A mutant of L-amino acid oxidase of Rhodococcus opaqueus and its application

Abstract

The present invention relates to a mutant of Rhodococcus opaqueus L-amino acid oxidase and its application, belonging to the field of bioengineering technology. The present invention overcomes the limitation of poor catalytic performance of wild-type RoLAAO and designs a mutant RoLAAO through maltoprotein engineering and combined mutation. Y226H / D227H / Y371L / A466C / W467A Its catalytic efficiency for five selected substrates is 9.2-82.8 times that of the wild type. It can use L-amino acids (L-Leu, L-Ile, L-Met, L-Val and L-Phe) as substrates to prepare a series of α-keto acids (α-ketoleucine, α-ketoisoleucine, α-ketomethionine, α-ketovaline and α-phenylpyruvate) on a large scale, with a corresponding yield of >101g / L and a conversion rate of >95%. The unit cell capacity for producing α-keto acids is 5.1-5.5g / g, laying the foundation for the industrial production of a variety of α-keto acids.

Description

Technical Field

[0001] The invention relates to a Rhodococcus opaque L-amino acid oxidase mutant and application thereof, belonging to the technical field of bioengineering. Background Art

[0002] α-keto acids are keto acids with a carboxyl group on the α-carbon atom. The combination of carboxyl and keto functional groups allows keto acid molecules to possess the salt- and ester-forming properties of carboxylic acids, as well as the hydroxylamine and hydrogenation-reduction reactions of ketones. In vivo, α-keto acids are crucial in amino acid metabolism. Due to their unique chemical and biological properties, α-keto acids are widely used in the pharmaceutical, food, feed, and cosmetic industries, particularly as important synthetic intermediates in the synthesis of organic compounds and biosynthesis.

[0003] Currently, the methods available for the industrial production of α-keto acids primarily include chemical and bioconversion methods. Bioconversion, however, outperforms chemical synthesis in terms of production safety, economic efficiency, and environmental protection, making it the preferred method for the industrial production of α-keto acids. Enzymatic conversion, a type of bioconversion method, utilizes L-amino acid oxidase (LAAO, EC1.4.3.2) to convert the substrate L-amino acid into α-keto acids. The oxidative deamination reaction of L-amino acids can be divided into two steps: first, the hydrogen on the amino acid Cα is transferred to FAD, converting the amino acid into an imino acid, which then decomposes into an α-keto acid and water. FADH2 is then oxidized by molecular oxygen to the reduced form FAD. Due to the low cost and short conversion cycle of the L-amino acid substrate used in the enzymatic conversion method, it has great industrial application value. However, the current large-scale production of α-keto acids using enzymatic conversion methods has the following drawbacks: (1) LAAO substrate spectra vary, requiring substrate spectrum determination for different types of LAAO; (2) LAAO with a broader substrate spectrum may have lower catalytic efficiency; and (3) LAAO-catalyzed α-keto acid synthesis is accompanied by the generation of H2O2. Therefore, it is urgent to address the issues of screening enzymes and LAAO catalytic efficiency in order to achieve large-scale application of enzymatic conversion methods for the production of α-keto acids.

[0004] Protein engineering is the most effective method to improve the properties of enzymes at the molecular level, such as expanding the substrate range, increasing enzyme activity and improving enzyme stability. Designing RoLAAO through protein engineering may solve the problem of its low catalytic efficiency. At present, the technology of using protein engineering to modify LAAO has made certain research progress. By simultaneously mutating multiple different key sites, the catalytic efficiency of LAAO for several specific substrates can be comprehensively improved. This multi-mutant modified RoLAAO has both a wider substrate spectrum and a catalytic efficiency far higher than that of the wild type. It is of great research value for its catalytic conversion of low-value L-amino acids to produce more expensive α-keto acids. However, a LAAO enzyme with both a wider substrate spectrum and a higher catalytic efficiency has not yet been found, which limits the large-scale preparation of α-keto acids. Summary of the Invention

[0005] The present invention provides a RoLAAO mutant that can efficiently prepare α-keto acids, and uses the RoLAAO mutant protein to catalyze L-Leu, L-Ile, L-Met, L-Val and L-Phe to prepare five α-keto acids (4-methyl-2-ketopentanoic acid (ketoleucine, α-KIC), 3-methyl-2-ketopentanoic acid (ketoisoleucine, α-KMV), 2-keto-4-methylthiobutyric acid (ketomethionine, α-KMTB), 3-methyl-2-ketobutyric acid (ketovaline, α-KIV) and 2-keto-3-phenylpropionic acid (phenylpyruvic acid, α-PPA)). The present invention also constructs a recombinant strain expressing the RoLAAO mutant. The recombinant strain has high production intensity and high catalytic stability when preparing α-keto acids, can reduce the amount of bacteria added, and greatly saves industrial production costs.

