A kind of amidohydrolase mutant and its application

Abstract

The present invention relates to the fields of enzyme engineering, synthetic biology and microbial fermentation technology, and in particular to an amidohydrolase mutant and its application. 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, its reaction activity for the large-volume substrate, indoleacetamide, is increased, thereby greatly improving the production efficiency of indoleacetic acid.

Description

Technical Field

[0001] The present 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, one of the earliest discovered plant hormones, is involved in many processes of plant growth and development, such as root and stem development and growth, organ aging, the formation and differentiation of vascular tissue, apical dominance, and tropism. The main type of auxin is indole-3-acetic acid (IAA), which is present 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 ) but not in plants. In this pathway, tryptophan monooxygenase (iaam) catalyzes the conversion of tryptophan to indole-3-acetamide (IAM); IAM is then hydrolyzed to IAA by indoleacetamide hydrolase (iaah). Therefore, constructing the IAM pathway in bacteria and enhancing the activity of key enzymes in the reaction through structural modification is a viable strategy to increase indoleacetic acid production.

[0005] Studies have reported that recombinant Escherichia coli whole cells catalyze the production of IAA through the tryptophan pathway and the indoleacetamide (IAM) pathway. However, due to the low activity of the amide hydrolase, there will be more residual IAM generated by the reaction, resulting in a low yield of IAA. An amide hydrolase RsAD was obtained in the early stage of laboratory screening, but RsAD also has such a problem 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, the 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 Rhodococcus sp.The amide hydrolase RsAD was mutated 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. Wild-type amidohydrolase (RsAD), a member of the amidase family, hydrolyzes the amide bond of indoleacetamide to produce indoleacetic acid. However, due to the narrow substrate channel, the enzyme exhibits low reactivity with large substrates. By mutating the amino acid residues that form the gating structure of RsAD, the present invention significantly enhances its enzymatic activity, significantly increasing IAA production when using indoleacetamide (IAM) 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. 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 the mutant is shown in SEQ ID NO.4.

[0009] The second technical solution provided by the present invention is a recombinant vector or recombinant strain containing the above mutant encoding gene;

[0010] Furthermore, the expression plasmid used in the recombinant vector is pETDuet;

[0011] Furthermore, the host cell used by the recombinant strain is Escherichia coli BL21 (DE3).

[0012] The third technical solution provided by the present invention is the use of the recombinant vector or recombinant strain described in the second technical solution, especially in the production of the amidohydrolase mutant described in the first technical solution.

[0013] The fourth technical solution provided by the present invention is the use of the amidohydrolase mutant described in the first technical solution, particularly in the synthesis of indoleacetic acid, and more particularly in catalyzing the hydrolysis of the amide bond of indoleacetamide to produce indoleacetic acid.

[0014] Beneficial effects:

[0015] 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, its reaction activity for the large-volume substrate, indoleacetamide, is increased, thereby greatly improving the production efficiency of indoleacetic acid. DETAILED DESCRIPTION

[0016] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods well known to those skilled in the art. In addition, the embodiments should be understood to be illustrative rather than limiting the scope of the present invention, and the spirit 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 components and dosages in these embodiments, without departing from the spirit and scope of the present invention, also fall within the scope of protection of the present invention.

[0017] 1. The following definitions are adopted in the present invention:

[0018] Amino acid and DNA sequences are nomenclature 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.

[0019] In the present invention, RsAD refers to Rhodococcus sp. The wild-type amidohydrolase RsAD The gene encodes wild-type amidohydrolase, L447A represents an amidohydrolase mutant, RsAD-L447A It is the gene encoding the L447A mutant. The specific information is as follows:

[0020]

[0021] 2. Some of the determination methods used in the present invention are as follows

[0022] (1) Determination of biomass concentration (OD): Take the fermentation liquid and measure the absorbance at 600 nm.

[0023] (2) Analysis and determination of indoleacetic acid:

[0024] Assay method and system: Take 500 μL of 200mM potassium phosphate (pH 7.0) as a buffer, add 50 μL of amidohydrolase RsAD and 10mM indole-3-acetamide, and react at 37°C for 20 minutes. Then take 200 μL of the reaction solution and add 100 μL of 1M H3PO4 to terminate the reaction. Add methanol (300 μL) to dilute and mix to prepare a sample, and centrifuge to pass through a membrane (0.22 μm).

[0025] HPLC detection conditions for indoleacetic acid: High-performance liquid chromatography (HPLC) using an Agilent Technologies 1260 Infinity ZORBAX Eclopse Plus C18 column (5 μm, 4.6 × 150 mm). Mobile phases were: (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 was at 245 nm, column temperature was 30°C, and elution conditions were: 30% mobile phase A, 70% mobile phase B, isocratic elution.

[0026] 3. Some of the culture media or solutions used in the present invention are as follows:

[0027] (1) LB solid medium (g / L): peptone 10, sodium chloride 10, yeast powder 5, agar 15, pH 7.0;

[0028] (2) LB medium (g / L): peptone 10%, sodium chloride 10%, yeast powder 5%, pH 7.0.

[0029] 4. Some of the culture conditions used in the present invention are as follows:

[0030] (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.

[0031] (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 liquid to make the initial OD of the bacteria 600 When the value reaches 0.1, culture at 37℃ and 220 r / min. 600 When the pH reached 0.6-0.8, 0.2 mM IPTG was added to induce gene expression and culture was continued at 28°C for 8-9 h. The absorbance of the cells was then measured at 600 nm.

