A method for efficiently expressing amidase in Rhodococcus and its application

Abstract

The present invention relates to a method for efficiently expressing amidase in Rhodococcus and its application. The present invention studies and finds amidase that can be efficiently expressed in Rhodococcus, and further improves its expression efficiency by promoter screening or optimization. The recombinant Rhodococcus obtained by the method of the present invention has significantly improved amidase activity obtained by fermentation culture, and significantly improved expression efficiency, laying a foundation for the industrial production of amidase.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a method for efficiently expressing amidase in rhodococcus and application thereof. Background Art

[0002] Amidase, also known as amide hydrolase, is a type of hydrolase that can catalyze the cleavage of amide bonds to generate corresponding carboxylic acids and ammonia. It is widely found in organisms such as bacteria (broad-spectrum amidase), mold (penicillin acylase), fungi (formamidase, acetamidase and broad-spectrum amidase), yeast (nicotinamidase and broad-spectrum amidase), plants (peptidases) and animals (arachidonylethanolamine amidase). Most amidases have a broad substrate spectrum and can efficiently catalyze the hydrolysis of various aliphatic, aromatic and heterocyclic amides. Amidase has great potential in the synthesis of chiral compounds such as chiral carboxylic acids, amide derivatives and optically pure amino acids due to its good chemical and stereoselectivity, and is increasingly receiving attention from the industry.

[0003] In 2020, Mochamed Chafik Bourkaib et al. found that the Streptomyces ambofaciens ) ATCC23877 The amidase 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 performances of Streptomyces ambofaciens aminoacylases. Enzyme and microbialtechnology 137(2020)109536.). At the same time, they also confirmed that the amidase Sam-AA is a wild strain S. ambofaciens ATCC23877 The main reason for possessing N-α-acylation activity.

[0004] S. ambofaciens ATCC23877 The strain grows slowly and the culture time is too long. S. ambofaciens 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 aminoacylase with simple operation and high production efficiency has become an urgent problem to be solved. Summary of the invention

[0005] In order to overcome the above disadvantages, the present invention provides a recombinant amidase gene and its application. The present invention uses the amidase efficiently expressed in Rhodococcus to further improve its expression efficiency by promoter optimization, thereby achieving efficient and specific expression of the amidase.

[0006] The invention provides a method for efficiently expressing amidase in rhodococcus, which comprises the steps of constructing an expression vector of an amidase gene and introducing it into rhodococcus to obtain a recombinant rhodococcus, and culturing the recombinant rhodococcus to express the amidase.

[0007] Specifically, the Rhodococcus is selected from ATCC 12674, GMCC 1.10360, DSM 44193, CGMCC 1.5301 or CGMCC 1.4904, all of which are commercially available.

[0008] More specifically, the amidase gene is derived from Streptomyces divaricata ( Streptomyces ambofaciens ).

[0009] In a specific example, the amino acid sequence encoded by the amidase gene is shown in SEQ ID NO: 1.

[0010] More preferably, the nucleotide sequence of the amidase gene is as shown in SEQ ID NO:2.

[0011] Further preferably, the promoter used for the expression of the amidase gene is P3, P4 or P5, and the nucleotide sequences thereof are shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 respectively.

[0012] The present invention provides the recombinant Rhodococcus obtained by the method.

[0013] The present invention further provides a method for preparing an acylase catalyst, which comprises culturing the recombinant rhodococcus through fermentation to obtain wet bacteria or crude enzyme solution or pure enzyme as a biocatalyst.

[0014] The invention also provides application of the recombinant rhodococcus in biocatalytic synthesis of octanoyl surfactant.

[0015] Specifically, the recombinant Rhodococcus is fermented and cultured, centrifuged, the supernatant is discarded, and the precipitate is collected to obtain wet bacteria as a whole-cell catalyst, and octanoic acid and amino acids are used as substrates to form a reaction system.

[0016] More specifically, the wet bacteria are used in an amount of 5-30 g / L in the reaction system, the caprylic acid concentration is 1.44-72 g / L, and the amino acids participate in the reaction in a saturated amino acid solution with a total volume of 60%-100%;

[0017] The reaction conditions are: the biocatalytic reaction is carried out at a temperature of 35-65°C, a pH of 5.5-8.5, and a stirring speed of 100-300 rpm; the amino acid is selected from glycine, arginine, glutamic acid, lysine and alanine.