[0006] The present invention provides a Rhodococcus opacus L-amino acid oxidase RoLAAO mutant, the amino acid sequence of the Rhodococcus opacus L-amino acid oxidase RoLAAO is shown in SEQ ID NO. 1, and the nucleotide sequence encoding the L-amino acid oxidase RoLAAO is shown in SEQ ID NO. 2. Furthermore, the L-amino acid oxidase RoLAAO is not limited to Rhodococcus opacus sources.

[0007] In one embodiment of the present invention, the mutant is obtained by mutating the tyrosine at position 226 to histidine relative to the RoLAAO parent. Y226H .

[0008] In one embodiment of the present invention, the mutant is obtained by mutating the 227th aspartic acid to histidine relative to the RoLAAO parent. D227H .

[0009] In one embodiment of the present invention, the mutant is obtained by mutating the 371st tyrosine to leucine relative to the RoLAAO parent. Y371L .

[0010] In one embodiment of the present invention, the mutant is obtained by mutating the 466th alanine to cysteine ​​relative to the RoLAAO parent. A466C .

[0011] In one embodiment of the present invention, the mutant is obtained by mutating the 467th tryptophan to alanine relative to the RoLAAO parent. W467A .

[0012] In one embodiment of the present invention, the mutant is obtained by mutating the tyrosine at position 226 to histidine and the aspartic acid at position 227 to histidine relative to the parent RoLAAO. Y226H / D227H .

[0013] In one embodiment of the present invention, the mutant is obtained by mutating the 371st tyrosine to leucine, the 466th alanine to cysteine, and the 467th tryptophan to alanine relative to the RoLAAO parent. Y371L / A466C / W467A .

[0014] In one embodiment of the present invention, the mutant is obtained by mutating the tyrosine at position 226 to histidine, the aspartic acid at position 227 to histidine, the tyrosine at position 371 to leucine, the alanine at position 466 to cysteine, and the tryptophan at position 467 to alanine relative to the RoLAAO parent. Y226H / D227H / Y371L / A466C / W467A .

[0015] In one embodiment of the present invention, the mutant RoLAAO Y226H 、RoLAAO D227H 、RoLAAO Y371L 、RoLAAO A466C 、RoLAAO W467A 、RoLAAO Y226H / D227H 、RoLAAO Y371L / A466C / W467A 、RoLAAO Y226H / D227H / Y371L / A466C / W467AThe amino acid sequences are shown as SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, and SEQ ID NO.17, respectively, and the nucleotide sequences encoding the mutants are shown as SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, and SEQ ID NO.18, respectively.

[0016] In one embodiment of the present invention, the mutant connects the MBP protein and the GS-Linker fragment, the amino acid sequence of the MBP protein and the GS-Linker fragment is shown in SEQ ID NO.19, and the nucleotide sequence of the gene encoding the MBP protein and the GS-Linker fragment is shown in SEQ ID NO.20.

[0017] In one embodiment of the present invention, when the MBP protein connected with the GS-Linker fragment is connected to the L-amino acid oxidase RoLAAO or the L-amino acid oxidase RoLAAO mutant, the first methionine of the L-amino acid oxidase RoLAAO is removed.

[0018] In the present invention, removing the first methionine of L-amino acid oxidase RoLAAO and simultaneously linking the MBP protein with the GS-Linker fragment is a conventional means to improve protein soluble expression and does not affect the activity of the mutant itself.

[0019] The present invention provides a method for obtaining the RoLAAO mutant, comprising the following steps:

[0020] (1) Determine the mutation site based on the amino acid sequence of the Rhodococcus opaque L-amino acid oxidase RoLAAO; design mutation primers for site-directed mutagenesis, and perform site-directed mutagenesis using a vector carrying the MBP tag and the RoLAAO gene as a template; and construct a plasmid containing the mutant gene.

[0021] (2) transforming the plasmid containing the mutant gene into host cells;

[0022] (3) Select positive clones for fermentation culture and purify the L-amino acid oxidase RoLAAO mutant.

[0023] In one embodiment of the present invention, the host cell is a bacterium.

[0024] In one embodiment of the present invention, the host cell is Escherichia coli.

[0025] The present invention provides a method comprising pET28a-MBP-RoLAAO Mutant Mutant strain BL21-MBP-RoLAAO containing the recombinant vector Mutant The mutant strain is based on E. coli BL21 (DE3) as the host and carries pET28a-MBP-RoLAAO Mutant , the pET28a-MBP-RoLAAO Mutant It is obtained by mutating one or more sites of RoLAAO through whole-plasmid PCR using pET28a-MBP-RoLAAO as a template.

[0026] The present invention provides a method for utilizing BL21-MBP-RoLAAO Mutant A method for preparing α-keto acid by a mutant strain, wherein the method uses L-amino acid as a reaction substrate, and BL21-MBP-RoLAAO Mutant The mutant strain was added to the reaction solution, and the reaction was carried out at pH 7.5-9.0, 25-35° C., 2000-3000 U / mL of catalase, and 180-240 rpm for 16-24 hours.

