Amide hydrolase mutant and application thereof

Abstract

The invention relates to the technical field of enzyme engineering, synthetic biology and microbial fermentation, in particular to an amide hydrolase mutant and application thereof. The substrate range of the amide hydrolase RsAD from the microorganism Rhodococcus sp. Is relatively narrow, and after the amino acid residue L447 in the substrate channel of the amide hydrolase RsAD is mutated into alanine, the reaction enzyme activity of the alanine on the large-volume substrate, namely indoleacetamide, is increased, so that the production efficiency of indoleacetic acid is greatly improved.

Description

Technical Field

[0001] The invention relates to the fields of enzyme engineering, synthetic biology and microbial fermentation technology, and in particular to an amidohydrolase mutant and application thereof. Background Art

[0002] Auxin is the earliest discovered plant hormone, which is involved in many processes of plant growth and development, such as the development and growth of roots and stems, aging of organs, formation and differentiation of vascular tissues, apical dominance, and tropism of plant growth. The main type of auxin is indole-3-acetic acid (IAA), which exists in higher plants as well as bacteria, fungi, and algae.

[0003] There are two main pathways for the biosynthesis of indoleacetic acid: the tryptophan pathway and the non-tryptophan pathway. The tryptophan pathway includes four branches: the indolepyruvate pathway, the indoleacetamide pathway, the indoleacetonitrile pathway, and the tryptamine pathway.

[0004] In microorganisms, especially bacteria, the indoleacetamide pathway is best understood, as it is expressed in Agrobacterium tumefaciens ( Agrobacterium tumefaciens ) and Pseudomonas ( Pseudomonas savastanoi ) and has not been found in plants. In this pathway, tryptophan monooxygenase (iaam) catalyzes the conversion of tryptophan into indole-3-acetamide (Indoleacetamide-IAM); IAM is then hydrolyzed to produce IAA by indoleacetamide hydrolase (Indole acetamidehydrolase-iaah). Therefore, it is a feasible strategy to construct the IAM pathway in bacteria and improve the enzyme activity of key enzymes in the reaction process by structural modification to increase the production of indoleacetic acid.

[0005] Studies have reported that IAA can be produced by catalytically using recombinant Escherichia coli whole cells via the tryptophan pathway and the indoleacetamide (IAM) pathway. However, due to the low activity of amide hydrolase, more IAM generated by the reaction will remain, resulting in a low yield of IAA. An amide hydrolase RsAD was screened in the laboratory in the early stage, but RsAD also has such problems at this stage. When RsAD reacts with amide compounds with larger molecular structures, its catalytic efficiency is lower than that of compounds with smaller molecular weight. This may be because the RsAD substrate channel is relatively narrow, which limits the binding of larger substrates. For example, its efficiency for acrylamide is much lower than that for propionamide. In addition, RsAD can also react with a variety of amide compounds, such as reacting with phenylacetyl to hydrolyze to produce phenylacetic acid, and hydrolyzing indole-3-acetonitrile to produce IAA. In this study, by reacting with amide compounds from microorganisms Rhodococcus sp.The amide hydrolase RsAD was mutated and modified to obtain the optimal mutant L447A. Compared with the wild type, the yield of IAA synthesized by the mutant was greatly improved. Summary of the invention

[0006] The present invention is derived from microorganisms Rhodococcus sp. The wild-type amidohydrolase, referred to as RsAD, belongs to the amidase family. Indoleacetamide undergoes amide bond hydrolysis to generate indoleacetic acid under the action of RsAD. However, due to the limitation of the narrow range of the substrate channel, the enzyme has low reaction activity for large-volume substrates. The present invention significantly improves its enzyme activity by mutating the amino acid residues that constitute the gating structure of RsAD, thereby greatly increasing the yield of IAA when indoleacetamide (IAM) is used as a substrate.

[0007] One of the technical solutions provided by the present invention is an amidohydrolase mutant, which is obtained by mutation of leucine at position 447 to alanine based on the wild-type amidohydrolase shown in SEQ ID NO.1, and the amino acid sequence of the amidohydrolase mutant is shown in SEQ ID NO.3.

[0008] The present invention also provides a gene encoding the amidohydrolase mutant described in one of the technical solutions, and the nucleotide sequence of the gene encoding is shown in SEQ ID NO.4.

[0009] The second technical solution provided by the present invention is a recombinant vector or recombinant strain comprising the above mutant encoding gene; Furthermore, the expression plasmid used in the recombinant vector is pETDuet; Furthermore, the host cell used by the recombinant strain is Escherichia coli BL21 (DE3).

[0010] The third technical solution provided by the present invention is the application of the recombinant vector or recombinant strain described in the second technical solution, especially in the production of the amide hydrolase mutant described in the first technical solution.

[0011] The fourth technical solution provided by the present invention is the application of the amidohydrolase mutant described in the first technical solution, especially in the synthesis of indoleacetic acid, and more particularly in catalyzing the hydrolysis of the amide bond of indoleacetamide to generate indoleacetic acid.