[0018] In a specific example, the wet bacteria are prepared as follows: the recombinant Rhodococcus is inoculated into a TSB liquid culture medium containing 100 μg / ml kanamycin resistance, cultured at 30°C, 200 rpm for 24 hours, and then inoculated into a fresh fermentation medium containing 100 μg / ml kanamycin resistance at a 2% (v / v) inoculation amount, and cultured at 30°C, 200 rpm for 48 hours; then centrifuged at 4°C, 8000 rpm for 20 minutes, discarded the supernatant, and collected the precipitate to obtain the wet bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Sam-AA_pET28a(+) plasmid map.

[0020] Figure 2 Sam-AA_pDD57 plasmid map.

[0021] Figure 3 Enzyme activities of each expression strain after promoter optimization. DETAILED DESCRIPTION

[0022] 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.

[0023] Example 1. Determination of enzyme activity of Sam-AA Escherichia coli expression strain

[0024] Sam-AA gene fragment (from Streptomyces divaricata ( Streptomyces ambofaciens ), NCBI No. WP_053128363.1) was synthesized by Nanjing GenScript Biotechnology Co., Ltd. and recombined into the pET28a (+) vector, namely Sam-aa_pET28a (+) plasmid. Its nucleotide sequence is shown in SEQ ID NO.2 (the encoded amino acid sequence is shown in SEQ ID NO: 1), and the plasmid map is shown in Figure 1 shown.

[0025] The Sam-AA_pET28a(+) plasmid was transferred into BL21(DE3) competent cells to obtain the Smm-aa Escherichia coli expression strain.

[0026] The Smm-AA Escherichia coli expression strain was inoculated into LB liquid medium containing 50 μg / ml kanamycin resistance, and cultured at 37°C, 200 rpm for 12 h. Then, it was inoculated into fresh LB liquid medium containing 50 μg / ml kanamycin resistance at a 1% (v / v) inoculation amount, and cultured at 37°C, 200 rpm until the bacterial OD 600 When the pH reaches 0.6-0.8, add IPTG with a final concentration of 0.1mM, and culture at 20℃, 200rpm for 15h. Centrifuge at 4℃, 8000rpm for 20min, discard the supernatant, collect the precipitate, and obtain the wet bacterial cells of recombinant Sam-aa Escherichia coli containing the recombinant plasmid. The bacterial cells can be directly used as biocatalysts.

[0027] Determination of octanoyl alanine synthase activity: 144 mg octanoic acid, 10 ml saturated alanine solution, 0.1 g wet bacteria, pH 8.0. React at 37°C for 24 hours. After the reaction, the reaction solution was analyzed by HPLC to obtain the octanoyl alanine synthase activity. The reaction and analysis were performed more than 3 times for each reaction system, and the data deviation caused by the operation was corrected.

[0028] 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℃; Flow rate: 1mL / min; Detection time: 15min.

[0029] Example 2: Determination of enzyme activity of Sam-aa Rhodococcus expression strain

[0030] The Sam-aa Escherichia coli expression strain has extremely low octanoylalanine synthetase activity, so the present invention seeks out an engineering bacterium Rhodococcus that is close to the source of Streptomyces as the Sam-AA expression strain in order to improve its enzyme activity.

[0031] The fragment was amplified by PCR using the Sam-AA_pET28a(+) plasmid in Example 1 as a template. The primers for PCR amplification of Sam-AA were: upstream primer 1 was 5'-attaagaaggagatatacatATGAGTGATTCAGGAACAGCT-3', and downstream primer 2 was 5'-aagctggatccagctcgaattcaCGACGCATCGATGAAACGGTC-3'.

[0032] 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 1.4 kb.

[0033] Obtaining the vector fragment: Use upstream primer 3: 5'-tgaattcgagctggatccagcttcctcgctcac-3' and downstream primer 4: 5'-atgtatatctccttcttaattaagcatgc-3' to amplify the pDD57 plasmid. The PCR system and reaction conditions are as described above.

[0034] The PCR product was purified and recovered by a purification and recovery kit. Finally, the two fragments were homologously recombined using the Novozymes one-step recombination kit. The reaction system was: 1 μL of gene fragment, 1 μL of vector fragment, 2 μL of 5× buffer, and 1 μL of recombinase. The reaction was carried out at 37°C for 30 min. The connected vector was transferred into the competent E. coli Top10 and the Sam-aa red ball expression plasmid Sam-AA_pDD57 was obtained by sequencing. The plasmid map is shown in the figure. Figure 2 shown.