[0027] In one embodiment of the present invention, the BL21-MBP-RoLAAO Mutant The amount of mutant strain added was a final concentration of 10 to 30 g / L.

[0028] The present invention provides a BL21-MBP-RoLAAO Mutant Application of mutant strains in the preparation of a series of α-keto acids.

[0029] Beneficial effects

[0030] The present invention provides a mutant RoLAAO of Rhodococcus opaque L-amino acid oxidase Y226H / Y227H / Y371L / A466C / W467A , can efficiently convert a series of L-amino acids (L-Leu, L-Ile, L-Met, L-Val or L-Phe) to produce a series of α-keto acids. Compared with the wild-type enzyme, the k cat Up-regulated by 4.4 times, K m Downgraded by 3.7 times, k cat / K m Upregulated 16.4-fold; k catalyzes L-Ile to produce ketoisoleucine cat Upregulated 7.5-fold, K m Downgraded by 5.3 times, k cat / K m Upregulated 39.9-fold; catalyzes L-Met to produce ketomethioninecat Increased by 3.1 times, K m Downgraded by 2.9 times, k cat / K m Upregulated 9.2-fold; catalyzes the production of ketovaline from L-Val cat Increased by 13.1 times, K m Downgraded by 6.3 times, k cat / K m Upregulated 82.8-fold; k catalyzes L-Phe to produce phenylpyruvate cat Upregulated 11.5-fold, K m Downgraded by 2.4 times, k cat / K m Increased by 28.1 times.

[0031] The mutant strain BL21-MBP-RoLAAO obtained by the present invention Y226H / Y227H / Y371L / A466C / W467A Five amino acid substrates can be efficiently converted. The addition amount of mutant bacteria is 20 g / L, the addition amount of substrate L-Leu, L-Ile, L-Met, L-Val or L-Phe is 110 g / L, the conversion cycle is 24 hours, and the yields of corresponding α-keto acids (ketoleucine, ketoisoleucine, ketomethionine, ketovaline, phenylpyruvate) are all greater than 101 g / L, and the substrate molar conversion rates are all higher than 95%.

[0032] The present invention provides a method for producing a oxidative enzyme using L-amino acid as substrate and a mutant RoLAAO of L-amino acid oxidase of Rhodococcus opacus. Y226H / Y227H / Y371L / A466C / W467A or mutant strain BL21-MBP-RoLAAO Y226H / Y227H / Y371L / A466C / W467A The method for preparing a series of α-keto acids using hydroxybenzoic acid as a catalyst effectively reduces production costs, and only uses water as a catalytic medium in the reaction, and has the advantages of mild reaction conditions, simple operation, high yield, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 :RoLAAO, MBP-RoLAAO and mutant RoLAAO Y226H / D227H / Y371L / A466C / W467A SDS-PAGE results, lanes 1-3 are BL21-pET28a-RoLAAO bacteria, fermentation supernatant and precipitate; lanes 4-6 are BL21-MBP-RoLAAO bacteria, supernatant and precipitate; lanes 7-9 are BL21-MBP-RoLAAO Y226H / D227H / Y371L / A466C / W467A Bacteria, fermentation liquid supernatant and precipitate;

[0034] Figure 2 :According to the 2,4-dinitrophenylhydrazine colorimetric method, the OD 520 The standard curve of α-keto acid standards was drawn based on the absorbance values ​​measured at ;

[0035] (A): Ketoleucine standard curve; (B): Ketoisoleucine standard curve; (C): Ketomethionine standard curve; (D): Ketovaline standard curve; (E): Phenylpyruvate standard curve; (F): Pyruvate standard curve;

[0036] Figure 3 : HPLC chromatograms of five representative substrate L-amino acids (L-Leu, L-Ile, L-Met, L-Val, L-Phe) and their corresponding α-keto acids;

[0037] (A-1): L-Leu standard sample; (A-2): Spectrum of the remaining amount of substrate in the L-Leu conversion supernatant; (A-3): Ketoleucine standard sample; (A-4): L-Leu conversion supernatant;

[0038] (B-1): L-Ile standard sample; (B-2): Spectrum of the remaining amount of substrate in the supernatant of L-Ile conversion; (B-3): Ketoisoleucine standard sample; (B-4): L-methionine conversion supernatant;

[0039] (C-1): L-Met standard; (E-2): Spectrum of the remaining amount of substrate in the L-Met conversion supernatant; (E-3): Ketomethionine standard; (E-4): L-Met conversion supernatant;

[0040] (D-1): L-Val standard sample; (D-2): L-Val conversion supernatant substrate residual amount spectrum; (D-3): Ketovaline standard sample; (D-4): L-Val conversion supernatant;

