was purified and recovered by a purification recovery kit, and then the two fragments were homologously recombined using the Novozyme one-step recombination kit. The reaction system was: 1 μL of gene fragment, 1 μL of vector fragment, 2 μL of 5× buffer, 1 μL of recombinase, and 1 μL of H 2 O 5μL. React at 37°C for 30min. Transform the ligated vector into E. coli Top10 competent cells and obtain the Sam-aa red sphere expression plasmid, i.e., pDD57-Sam-aa, by sequencing.

[0038] The above Sam-aa red sphere expression plasmid was electroporated into competent cells of Rhodococcus ATCC 12674 to obtain the Sam-aa expression strain.

[0039] Example 2: Protein expression of Sam-aa

[0040] The expression strain of Sam-aa obtained in Example 1 was inoculated into TSB liquid medium (tryptone 17 g / L, soy peptone 3 g / L, sodium chloride 5 g / L, glucose 2.5 g / L, dipotassium hydrogen phosphate 2.5 g / L) containing 100 μg / ml kanamycin resistance, cultured at 30°C, 200 rpm for 16 h, and then inoculated into fresh fermentation medium (glucose 5 g / L, yeast extract 3 g / L, tryptone 3 g / L, malt extract 3 g / L, dipotassium hydrogen phosphate 0.5 g / L, potassium dihydrogen phosphate 0.5 g / L, magnesium sulfate 1 g / L, urea 1 g / L, pH adjusted to 7) at a 5% (v / v) inoculation amount, cultured at 30°C, 200 rpm for 48 h. Centrifuged at 4°C, 8000 rpm for 20 min, discarded the supernatant, and collected the precipitate to obtain wet cells.

[0041] Example 3: Construction of Sam-aa mutant library

[0042] Using the plasmid pDD57-Sam-aa constructed in Example 1 as a template, error-prone PCR was performed. Error-prone PCR was performed under the action of upstream primer 1 and downstream primer 2. PCR reaction system (total volume 50 μL): 10×Taq DNA polymerase Buffer 5 μL, 10 mM dNTP mixture (2.5 mM each of dATP, dCTP, dGTP and dTTP) 4 μL, 10 mM dCTP 0.8 μL, 10 mM dTTP 0.8 μL, 1 μL each of primer 1 and primer 2 with a concentration of 10 μM, 2 μL of plasmid template, 1 μL of R-Taq enzyme, Mg 2+ (25mM) 4μL, Mn 2+ (0.5mM) 4μL, deionized water to 50μL. The PCR amplification program is: 95℃ pre-denaturation for 5min; enter the cycle (95℃ denaturation for 30s; 56℃ annealing for 20s; 72℃ extension for 1min), a total of 25 cycles, 72℃ final extension for 10min. After purifying the error-prone PCR product, it was homologously recombined with the vector fragment obtained in Example 1. The specific experimental method was the same as in Example 1. The homologous recombination product was finally transferred into the competent cells of Rhodococcus ATCC 12674, and cultured at 30°C for 48h to obtain the Sam-aa mutant library.

[0043] Example 4: High-throughput screening of the Sam-aa mutant library

[0044] 1000 single clones were selected from the Sam-aa mutant library obtained in Example 3 and inoculated into a 96-well plate to culture the single clones. Each single clone contained 500uLTSB liquid culture medium and cultured at 30°C, 200rpm for 16h. Then, the inoculation amount was inoculated into a fresh fermentation medium at 5% (v / v) and cultured at 30°C, 200rpm for 48h. The mixture was centrifuged at 5000rpm for 5min, the supernatant was discarded, and the precipitate was collected to obtain 1000 mutant wet cells.

[0045] The above wet cells were used for octanoylalanine biosynthesis. The final concentration composition and catalytic conditions of the catalytic system (200 μL) were as follows: 2.88 mg of octanoic acid, 200 μL of saturated alanine, pH 8.0 were added to the wet cells. Reaction conditions: temperature 37°C, 800 rpm, reaction time 24 h. After the reaction was completed, the mixture was shaken and mixed evenly, and the reaction solution was analyzed by HPLC to obtain the octanoylalanine synthase activity.

