CN109576234B

CN109576234B - Leucine-5-hydroxylase mutant and application thereof

Info

Publication number: CN109576234B
Application number: CN201811596777.3A
Authority: CN
Inventors: 秦慧民; 路福平; 孙登岳; 李玉; 毛淑红
Original assignee: Tianjin University of Science and Technology
Current assignee: Tiankeda Tianjin Science Park Co ltd
Priority date: 2018-12-26
Filing date: 2018-12-26
Publication date: 2021-05-07
Anticipated expiration: 2038-12-26
Also published as: CN109576234A

Abstract

The invention belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to a leucine-5-hydroxylase mutant with improved activity and application thereof. The invention obtains the leucine-5-hydroxylase coding gene from Nostoc punctiform NIES-2108 by a molecular biology means, obtains the gene of a coding mutant by adopting reverse PCR to carry out site-directed mutagenesis, and then obtains the mutant V77A of the leucine-5-hydroxylase by carrying out heterologous expression on the gene of the coding mutant. When leucine and methionine are used as substrates, the relative enzyme activity of the mutant is respectively increased by 69.53 percent and 23.26 percent compared with that of the wild type. The screened variant with improved activity can more efficiently hydroxylate the remote-end carbon position 5 of leucine, can catalyze methionine to generate methionine sulfoxide, and provides wide prospect for enzymatic catalytic production of industrial drug intermediates.

Description

Leucine-5-hydroxylase mutant and application thereof

The technical field is as follows:

the invention belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to a leucine-5-hydroxylase mutant with improved activity and application thereof.

Background art:

hydroxylated amino acids are widely used in the synthesis of multifunctional biomacromolecules, medicines and fine chemicals because they can be used as chiral precursors, intermediates and end products in the synthesis of various chemical materials and medical drugs. The hydroxy amino acid can be used as an intermediate for drug synthesis in the medical field, and also can be widely used in a plurality of fields such as feed additives, food fortifiers, spices and the like, for example, the L-beta-hydroxy alanine can be used as an animal feed additive besides amino acid weight-reducing beverage and sports beverage in the food field; l-hydroxyproline is often used as a nutrition enhancer and a flavoring agent in fruit juice, nutritional beverages and cold beverages; L-beta-hydroxy-L-amino-acid is also an additive, which is commonly used in the food and feed fields, and in addition, 5-hydroxy-L-amino-acid is an important synthetic precursor of 4-methyl proline and is important in the synthesis of antibiotic light grisein.

At present, the synthetic methods of the hydroxylated leucine mainly comprise chemical synthesis and enzymatic biosynthesis. Microbial conversion is catalyzed by the relevant enzyme systems in the body of the microorganism to produce the desired product. The hydroxylation of the far-end carbon position of the amino acid is realized, and the chemical process has a plurality of defects, including complex process route, harsh reaction conditions, more byproducts, low yield and the like. Enzymatic catalysis provides a more efficient strategy for the development of many drug intermediates due to its important advantages of strong specificity, mild reaction conditions, high yield, etc.

Nostoc punctiform NIES-2108 derived leucine-5-hydroxylase (LEH, Genbank serial number RCJ32143.1), or leucine-5-dioxygenase, has the function of oxidizing and dehydrogenating the carbon position of leucine 5 to form hydroxyl, is a key enzyme for realizing the remote C-H functionalization of leucine, and can perform sulfonation reaction on methionine to generate methionine sulfoxide. It contains 795bp open reading frame and encodes 265 amino acids.

The wild leucine-5-hydroxylase gene LEH is transformed into E.coli BL21(DE3) for heterologous expression, and the supernatant is purified by Ni affinity chromatography and ion exchange chromatography and then is subjected to enzymological property analysis, so that the result shows that the optimum temperature for leucine reaction is 30 ℃, the optimum pH is 7.4, the specific enzyme activity is 1041.67U/mg, the optimum temperature for methionine reaction is 30 ℃, the optimum pH is 7.4 and the specific enzyme activity is 822.92U/mg, which is still different from industrial application, so that the enzyme activity is improved by modifying the wild type molecule, and the popularization of the leucine-5-hydroxylase in the industrial production of drug intermediates is facilitated.