[0041] (E-1): L-Phe standard sample; (E-2): Spectrum of the remaining amount of substrate in the supernatant of L-phenylalanine conversion; (E-3): Phenylpyruvic acid standard sample; (E-4): L-phenylalanine conversion supernatant. DETAILED DESCRIPTION

[0042] Gene source: The biological enzyme RoLAAO gene involved in this patent is derived from Rhodococcus opacus. Based on the LAAO gene sequence from Rhodococcus opacus disclosed in the NCBI database, Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize the gene and perform codon optimization. The resulting gene was named RoLAAO, and its nucleotide sequence is shown in SEQ ID NO.2. The amino acid sequence of the L-amino acid oxidase RoLAAO encoded by the gene is shown in SEQ ID NO.1.

[0043] The pET28a(+) plasmid was purchased from Novagen (Madison, WI, USA). The host cell used was E. coli BL21(DE3). Restriction enzymes, the ClonExpress II One-Step Cloning Kit, and Primer Star Max were purchased from TaKaRa (Dalian, China). Standards were purchased from SIGMA. RoLAAO mutants were all molecularly engineered, and all other reagents were commercially available.

[0044] Prepare LB medium: 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, and sterilize at 121°C for 20 min.

[0045] Prepare TB fermentation medium: tryptone 12 g / L, Angel Yeast FM 802 24 g / L, glycerol 4 g / L, KH2PO4 2.31 g / L and K2HPO4 12.31 g / L.

[0046] Prepare pH 6.0 sodium phosphate buffer: 20 mmol / L Tris-HCl buffer. For the specific formula, see the "Industrial Microbiology Experimental Technology Manual" (China Light Industry Press, edited by Zhuge Jian).

[0047] Preparation of standard curves for five α-keto acids: Weigh 10 mg each of ketoleucine (α-KIC), ketoisoleucine (α-KMV), ketovaline (α-KIV), ketomethionine (α-KMTB), phenylpyruvic acid (α-PPA), and pyruvic acid (α-PA), and dissolve them in 10 mL of ddH2O. Prepare the concentration gradient of each standard according to Table 6. After thorough mixing, take 75 μL of the standard, add 150 μL of 20 mM 2,4-dinitrophenylhydrazine, and let it stand in the dark for 15 minutes. Add 3 mL of 0.8 M NaOH solution, and then transfer 200 μL to the enzyme label strip. Use an enzyme reader to detect the absorbance at a wavelength of 520 nM. According to the above method, the standard curves of the five α-keto acids (ketoleucine, ketoisoleucine, ketovaline, ketomethionine and phenylpyruvic acid) were prepared. The corresponding standard curves can be seen. Figure 2 .

[0048] Table 1 Preparation of standard products

[0049]

[0050] Determination of α-keto acids by 2,4-dinitrophenylhydrazine colorimetric method and determination of L-amino acids and α-keto acids by HPLC: For detailed steps, please refer to the reference ChemCatChem, 2021, 13(21): 4557-4566 (Enhanced catalytic efficiency of L-amino acid deaminase achieved by a shorter hydride transfer distance)

[0051] Relative activity determination method: The relative activity was determined using the 2,4-dinitrophenylhydrazine colorimetric method. The relative activity was calculated based on the amount of α-keto acid produced by different L-amino acid substrates under the same catalytic conditions. The relative activity of the enzyme corresponding to the substrate group with the highest catalytic activity of the parent enzyme was defined as 100%, and the relative activities of other enzymes were compared with this.

[0052] Example 1 Construction of a recombinant strain producing RoLAAO based on MBP-tagged protein

[0053] 1. Construction, induced expression, and SDS-PAGE verification of the RoLAAO parent strain

[0054] The RoLAAO gene was ligated to the pET-28a vector using BamHI and HindIII as restriction sites, and then transformed into E. coli BL21 (DE3). The resulting recombinant bacteria was named BL21-pET-28a-RoLAAO.

[0055] The recombinant bacteria BL21-pET-28a-RoLAAO were inoculated into LB seed medium and cultured at 200 rpm and 37 °C for 8-12 h. 2% inoculum was inoculated into shake flask fermentation medium and cultured at 220 rpm and 37 °C until OD 600 = about 0.6 to 0.8, add IPTG with a final concentration of 0.2 mM for induction, and the induction conditions are 200 rpm, 16°C for 16 to 20 h.

[0056] The cells in the fermentation broth were collected and ultrasonically disrupted. The suspension after disruption was centrifuged and SDS-PAGE was performed on BL21-pET-28a-RoLAAO, the supernatant of the disruption broth, and the precipitate of the disruption broth. The results showed that most of the RoLAAO protein was in the precipitate and the soluble expression was very poor ( Figure 1 ), and subsequently introduced the existing maltose binding protein in the laboratory to promote protein soluble expression to enhance the expression of the parent enzyme RoLAAO in the supernatant, thereby improving the catalytic performance of RoLAAO on L-amino acid substrates.