[0046] Analysis conditions: Chromatographic column: C18, 4.6mm×250mm, 5μm; Detection wavelength: ultraviolet (UV) = 200nm; Mobile phase: 50% acetonitrile and 50% 0.05% trifluoroacetic acid aqueous solution; Column temperature: 30°C; Flow rate: 1mL / min; Detection time: 15min.

[0047] By comparing the relative values ​​of enzyme activities of mutants and wild-type, a total of 18 superior mutants were screened and obtained. The results are shown in Table 1.

[0048] Table 1. Acylase mutant information

[0049]

[0050] From the results shown in Table 1, it can be seen that the octanoylalanine synthetase activity of Sam-aa acylase can be improved by mutations of A156S, V158G, V183I, V183L, F186T, F186Y, F186K, R195I, A330E, Y379V, L381M, L381I, G384A, G384K, T385G, A412M, A412P, and T415Y alone.

[0051] Example 5: Obtaining the acylase mutant (20)

[0052] The recombinant plasmid pDD57-Sam-aa constructed in Example 1 was used as a template for full plasmid PCR amplification. The amount of each component added to the PCR system (total volume 50 μL): PrimerStar 25 μL, ddH 2 O 20μL, primers 1-F and 1-R 1.5μL each, template 2uL. The PCR amplification program is: 98℃ pre-denaturation for 2min; enter the cycle (98℃ denaturation for 10s; 56℃ annealing for 15s; 72℃ extension for 70s), a total of 25 cycles, 72℃ final extension for 5min. After the PCR reaction is completed, add DpnI 1μL, digest at 37℃ for 30min. Then, transfer into the competent cells of Rhodococcus ATCC 12674 to obtain mutant (20), DNA sequencing to determine the acylase gene sequence, and mutant (20) is obtained by the method in Example 2 to obtain wet bacteria (20).

[0053] The wet bacteria (20) was subjected to the octanoyl alanine synthase activity assay. Synthesis reaction system (10 mL): 144 mg of octanoic acid, 10 ml of saturated alanine solution, 0.1 g of wet bacteria, pH values ​​of 6.0 and 8.0, respectively. The reaction was carried out at 37°C for 24 h. After the reaction was completed, the octanoyl alanine synthase activity was evaluated according to the method recorded in Example 4. The results of the relative value of the enzyme activity relative to the wild type are shown in Table 2.

[0054] Example 6: Obtaining the acylase mutant (21)

[0055] The recombinant plasmid pDD57-Sam-aa constructed in Example 1 was used as a template for PCR amplification. The amount of each component added to the PCR system (total volume 50 μL): PrimerStar 25 μL, ddH 2 O 20μL, primers 2-F and 2-R 1.5μL each, template 2uL. PCR amplification program: 98℃ pre-denaturation 2min; enter the cycle (98℃ denaturation 10s; 56℃ annealing 15s; 72℃ extension 10s), a total of 25 cycles, 72℃ final extension 5min. After purifying the PCR product, use the product as a primer and pDD57-Sam-aa as a template for large primer PCR to obtain large primer PCR products. PCR system: primer 3μL, plasmid template 2μL, PrimerStar 25μL, ddH2O 20μL. PCR amplification program: 98℃ pre-denaturation 2min; enter the cycle (98℃ denaturation 10s; 56℃ annealing 15s; 72℃ extension 60s), a total of 25 cycles, 72℃ final extension 5min. After the PCR reaction is completed, add DpnI 1μL and digest at 37℃ for 30min. Then, the mutant (21) was transformed into competent cells of Rhodococcus sp. ATCC 12674 to obtain the mutant (21). The acylase gene sequence was determined by DNA sequencing. The mutant (21) was obtained by the method in Example 2 to obtain wet cells (21).

[0056] The evaluation was performed according to the octanoylalanine synthetase activity detection method described in Example 5. The results of the relative values ​​of the enzyme activity relative to the wild type are shown in Table 2.

[0057] Example 7: Obtaining the acylase mutant (22)

[0058] According to the method described in Example 6, the primers were replaced with primers 3-F and 3-R, and the acylase mutant (22) expressing the activity shown in Table 2 was obtained. The activity of octanoylalanine synthetase was evaluated according to the method for detecting the activity of octanoylalanine synthetase described in Example 5. The results of the relative value of the enzyme activity relative to the wild type are shown in Table 2.