The invention content is as follows:

in order to solve the technical problems, the invention provides a leucine-5-hydroxylase mutant and preparation and application thereof.

The technical route for realizing the invention is summarized as follows:

a coding gene of leucine-5-hydroxylase (hereinafter, wild type, WT) derived from Nostoc punctiform NIES-2108 is obtained by a molecular biological means, the gene of a coding mutant is obtained by adopting inverse PCR (polymerase chain reaction) to carry out site-directed mutagenesis, and then the gene of the coding mutant is heterologously expressed to obtain the mutant V77A of the leucine-5-hydroxylase.

The following definitions are used in the present invention:

1. nomenclature for amino acid and DNA nucleic acid sequences

The accepted IUPAC nomenclature for amino acid residues is used, in the form of a three letter code. DNA nucleic acid sequences employ the accepted IUPAC nomenclature.

2. Identification of leucine-5-hydroxylase mutants

"amino acid substituted at the original amino acid position" is used to indicate a mutated amino acid in the leucine-5-hydroxylase mutant. For example Val77Ala, which means that the amino acid at position 77 has been replaced by Val of the wild-type leucine-5-hydroxylase to Ala, the numbering of the position corresponding to the numbering of the amino acid sequence of the wild-type leucine-5-hydroxylase of SEQ ID No. 1.

In the present invention, LEH represents a gene encoding wild-type leucine-5-hydroxylase, and mLEH represents a gene encoding mutant leucine-5-hydroxylase V77A, and the information is shown in the following table.

Leucine-5-hydroxylase	Amino acid mutation site	Site of gene mutation
			Wild type	——	——
V77A	Val77Ala	T230→C

The amino acid sequence of the wild leucine-5-hydroxylase is shown in a sequence table SEQ ID NO. 1;

the nucleotide sequence of the wild leucine-5-hydroxylase is shown in a sequence table SEQ ID NO. 3;

the amino acid sequence of the mutant V77A is shown in a sequence table SEQ ID NO. 2;

the nucleotide sequence of the mutant V77A is shown in a sequence table SEQ ID NO.4, and the sequence is a sequence optimized by an escherichia coli codon;

the invention also provides a recombinant vector and a recombinant bacterium carrying the mutant and/or the coding gene.

Preferably, the expression vector of the recombinant vector is pET28a (+);

preferably, the host cell of the recombinant bacterium is escherichia coli BL21(DE 3);

the experimental scheme of the invention is as follows:

1. the mutant V77A encoding gene is obtained, comprising the following steps:

(1) the gene of leucine-5-hydroxylase from Nostoc is constructed on a pET28a (+) expression vector after codon optimization (SEQ ID NO.3) to obtain LEH-pET28 a;

(2) and (2) designing a primer by taking the wild type recombinant vector LEH-pET28a in the step (1) as a template, and obtaining a mutant coding gene mLEH through reverse PCR site-specific mutagenesis, wherein the nucleotide sequence is shown as SEQ ID NO. 4. The primers are as follows:

2. the invention also provides a production method of the mutant V77A, which comprises the following steps:

(1) the correctly verified mutant recombinant vector (mLEH-pET28a) was transformed into E.coli BL21(DE3) by transformation, inoculated into LB medium (containing 50-70. mu.g/mL kanamycin), and cultured overnight at 37 ℃ at 220 r/min; inoculated in LB medium (containing 50-80. mu.g/mL kanamycin) at an inoculum size of 1%, to be the bacterial concentration OD₆₀₀Adding IPTG (isopropyl thiogalactoside) with the final concentration of 1mM at 16 deg.C and under 180r/min for 18-20 h;

(2) and (3) centrifuging, collecting the thalli, carrying out ultrasonic crushing after resuspension, and carrying out high-speed centrifugation at low temperature to obtain a supernatant, namely the supernatant containing the leucine-5-hydroxylase mutant V77A.

(3) Subjecting the high-speed centrifugation supernatant to Ni-NTA affinity chromatography and gel filtration chromatography (GE-Superdex 200 Increate 10/300 GL)^TM) After purification, leucine-5-hydroxylase mutant V77A was obtained.