[0057] 2. Construction of MBP-tagged protein and RoLAAO co-expression strain

[0058] Using the plasmid containing the MBP gene as a template (plasmid information is disclosed in Rational design ofphospholipase D to improve the transphosphatidylation activity forphosphatidylserine synthesis[J].Journal of Agricultural and Food Chemistry, 2022, 70(22):6709-6718.), PCR experiments were performed using MBP-S and MBP-A shown in Table 2 as primers (the reaction system is shown in Table 3). The PCR reaction conditions were: ①98℃30s; ②98℃10s; ③55℃15s; ④72℃2000bp / min; ⑤cycle ②~④34 times; ⑥72℃10min; ⑦keep warm at 12℃. The PCR product was incubated at 37°C for 30-45 min to digest the plasmid template (digestion system: DpnI quick 0.3 μL, 8.7 μL of the above PCR product, 1 μL of 10×T Buffer) to obtain the MBP-GS linker fragment.

[0059] PCR experiments were performed using the pET-28a-RoLAAO vector as a template and ZT-MBP-RoLAAO-S and ZT-MBP-RoLAAO-A shown in Table 2 as primers (the reaction system is shown in Table 3). The PCR reaction conditions were the same as those for the above-mentioned method for constructing the MBP-GS linker fragment. The plasmid template was digested using the method described above for constructing the MBP-GS linker fragment to obtain a linear fragment of the pET-28a-RoLAAO vector.

[0060] Table 2 Primer sequences for constructing E. coli-pET-28a-MBP-RoLAAO

[0061]

[0062]

[0063] Table 3 Primer Star Max system table

[0064]

[0065] The MBP-GS linker fragment was ligated with the linear fragment of the pET-28a-RoLAAO vector using the ClonExpress II One-Step Cloning Kit to obtain the MBP-tagged vector pET-28a-MBP-RoLAAO. The ClonExpress II One-Step Cloning Kit is shown in Table 4. The reaction conditions were 37°C for 30-45 minutes.

[0066] Table 4 ClonExpress II One Step Cloning Kit Recombination System

[0067]

[0068] The above pET-28a-MBP-RoLAAO vector was introduced into E. coli BL21 (DE3) competent cells by chemical transformation. The specific steps of the chemical transformation method are as follows:

[0069] (1) Introduce 10 μL of homologous recombination product into 100 μL of E. coli BL21 (DE3) competent cells;

[0070] (2) Ice bath for 15-30 minutes;

[0071] (3) Heat shock in a 42°C water bath for 90 seconds, then quickly place in ice and let stand for 3-5 minutes;

[0072] (4) Add 800 μL of resistance-free LB medium, mix well, and incubate at 37°C, 200 rpm for 45 min-1 h;

[0073] (5) Centrifuge at 4000 rpm for 3 min to collect the bacteria;

[0074] (6) Remove the supernatant and mix the remaining 100-200 μL by pipetting. Apply the mixture to a plate containing 0.05 mg / mL kanamycin resistance and incubate at 37°C for about 12 h.

[0075] (7) Single clones were picked and placed in LB containing 0.05 mg / mL kanamycin resistance. After constant culture at 200 rpm and 37°C for 12 h, they were sent to the company for sequencing. The ones with correct sequencing were positive transformants and named BL21-MBP-RoLAAO.

[0076] Example 2 Construction of mutant strains

[0077] 1. Construction of single mutant strains

[0078] RoLAAO was designed using the pET-28a-MBP-RoLAAO vector as a template.Y226H 、RoLAAO D227H 、RoLAAO Y371L 、RoLAAO A466C , and RoLAAO W467A The mutants were constructed by whole plasmid PCR using primers targeting the mutation sites. The primers used are shown in SEQ ID NO. 25-34 in Table 5. The PCR system and steps were the same as those in Example 1. After the whole plasmid PCR was completed, the PCR product was digested with DpnIquick digestion enzyme and then transformed into E. coli BL21 (DE3) competent cells. After culture on kanamycin-resistant plates, positive transformants were selected for sequencing verification to obtain BL21-MBP-RoLAAO. Y226H BL21-MBP-RoLAAO D227H BL21-MBP-RoLAAO Y371L BL21-MBP-RoLAAO A466C , and BL21-MBP-RoLAAO W467A Mutant strains.

[0079] Table 5 Mutation primer sequences

[0080]

[0081] 2. Construction of double, triple, and pentamutant mutant strains

[0082] (1) In the mutant BL21-MBP-RoLAAO Y226H Based on pET-28a-MBP-RoLAAO Y226H As a template, the double mutant was constructed by whole plasmid PCR using the mutation primers D227H-S and D227H-A. The primers used are shown in SEQ ID NO.27 and SEQ ID NO.28 in Table 5. The other construction methods were the same as those for the single mutant, and the double mutant BL21-MBP-RoLAAO was obtained. Y226H / D227H strains.