[0059] Example 8: Obtaining the acylase mutant (23)

[0060] According to the method described in Example 6, the primers were replaced with primers 4-F and 4-R, and the acylase mutant (23) expressing the activity shown in Table 2 was obtained. The activity of octanoylalanine synthetase was evaluated according to the method for detecting the activity of octanoylalanine synthetase described in Example 5. The results of the relative value of the enzyme activity relative to the wild type are shown in Table 2.

[0061] Example 9: Obtaining the acylase mutant (24)

[0062] According to the method described in Example 6, the primers were replaced with primers 5-F and 5-R, and the acylase mutant (24) expressing the activity shown in Table 2 was obtained. The activity of octanoylalanine synthetase was evaluated according to the method for detecting the activity of octanoylalanine synthetase described in Example 5. The results of the relative value of the enzyme activity relative to the wild type are shown in Table 2.

[0063] Example 10: Obtaining the acylase mutant (25)

[0064] According to the method described in Example 6, the primers were replaced with primers 6-F and 6-R, and the acylase mutant (25) expressing the activity shown in Table 2 was obtained. The activity of octanoylalanine synthetase was evaluated according to the method for detecting the activity of octanoylalanine synthetase described in Example 5. The results of the relative value of the enzyme activity relative to the wild type are shown in Table 2.

[0065] Example 11: Obtaining the acylase mutant (26)

[0066] According to the method described in Example 6, the primers were replaced with primers 7-F and 7-R, and the acylase mutant (26) expressing the activity shown in Table 2 was obtained. The activity of octanoylalanine synthetase was evaluated according to the method for detecting the activity of octanoylalanine synthetase described in Example 5. The results of the relative value of the enzyme activity relative to the wild type are shown in Table 2.

[0067] Example 12: Obtaining the acylase mutant (27)

[0068] According to the method described in Example 6, the primers were replaced with primers 8-F and 8-R, and the acylase mutant (27) expressing the activity shown in Table 2 was obtained. The activity of octanoylalanine synthetase was evaluated according to the method for detecting the activity of octanoylalanine synthetase described in Example 5. The results of the relative value of the enzyme activity relative to the wild type are shown in Table 2.

[0069] Example 13: Obtaining the acylase mutant (28)

[0070] The recombinant plasmid pDD57-Sam-aa constructed in Example 1 was used as a template for full plasmid PCR amplification. The amount of each component added to the PCR system (total volume 50 μL): PrimerStar 25 μL, ddH 2 O 20μL, primers 9-F and 9-R 1.5μL each, template 2uL. PCR amplification program is: 98℃ pre-denaturation for 2min; enter the cycle (98℃ denaturation for 10s; 56℃ annealing for 15s; 72℃ extension for 70s), a total of 25 cycles, 72℃ final extension for 5min. After the PCR reaction is completed, add DpnI 1μL and digest at 37℃ for 30min. Then, transfer into the competent cells of Rhodococcus ATCC 12674 to obtain the mutant (28).

[0071] The evaluation was performed according to the octanoylalanine synthetase activity detection method described in Example 5. The results of the relative values ​​of the enzyme activity relative to the wild type are shown in Table 2.

[0072] Table 2. Acylase mutant information

[0073]

[0074] From the results shown in Table 2, it can be seen that a residue mutation group consisting of two mutations selected from the amino acid residue mutations such as A156S, V158G, V183I, V183L, F186T, F186Y, F186K, R195I, A330E, Y379V, L381M, L381I, G384A, G384K, T385G, A412M, A412P, and T415Y can also improve the octanoylalanine synthetase activity of Sam-aa acylase.

[0075] Example 14: Obtaining the acylase mutant (29)

[0076] According to the method described in Example 13, the primers were replaced with primers 10-F and 2-R, and the template was replaced with the recombinant plasmid obtained in Example 5, and the acylase mutant (29) expressing the activity shown in Table 3 was obtained. The activity of octanoylalanine synthetase was evaluated according to the method for detecting the activity of octanoylalanine synthetase described in Example 5. The results of the relative value of the enzyme activity relative to the acylase (20) are shown in Table 3.