[0032] In the present invention, the amino acid sequence of the wild-type amidohydrolase RsAD is shown in SEQ ID NO.1:

[0033] MATIRPDDKAIDAAARHYGITLDKTARLEWPALIDGALGSYDVVDQLYADEATPPTTSREHAVPSASENPLSAWYVTTSIPPTSDGVLTGRRVAIKDNVTVAGVPMMNGSRTVEGFTPSRDATVVTRLLAAGATVAGKAVCEDLCFSGSSFTPASGPVRNPWDRQREAGGSSGGSAALVANGDVDFAIGGDQGGSIRIPAAFCGVVGHKPTFGLVPYTGAFPIERTIDHLGPITRTVHDAALMLSVIAGRDGNDPRQADSVEAGDYLSTLDSDVDGLRIGIVREGFGHAVSQPEVDDAVRAAAHSLTEIGCTVEEVNIPWHLHAFHIWNVIATDGGAYQMLDGNGYGMNAEGLYDPELMAHFASRRIQHADALSETVKLVALTGHHGITTLGGASYGKARNLVPLARAAYDTALRQFDVLVMPTLPYVASELPAKDVDRATFITKALGMIANTAPFDVTGHPSLSVPAGLVNGLPVGMMITGRHFDDATVLRVGRAFEKLRGAFPTPAERASNSAPQLSPAHHHHHH*

[0034] In the present invention, the amino acid sequence of the amide hydrolase mutant L447A is as shown in SEQ ID NO.3:

[0035] *

[0036] The present invention is further explained below through specific examples.

[0037] Example 1: Construction of an amino acid residue saturation mutant at position L447 in RsAD

[0038] Will come from Rhodococcus sp. The nucleic acid sequence of the amidohydrolase RsAD (SEQ ID NO. 2) was integrated into the petDuet plasmid and named petDuet-RsAD as a template for constructing mutants.

[0039] 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:

[0040] (1) Design of mutation primers

[0041] V-Fwd (5'-CAAAGCTGCGGGTATGATCGCTAACACCGCTCCGTT-3'),

[0042] V-Rev (5'-TCATACCCGCAGCTTTGGTGATGAAGGTAGCACGGTCAACG-3');

[0043] (2) PCR amplification conditions are: 98°C for 10 seconds, 55°C for 2 minutes, and 72°C for 45 seconds. Amplification was performed using the petDuet-RsAD plasmid vector as a template. The amplified product was digested with Dpn1 to remove the methylated template plasmid. The digested product was then used for recombinant ligation. The reaction product was then transformed into Escherichia coli DH5α and plated onto 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 had mutated to alanine GCG, the mutant was successfully constructed, and the plasmid petDuet-RsAD-L447A was obtained.

[0044] The other mutants and mutant plasmids were obtained using the same method as above, simply by replacing the primers.

[0045] Example 2: Preparation and expression of amidohydrolase RsAD and its mutants

[0046] The plasmid petDuet-RsAD, the mutant plasmid petDuet-RsAD-L447A, and other plasmids expressing mutants at the L447 locus were transformed into Escherichia coli BL21(DE3) cells. The cells were cultured in LB medium at 37°C to an OD600 of 0.6–0.8, followed by induction with 0.02% IPTG at 28°C for 8–9 hours. The harvested cells were suspended in buffer A (50 mM PBS, pH 7.4), disrupted by sonication, and centrifuged to remove cell debris.

[0047] The supernatant was purified using an AKTA protein purifier. During purification, the protein was linearly eluted for 30 minutes using elution buffer B (20 mM PBS, pH 7.4; 100 mM NaCl; 500 mM imidazole) on a nickel column (HisTrap™ HP5Ml). The target protein peak was collected. During desalting, the protein was eluted using a HiPrep™ 26 / 10 Desalting column using buffer A (50 mM PBS, pH 7.4). The collected protein peak was the desalted protein solution, and protein concentration was determined using the BCA assay.

[0048] The samples obtained in each stage were tested for protein expression by SDS polyacrylamide gel electrophoresis. The results showed that the protein was expressed normally before and after mutation, and the purified protein retained a high purity of the target protein and could be used for subsequent enzyme activity determination experiments.

[0049] Example 3: RsAD enzyme activity determination

[0050] To 500 μL of 200 mM potassium phosphate (pH 7.0) as a buffer solution, 50 μL of amidohydrolase (the purified enzyme obtained in Example 2: wild type or L447A mutant or other mutant) and a substrate indole-3-acetamide at a final concentration of 10 mM were added. The mixture was shaken at 37°C and 200 rpm for 20 min. Afterwards, 200 μL of the reaction solution was added to 100 μL of 1 M H 3 PO 4 to terminate the reaction. The mixture was diluted and mixed with 300 μL of methanol to prepare a sample. The sample was centrifuged and passed through a membrane (0.22 μm). Indoleacetic acid was then determined by HPLC using a ZORBAX Eclopse Plus C18 column (5 μm, 4.6 × 150 mm).

[0051] Enzyme activity was characterized by the amount of enzyme required to generate 1 μmol of IAA in a 1-minute reaction. The results of enzyme activity detection and calculation are shown in Table 1. It can be seen that multiple mutants at the L447 site all showed enhanced enzyme activity compared to the wild type, with mutant L447A showing the best effect, with its enzyme activity increased approximately 9-fold compared to the wild type, reaching 2739.53 μmol / min / mg.

[0052] Table 1

[0053]

[0054] Although the present invention has been disclosed 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 in the wild-type amidohydrolase shown in SEQ ID NO.

1. The amino acid sequence of the amidohydrolase 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, wherein The nucleotide sequence of the coding gene is shown in SEQ ID NO.

4.

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

5. The recombinant vector according to claim 4, wherein 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. The use of the amidohydrolase mutant according to claim 1, characterized in that: It is used in catalyzing the hydrolysis of the amide bond of indoleacetamide to generate indoleacetic acid.

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