[0083] (2) In the mutant BL21-MBP-RoLAAO W467A Based on pET-28a-MBP-RoLAAO W467AAs templates, double mutants were constructed by whole plasmid PCR using mutant primers Y371-S, Y371-A and A466-S, A466-A, respectively. The primers used are shown in SEQ ID NO.29, SEQ ID NO.30 and SEQ ID NO.31, SEQ ID NO.32. The other construction methods were the same as those for the single mutants, and the double mutant BL21-MBP-RoLAAO was prepared. Y371L / W467A BL21-MBP-RoLAAO A466C / W467A strains.

[0084] (3) In the double mutant BL21-MBP-RoLAAO Y371L / W467A Based on pET-28a-MBP-RoLAAO Y371L / W467A As a template, the triple mutant was constructed by whole plasmid PCR using the mutation primers A466-S and A466-A. The primers used are shown in SEQ ID NO.31 and SEQ ID NO.32. The other construction methods were the same as those for the single mutant. The triple mutant BL21-MBP-RoLAAO was prepared. Y371L / A466C / W467A strains.

[0085] (4) In the triple mutant BL21-MBP-RoLAAO Y371L / A466C / W467A Based on pET-28a-MBP-RoLAAO Y371L / A466C / W467A As a template, the five-mutant mutant was constructed by whole-plasmid PCR using the mutation primers Y226HD227H-S and Y226HD227H-A. The primers used are shown in SEQ ID NO.35 and SEQ ID NO.36. The other construction methods were the same as those for the single-mutant mutant. The five-mutant mutant BL21-MBP-RoLAAO was prepared. Y226H / D227H / Y371L / A466C / W467A strains.

[0086] Example 3 Screening of mutants

[0087] 1. Determination of relative activity of L-amino acids catalyzed by RoLAAO parent enzyme

[0088] The RoLAAO parent enzymes described below are all enzymes expressed by the parent strain BL21-pET-28a-RoLAAO without MBP tag modification. The relative activity of catalytic L-amino acids was determined using BL21-pET-28a-RoLAAO bacteria. The reaction system contained 20 g / L of bacteria, 100 g / L of L-amino acid substrate (L-Leu, L-Ile, L-Met, L-Val, L-Phe or L-Ala), the reaction temperature was 30 ° C, the pH was 8.0, the commercial catalase addition amount was 2200 U / mL, the conversion time was 24 h, and the rotation speed was 220 rpm. After the conversion reaction, the activity was detected using the 2,4-dinitrophenylhydrazine colorimetric method. A portion of the diluted conversion solution was added to 20% trichloroacetic acid; after centrifugation at 12,000 rpm for 10 minutes, 500 μL of the supernatant was taken and added to 100 μL of 20 mM 2,4-dinitrophenylhydrazine solution. The solution was allowed to stand for 15 minutes, and 2 mL of 0.8 M NaOH solution was added. The mixture was mixed and allowed to stand for 10 minutes. The OD was measured at 520 nm. 520 Value. Figure 2 The standard curve was used to calculate the corresponding α-keto acid production. The results showed that the RoLAAO parent enzyme had the strongest activity in catalyzing L-Ala, and its relative activity was defined as 100%. In comparison, the relative activities of the above parent enzymes for L-Leu, L-Ile, L-Met, L-Val and L-Phe were 68.1%, 45.2%, 72.4%, 32.3% and 53.1%, respectively.

[0089] 2. Determination of relative activity of mutant enzymes in catalytic L-amino acid reactions

[0090] The correctly sequenced mutant strain BL21-MBP-RoLAAO Mutant Inoculate into LB seed medium, culture at 200 rpm and 37 ° C for 8-12 h, inoculate into shake flask fermentation medium at 2% inoculum, and culture at 220 rpm and 37 ° C until OD 600 = 0.6-0.8, IPTG was added to a final concentration of 0.2 mM for induction at 200 rpm and 16°C for 16-20 h. The induced cells were transformed with five substrates (L-Leu, L-Ile, L-Met, L-Val, or L-Phe).

[0091] The mutant strain was subjected to the same transformation method as the above-mentioned RoLAAO parent strain catalyzing the conversion of L-amino acids to five substrates (L-Leu, L-Ile, L-Met, L-Val or L-Phe). After the conversion reaction, the relative activity of L-amino acids catalyzed by the above-mentioned RoLAAO parent strain was determined by 2,4-dinitrophenylhydrazine (DNP) colorimetric method, and the activity was determined according to the above-mentioned RoLAAO parent strain catalytic activity. Figure 2 The standard curve of the corresponding α-keto acid production was calculated, and the results are shown in Table 6. The results show that the mutant RoLAAO Y226H / D227H / Y371L / A466C / W467A The catalytic effect on the five substrates was the best.