[0035] Sam-aa red sphere expression plasmid Sam-AA_pDD57 was transferred into ATCC 12674, GMCC 1.10360, DSM44193, CGMCC 1.5301 and CGMCC 1.4904 (available commercially) red sphere expression strains. It was cultured in TSB liquid medium at 30°C and 220rpm for 24h. It was transferred to fermentation medium (glucose 30g / L, yeast extract 5g / L, tryptone 3g / L, malt extract 3g / L, dipotassium hydrogen phosphate 0.5g / l, potassium dihydrogen phosphate 0.5g / L, magnesium sulfate 1g / L, urea 1g / L, pH adjusted to 7) at an inoculum of 2%, and cultured at 30°C and 220rpm for 48h. The whole cells were collected by centrifugation at 8000rpm to obtain the wet cells of the recombinant Sam-aa red sphere containing the expression recombinant plasmid.

[0036] According to the method for determining the activity of octanoylalanine synthetase described in Example 1, the above wet cells were subjected to enzyme activity determination, and the results of enzyme activity relative to Escherichia coli BL21 (DE3) are shown in Table 1.

[0037] Table 1 Comparison of enzyme activities of different expression strains

[0038]

[0039] As can be seen from Table 1, the octanoylalanine synthetase activity of Sam-aa in Rhodococcus is significantly higher than that of BL21(DE3), indicating that the expression efficiency of Sam-aa in Rhodococcus is significantly higher than that of BL21(DE3).

[0040] Example 3, Rhodococcus promoter optimization

[0041] In order to further improve the activity of Sam-aa octanoylalanine synthetase, a series of promoter optimizations were performed on the expression vector.

[0042] Artificially synthesized promoter fragments P1, P2, P3, P4 and P5, whose base sequences are shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 respectively. The homologous recombination method was used to connect to the Sam-aa red sphere expression plasmid Sam-AA_pDD57, and the expression plasmid containing promoters P1, P2, P3, P4 and P5 was successfully constructed, and then transferred into the ATCC 12674 red sphere expression strain. According to the method shown in Example 2, the octanoyl alanine synthetase activity of Sam-aa in red sphere was determined. Its relative enzyme activity relative to P0 is shown in Figure 3 As shown in FIG. 1 , P0 refers to the Sam-aa ATCC 12674 red ball expression strain without promoter optimization. Figure 3 The results show that the P3, P4 and P5 promoters can significantly increase the octanoylalanine synthetase activity of Sam-aa in Rhodococcus, indicating that the P3, P4 and P5 promoters can significantly increase the expression efficiency of Sam-aa in Rhodococcus.

Claims

1. A method for expressing amidase in Rhodococcus, characterized in that: The amidase gene expression vector is introduced into Rhodococcus to obtain a recombinant Rhodococcus, and the amidase is obtained by culturing the recombinant Rhodococcus for expression; The promoters used for the expression of the amidase gene are P3 and P5 promoters, and their nucleotide sequences are shown in SEQ ID NO.5 and SEQ ID NO.7 respectively; The amidase gene is derived from Streptomyces divaricata ( Streptomyces ambofaciens ).

2. The method according to claim 1, characterized in that The Rhodococcus is selected from the group consisting of ATCC 12674, CGMCC1.10360, DSM 44193, CGMCC 1.5301 or CGMCC 1.4904.

3. The method according to claim 1, characterized in that The amino acid sequence encoded by the amidase gene is shown in SEQ ID NO:

1.

4. The method according to claim 1, characterized in that The nucleotide sequence of the amidase gene is shown in SEQ ID NO:

2.

5. The recombinant Rhodococcus obtained by the method according to any one of claims 1 to 4.

6. A method for preparing an acylase catalyst, characterized in that The recombinant Rhodococcus as claimed in claim 5 is cultured by fermentation to obtain wet bacteria or crude enzyme solution or pure enzyme as a biocatalyst.

7. Use of the recombinant Rhodococcus according to claim 5 in biocatalytic synthesis of octanoyl surfactants, characterized in that: The recombinant rhodococcus is fermented and cultured, centrifuged, the supernatant is discarded, and the precipitate is collected to obtain wet bacteria as a whole-cell catalyst, and octanoic acid and alanine are used as substrates to form a reaction system.

8. The use according to claim 7, characterized in that The wet bacteria are used in the reaction system in an amount of 5-30 g / L, the caprylic acid concentration is 1.44-72 g / L, and the alanine is reacted in a saturated alanine solution with a total volume of 60%-100%; The reaction conditions are: the biocatalytic reaction is carried out at a temperature of 35-65°C, a pH of 5.5-8.5, and a stirring speed of 100-300 rpm.

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