[0012] Beneficial effects: From microorganisms Rhodococcus sp. The substrate range of the amidohydrolase RsAD is relatively narrow. After the amino acid residue L447 in the substrate channel of the amidohydrolase RsAD is mutated to alanine, the reaction activity of the amidohydrolase RsAD for the large-volume substrate, indoleacetamide, is increased, thereby greatly improving the production efficiency of indoleacetic acid. DETAILED DESCRIPTION

[0013] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0014] 1. The following definitions are used in the present invention: Amino acid and DNA sequences are referred to using the generally accepted IUPAC nomenclature for amino acid residues, using the three-letter / one-letter code format. DNA sequences use the generally accepted IUPAC nomenclature.

[0015] In the present invention, RsAD refers to Rhodococcus sp. The wild-type amidohydrolase RsAD The gene encodes a wild-type amidohydrolase, L447A represents an amidohydrolase mutant, RsAD-L447A It is the coding gene of L447A mutant. The specific information is as follows:

[0016] 2. Some of the determination methods used in the present invention are as follows (1) Determination of biomass concentration (OD): Take the fermentation liquid and measure the absorbance at 600 nm.

[0017] (2) Analysis and determination of indoleacetic acid: Determination method and system: Take 500 μL of 200mM potassium phosphate (pH 7.0) as buffer, add 50 μL of amide hydrolase RsAD and 10mM indole-3-acetamide, react at 37°C for 20 minutes, then take 200μL of reaction solution and add 100μL 1M H3PO4 to terminate the reaction, add methanol (300μL) to dilute and mix to make a sample, and centrifuge through a membrane (0.22 μm).

[0018] HPLC detection conditions for indoleacetic acid: High performance liquid chromatography (HPLC) Agilent Technologies 1260Infinity, ZORBAX Eclopse Plus C18 column (5 μm, 4.6×150 mm). Mobile phase: (A): 0.1% (v / v) trifluoroacetic acid (TFA) in deionized water (ddH2O); (B): 0.1% (v / v) TFA in acetonitrile (CH3CN), flow rate 1 mL min -1 , detection at 245 nm, column temperature 30 °C. Elution conditions: 30% mobile phase A; 70% mobile phase B isocratic elution.

[0019] 3. Some of the culture media or solutions used in the present invention are as follows: (1) LB solid medium (g / L): peptone 10, sodium chloride 10, yeast powder 5, agar 15, pH 7.0; (2) LB medium (g / L): peptone 10, sodium chloride 10, yeast powder 5, pH 7.0.

[0020] 4. Some of the culture conditions used in the present invention are as follows: (1) Seed culture: Inoculate a single clone into 4 mL of LB medium, add ampicillin (100 μg / mL) according to the resistance of the transferred plasmid, and culture at 37°C, 220 rpm for 8-10 hours.

[0021] (2) Fermentation culture: Add 50 mL of LB medium to a 250 mL shake flask, first transfer the plasmid resistance, i.e. ampicillin (100 μg / mL), and then inoculate the seed solution to make the initial OD of the bacteria 600 When the value reaches 0.1, culture at 37°C and 220 r / min. 600 When the pH reached 0.6-0.8, 0.2 mM IPTG was added to induce gene expression, and the culture was continued at 28°C for 8-9 h. The absorbance of the cells was then measured at 600 nm.

[0022] In the present invention, the amino acid sequence of the wild-type amidohydrolase RsAD is shown in SEQ ID NO.1: MATIRPDDKAIDAAARHYGITLDKTARLEWPALIDGALGSYDVVDQLYADEATPPTTSREHAVPSASENPLSAWYVTTSIPPTSDGVLTGRRVAIKDNVTVAGVPMMNGSRTVEGFTPSRDATVVTRLLAAGATVAGKAVCEDLCFSGSSFTPASGPVRNPWDRQREAGGSSGGSAALVANGDVDFAIGGDQGGSIRIPAAFCGVVGHKPTFGLVPYTGAFPIERTIDHLGPITRTVHDAALMLSVIAGRDGNDPRQADSVEAGDYLSTLDSDVDGLRIGIVREGFGHAVSQPEVDDAVRAAAHSLTEIGCTVEEVNIPWHLHAFHIWNVIATDGGAYQMLDGNGYGMNAEGLYDPELMAHFASRRIQHADALSETVKLVALTGHHGITTLGGASYGKARNLVPLARAAYDTALRQFDVLVMPTLPYVASELPAKDVDRATFITKALGMIANTAPFDVTGHPSLSVPAGLVNGLPVGMMITGRHFDDATVLRVGRAFEKLRGAFPTPAERASNSAPQLSPAHHHHHH* In the present invention, the amino acid sequence of the amide hydrolase mutant L447A is as shown in SEQ ID NO.3: * The present invention is further explained below by means of specific examples.

[0023] Example 1: Construction of amino acid residue saturation mutants at position L447 in RsAD Will come from Rhodococcus sp. The nucleic acid sequence of the amide hydrolase RsAD (SEQ ID NO. 2) was integrated into the petDuet plasmid and named petDuet-RsAD as a template for constructing mutants.