[0092] Table 6 Shake flask test results of representative mutants transforming five substrate L-amino acids to produce corresponding α-keto acids

[0093]

[0094] Example 4: Expression and purification of parent enzyme and mutant enzyme

[0095] The parent strain BL21-pET-28a-RoLAAO was recombined with the mutant strain BL21-MBP-RoLAAO prepared in Example 2 Mutant Positive transformants were inoculated into LB medium and cultured at 37°C until OD 600 When the pH is 0.6-0.8, add IPTG to a final concentration of 0.2 mM to induce enzyme expression. The induction temperature is 16°C and the induction time is 18-22 hours to obtain a fermentation broth. The fermentation broth is centrifuged at 8000 rpm at 4°C for 8 minutes to collect the bacteria. Add 10 mL of binding solution A (20 mM Tris-HCl, 0.5 mM NaCl, 20 mM imidazole, pH adjusted to 8.0 with HCl) to fully resuspend the bacteria. Then place the centrifuge tube in an ice bath and place it in an ultrasonic cell disruptor. The ultrasonic disruption conditions are: 4 seconds on time, 6 seconds interval, for a total of 15 minutes. The resulting disrupted liquid is subjected to low-temperature high-speed centrifugation at 10000 rpm at 4°C for 30 minutes to obtain a crude enzyme solution. Filter through a 0.22 μm microporous filter membrane and set aside.

[0096] Prepare the nickel ion affinity chromatography column. First, use a constant flow pump at 4°C to pump ultrapure water into the column to rinse the column (about 6 to 12 column volumes), then balance the column environment with 10 mL of binding solution A. When the effluent at the bottom of the column is consistent with the pH value of the low-salt concentration buffer pumped into the column (about 5 column volumes of buffer are required), add the obtained membrane-permeable crude enzyme solution to the column. First, rinse the impurities with binding solution A to the baseline equilibrium, and then elute with eluent B (20mM Tris-HCl, 0.5mMNaCl, 500mM imidazole). Collect the eluate at the absorption peak, determine the enzyme activity, and obtain the target protein that has achieved electrophoretic purity.

[0097] Example 5: Determination of kinetic parameters of the parent enzyme and the best mutant

[0098] In order to evaluate the mutants, the present invention used the DNT colorimetric method to determine the RoLAAO parent enzyme and the best mutant RoLAAO Y226H / Y227H / Y371L / A466C / W467AKinetic parameters at 30°C. The test process is as follows: 250 μL of the enzyme solution obtained in Example 4 was mixed with 2.5 mL of L-amino acid (L-Leu, L-Ile, L-Met, L-Val or L-Phe) with a concentration gradient of 0-100 mM, and the reaction was shaken at 30°C for 10 minutes. 20% trichloroacetic acid was added to terminate the enzyme reaction; 500 μL of supernatant was centrifuged and added with 100 μL of 20 mM 2,4-dinitrophenylhydrazine solution, and the mixture was allowed to stand for 15 minutes. 2 mL of 0.8 M NaOH solution was added, the mixture was mixed and allowed to stand for 10 minutes, and the OD was measured at 520 nm. 520 According to the measured OD 520 The value was substituted into the α-keto acid standard curve to calculate the concentration of the corresponding α-keto acid (ketoleucine, ketoisoleucine, ketomethionine, ketovaline and phenylpyruvic acid), and the corresponding enzymatic parameter k was calculated. cat , K m and k cat / K m value.

[0099] As shown in Table 7, the best mutant RoLAAO Y226H / Y227H / Y371L / A466C / W467A Compared with RoLAAO, the k cat Up-regulated by 4.4 times, K m Downgraded by 3.7 times, k cat / K m Upregulated 16.4-fold; k catalyzes L-Ile to produce ketoisoleucine cat Upregulated 7.5-fold, K m Downgraded by 5.3 times, k cat / K m Upregulated 39.9-fold; catalyzes L-Met to produce ketomethionine cat Increased by 3.1 times, K m Downgraded by 2.9 times, k cat / K m Upregulated 9.2-fold; catalyzes the production of ketovaline from L-Val cat Increased by 13.1 times, K m Downgraded by 6.3 times, k cat / K m Upregulated 82.8-fold; k catalyzes L-Phe to produce phenylpyruvate cat Upregulated 11.5-fold, K m Downgraded by 2.4 times, k cat / K m Increased by 28.1 times.