[0077] Example 15: Obtaining the acylase mutant (30)

[0078] According to the method described in Example 13, the primers were replaced with primers 6-F and 10-R, and the template was replaced with the recombinant plasmid obtained in Example 5, and the acylase mutant (30) expressing the activity shown in Table 3 was obtained. The activity of octanoylalanine synthetase was evaluated according to the method for detecting the activity of octanoylalanine synthetase described in Example 5. The results of the relative value of the enzyme activity relative to the acylase (20) are shown in Table 3.

[0079] Example 16: Obtaining the acylase mutant (31)

[0080] According to the method described in Example 13, the primers were replaced with primers 11-F and 11-R, and the template was replaced with the recombinant plasmid obtained in Example 5, and the acylase mutant (31) expressing the activity shown in Table 3 was obtained. The activity of octanoylalanine synthetase was evaluated according to the method for detecting the activity of octanoylalanine synthetase described in Example 5. The results of the relative value of the enzyme activity relative to the acylase (20) are shown in Table 3.

[0081] Example 17: Obtaining the acylase mutant (32)

[0082] According to the method described in Example 13, the primers were replaced with primers 12-F and 12-R, and the template was replaced with the recombinant plasmid obtained in Example 5, and the acylase mutant (32) expressing the activity shown in Table 3 was obtained. The activity of octanoylalanine synthetase was evaluated according to the method for detecting the activity of octanoylalanine synthetase described in Example 5. The results of the relative value of the enzyme activity relative to the acylase (20) are shown in Table 3.

[0083] Example 18: Obtaining the acylase mutant (33)

[0084] According to the method described in Example 13, the primers were replaced with primers 13-F and 13-R, and the template was replaced with the recombinant plasmid obtained in Example 5, and the acylase mutant (33) expressing the activity shown in Table 3 was obtained. The activity of octanoylalanine synthetase was evaluated according to the method for detecting the activity of octanoylalanine synthetase described in Example 5. The results of the relative value of the enzyme activity relative to the acylase (20) are shown in Table 3.

[0085] Table 3. Acylase mutant information

[0086]

[0087] From the results shown in Table 3, it can be seen that a residue mutation group consisting of three mutations selected from the amino acid residue mutations such as A156S, V158G, V183I, V183L, F186T, F186Y, F186K, R195I, A330E, Y379V, L381M, L381I, G384A, G384K, T385G, A412M, A412P, and T415Y can also improve the octanoylalanine synthetase activity of Sam-aa acylase.

Claims

1. A mutant of an acylase derived from Streptomyces, characterized in that: Its amino acid sequence only has mutations in the following sites based on the amino acid sequence shown in SEQ ID NO.2: A156S, V183I, V183L, F186T, F186Y, F186K, R195I, A330E, Y379V, L381M, L381I, G384A, G384K, A412M, A412P, F415Y, A156S / V183I, V183L / A412M, F186T / R195I, F186K / A412P, R195I / F415Y, A330E / T385G or L381M / G384K.

2. A nucleic acid encoding the acylase mutant according to claim 1.

3. A recombinant expression vector containing the encoding nucleic acid as claimed in claim 2.

4. A genetically engineered bacterium containing the encoding nucleic acid according to claim 2 or the recombinant expression vector according to claim 3.

5. The genetically engineered bacterium according to claim 4, characterized in that It is Rhodococcus.

6. Use of the acylase mutant according to claim 1, or a nucleic acid encoding the mutant, in the preparation of octanoylalanine.

7. A method for preparing octanoylpropylamino acid, characterized in that: The method comprises using the genetically engineered bacteria as claimed in claim 4 or 5 as a whole-cell catalyst and octanoic acid and alanine as substrates to synthesize octanoylalanine.

8. The preparation method according to claim 7, characterized in that: The method specifically comprises the following steps: centrifuging the fermented bacterial liquid, discarding the supernatant, and collecting the precipitate to obtain wet bacterial cells; forming a reaction system with a wet bacterial cell dosage of 5-30 g / L, an octanoic acid concentration of 1.44-72 g / L, and a 60%-100% saturated alanine solution; and performing a biocatalytic reaction under the reaction conditions of a temperature of 35-65°C, a pH of 5.5-8.5, and a stirring speed of 100-300 rpm to obtain octanoyl alanine.

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