[0024] Taking the construction of mutant L447-A as an example, the codon CTG encoding leucine at position 447 was mutated to alanine GCG through PCR amplification with mutant primers. The specific operation is as follows: (1) Design of mutation primers V-Fwd (5'-CAAAGCTGCGGGTATGATCGCTAACACCGCTCCGTT-3'), V-Rev (5'-TCATACCCGCAGCTTTGGTGATGAAGGTAGCACGGTCAACG-3'); (2) PCR amplification conditions: 98℃10s, 55℃2min, 72℃45s. The petDuet-RsAD plasmid vector was used as a template for amplification. The amplified product was digested with Dpn1 to remove the methylated template plasmid. The digested product was then used for recombination ligation. The reaction product was then transformed into Escherichia coli DH5α and plated on a plate containing ampicillin resistance. Finally, a single clone was selected for sequencing verification. If the sequencing results showed that the leucine codon CTG at position 447 mutated to alanine GCG, it proved that the mutant was successfully constructed and the plasmid petDuet-RsAD-L447A was obtained.

[0025] The other mutants and mutant plasmids were obtained by the same method as above, just by replacing the primers.

[0026] Example 2: Preparation and expression of amidohydrolase RsAD and mutants Plasmid petDuet-RsAD, mutant plasmid petDuet-RsAD-L447A, and other mutant plasmids at the L447 site were transformed into E. coli BL21 (DE3) cells. The cells were cultured in LB medium at 37°C until OD600 was 0.6-0.8, and then 0.02% IPTG was added at 28°C to induce expression for 8-9 hours. The collected cells were suspended in buffer A (50 mM PBS, pH = 7.4), disrupted by sonication, and then centrifuged to remove cell debris.

[0027] The supernatant was purified by AKTA protein purification. During the purification process, the nickel column (HisTrapTMHP5Ml) was linearly eluted with elution buffer B (20 mM PBS, pH=7.4; 100 mM NaCl; 500 mM imidazole) for 30 min, and the target protein peak was collected. During the desalting process, the HiPrepTM26 / 10Desalting column was eluted with buffer A (50 mM PBS, pH=7.4), and the protein peak collected was the desalted protein solution. The protein concentration was determined by the BCA method.

[0028] The samples obtained in the above stages were tested for protein expression by SDS polyacrylamide gel electrophoresis. The results showed that the proteins were expressed normally before and after mutation, and the purified proteins retained a high purity of the target protein and could be used for subsequent enzyme activity determination experiments.

[0029] Example 3: RsAD enzyme activity determination 500 μL of 200 mM potassium phosphate (pH 7.0) was taken as a buffer, 50 μL of amide hydrolase (the purified enzyme obtained in Example 2: wild type or L447A mutant or other mutants) and a substrate indole-3-acetamide at a final concentration of 10 mM were added thereto, and the reaction was carried out at 37°C on a shaker at 200 r / min for 20 min. After that, 200 μL of the reaction solution was taken and 100 μL of 1M H3PO4 was added to terminate the reaction. 300 μL of methanol was added to dilute and mix to prepare a sample, which was centrifuged and passed through a membrane (0.22 μm). Indoleacetic acid was determined by HPLC analysis equipped with a ZORBAX EclopsePlus C18 column (5 μm, 4.6×150 mm).

[0030] The enzyme activity was characterized by the amount of enzyme required to generate 1 μmol IAA in 1 min of reaction, and the results of enzyme activity detection and calculation are shown in Table 1. It can be seen that the enzyme activity of multiple mutants at the L447 site was improved compared with the wild type, among which the mutant L447A had the best effect, and its enzyme activity was increased by about 9 times compared with the wild type, reaching 2739.53 μmol / min / mg.

[0031] Table 1

[0032] Although the present invention has been disclosed as above in terms of preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various changes, modifications, substitutions and variations in form and details to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. An amidohydrolase mutant, characterized in that: The mutant is obtained by mutation of leucine at position 447 to alanine on the basis of the wild-type amide hydrolase shown in SEQ ID NO.

1. The amino acid sequence of the amide hydrolase mutant is shown in SEQ ID NO.

3.

2. A gene encoding the amidohydrolase mutant according to claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence of the coding gene is shown in SEQ ID NO.

4.

4. A recombinant vector or recombinant strain comprising the coding gene according to claim 2.

5. The recombinant vector according to claim 4, characterized in that The expression plasmid used in the recombinant vector is pETDuet.

6. The recombinant strain according to claim 4, characterized in that The host cell used by the recombinant strain is Escherichia coli BL21 (DE3).

7. Use of the recombinant vector or recombinant strain according to claim 4 in producing the amidohydrolase mutant according to claim 1.

8. Use of the amidohydrolase mutant according to claim 1 in the synthesis of indoleacetic acid.

9. The use according to claim 8, characterized in that It is used in catalyzing the hydrolysis of the amide bond of indoleacetamide to generate indoleacetic acid.

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