[0100] Table 7 RoLAAO parent enzyme and best mutant RoLAAO Y226H / Y227H / Y371L / A466C / W467A Kinetic parameters

[0101]

[0102] Example 6: Scaled production of a series of α-keto acids using the best mutants

[0103] To further verify the catalytic performance of the best mutant, we used RoLAAO Y226H / Y227H / Y371L / A466C / W467A Under the conversion conditions in Example 3, five substrate L-amino acids (L-Leu, L-Ile, L-Met, L-Val, or L-Phe) were prepared on a large scale in a shake flask to generate the corresponding α-keto acids. The conversion results showed that with 110 g / L L-amino acid (L-Leu, L-Ile, L-Met, L-Val, or L-Phe) as the substrate, the wet cell addition amount was 20 g / L, and the conversion cycle was 24 h. As shown in Table 8, the yields of the corresponding α-keto acids (ketoleucine, ketoisoleucine, ketomethionine, ketovaline, and phenylpyruvate) were all >101 g / L, and the substrate molar conversion rates were all higher than 95%.

[0104] Table 8 Best mutant RoLAAO Y226H / Y227H / Y371L / A466C / W467A Scaled Preparation Summary Table

[0105]

[0106] Comparative Example 1: Determination of kinetic parameters of other representative mutants

[0107] Referring to the determination method in Example 5, the kinetic parameters of other representative mutants for L-Leu, L-Ile, L-Met, L-Val or L-Phe were determined, including the double mutant RoLAAO Y226H / D227H and triple mutant RoLAAO Y371L / A466C / W467A Enzyme parameter k cat , K m and k cat / K m For details, see Table 9.

[0108] Table 9 Double mutant RoLAAO Y226H / D227H and triple mutant RoLAAO Y371L / A466C / W467A Kinetic parameters

[0109]

[0110]

[0111] Comparative Example 2: Parent enzyme converts 110 g / L substrate to produce a series of α-keto acids

[0112] The RoLAAO parent enzyme was used to convert five substrate L-amino acids (L-Leu, L-Ile, L-Met, L-Val or L-Phe) into corresponding α-keto acids in a shake flask under the conversion conditions in Example 3. The conversion results showed that when 110 g / L L-amino acid (L-Leu, L-Ile, L-Met, L-Val or L-Phe) was used as the substrate, the wet bacteria addition amount was 20 g / L, and the conversion cycle was 24 h, a large amount of substrate residue was found, and the conversion rate was generally low. The corresponding α-keto acids (ketoleucine, ketoisoleucine, ketomethionine, ketovaline, phenylpyruvate) yields and substrate molar conversion rates can be seen in Table 10.

[0113] Table 10 Summary of large-scale preparation of α-keto acids by RoLAAO parent enzyme

[0114]

[0115] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A Rhodococcus opaque L-amino acid oxidase Ro LAAO mutant, characterized in that The mutant is one of the following: (a) The tyrosine at position 226 of the amino acid sequence shown in SEQ ID NO. 1 was mutated to histidine and the aspartic acid at position 227 was mutated to histidine to obtain a mutant Ro LAAO Y226H / D227H ; (b) mutating the tyrosine at position 226 of the amino acid sequence shown in SEQ ID NO. 1 to histidine, the aspartic acid at position 227 to histidine, the tyrosine at position 371 to leucine, the alanine at position 466 to cysteine, and the tryptophan at position 467 to alanine to obtain a mutant Ro LAAO Y226H / D227H / Y371L / A466C / W467A .

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

3. A vector carrying the gene according to claim 2.

4. The carrier according to claim 3, characterized in that A gene carrying the maltose binding protein MBP gene and the mutant according to claim 2, wherein the MBP gene and the mutant gene are connected via a GS-Linker.

5. A recombinant microbial cell or recombinant bacterium containing the vector according to claim 4.

6. A method for preparing an α-keto acid, characterized in that: The method uses L-amino acid as substrate, The mutant according to claim 1 or the recombinant bacterium according to claim 5 is used as a catalyst; The α-keto acid is 4-methyl-2-ketopentanoic acid, 3-methyl-2-ketopentanoic acid, α-keto-γ-methylthiobutyric acid, 3-methyl-2-ketobutyric acid or 2-keto-3-phenylpropionic acid, and the substrate is L-Leu, L-Ile, L-Met, L-Val or L-Phe.

7. The method for preparing α-keto acid according to claim 6, characterized in that: The mutant was added at a final concentration of 10-30 g / L, and the reaction was carried out at pH 7.5-9.0, 25-35°C, 2000-3000 U / mL of catalase, and 180-240 rpm for 16-24 h.

8. Use of the mutant according to claim 1 or the recombinant bacterium according to claim 5 in preparing α-keto acid using L-amino acid as substrate, characterized in that: The α-keto acid is 4-methyl-2-ketopentanoic acid, 3-methyl-2-ketopentanoic acid, α-keto-γ-methylthiobutyric acid, 3-methyl-2-ketobutyric acid or 2-keto-3-phenylpropionic acid, and the substrate is L-Leu, L-Ile, L-Met, L-Val or L-Phe.

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