3. The invention also provides application of the leucine-5-hydroxylase mutant V77A;

further, the leucine-5-hydroxylase mutant V77A is applied to hydroxylation reaction of a far-end No.5 carbon position by taking leucine as a substrate;

further, the leucine-5-hydroxylase mutant V77A is applied to sulfonation reaction with methionine as a substrate;

has the advantages that:

the wild leucine-5-hydroxylase gene is transformed through site-directed mutagenesis, and the activity of the obtained mutant V77A on the hydroxylation reaction of the leucine 5 carbon site is improved compared with that of the wild type. The specific enzyme activity of the mutated leucine-5-hydroxylase mutant V77A to substrate leucine is 1765.94U/mg, which is increased by 69.53% compared with the wild type; the specific enzyme activity to the methionine of the substrate is 1017.13U/mg, which is increased by 23.26% compared with the wild type.

The leucine-5-hydroxylase mutant has greatly improved specific enzyme activity to leucine and methionine, and provides theoretical basis and technical guarantee for efficient catalytic production of antibiotic drug intermediates by a biological enzyme method in the future.

Description of the drawings:

FIG. 1 is agarose gel electrophoresis of wild type and mutant genes

Wherein, M: a standard Marker; 1, LEH wild type; 2: a mutant gene mLEH;

FIG. 2 shows the wild type and mutant SDS-PAGES of leucine-5-hydroxylase

Wherein, M is a standard protein Marker; 1, wild type; 2 mutant V77A;

FIG. 3 is a schematic diagram of the leucine and methionine catalyzed process by leucine-5-hydroxylase

Wherein, a shows that leucine is catalyzed by leucine-5-hydroxylase; leucine-5-hydroxylase catalyzes methionine schematic;

FIG. 4 is a HPLC detection chart of a reaction product with leucine as a substrate;

FIG. 5 is a HPLC check chart of reaction products with methionine as a substrate (in which methionine sulfoxide standard is added to the control group).

The specific implementation mode is as follows:

the process of the invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.

The invention discloses a leucine-5-hydroxylase mutant and a construction method and application thereof, belonging to the technical field of genetic engineering and enzyme engineering. The leucine-5-hydroxylase (LEH) from Nostoc is subjected to site-directed mutagenesis modification to obtain mutants comprising: I70G, I75A, I75F, V77A, V77I, V77L. Through activity screening, the mutant with improved enzyme activity is V77A, and the activity on leucine and methionine is improved by 69.53 percent and 23.26 percent respectively compared with the wild type. The leucine-5-hydroxylase mutant provided by the invention is beneficial to popularization of the industrial application of the mutant in drug intermediate synthesis.

The following will be further described by way of specific examples.

Example 1 acquisition of the Gene mLEH encoding leucine-5-hydroxylase mutant V77A

Obtaining mutant V77A: the gene of wild leucine-5-hydroxylase derived from Nostoc is constructed in a pET28a (+) expression vector (Genbank sequence number RCJ32143.1 of wild leucine-5-hydroxylase) after codon optimization (SEQ ID NO.3) to obtain LEH-pET28 a; carrying out reverse PCR site-directed mutagenesis by taking LEH-pET28a plasmid as a template and V77A _ F (SEQ ID NO.5) and V77A _ R (SEQ ID NO.6) as primers; this example experiment used the KOD-Plus mutant kit (purchased from Toyobo (Shanghai) Biotech Co., Ltd.):

(1) the PCR reaction system is as follows:

stencil (53 ng/. mu.L)	1μL
		Primer F (10 pmol/. mu.L)	1.5μL
Primer R (10 pmol/. mu.L)	1.5μL
		2mM dNTP	5μL
10×Buffer for iPCR	5μL
		KOD-Plus	1μL
ddH₂O	35μL

And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 2 min; denaturation at 98 ℃ for 10 sec; extension at 68 ℃ for 7.5 min; after 8 cycles, the cells were stored at 4 ℃. The band of interest was detected by 0.8% agarose gel electrophoresis.

(2) Digestion of template in PCR products

mu.L of Dpn I was added to the PCR reaction solution (50. mu.L), and after gently mixing, the mixture was reacted at 37 ℃ for 1 hour.

(3) The PCR product is cyclized by itself, and the reaction system is as follows:

dpn I-treated PCR product	5μL
		Ligation high	5μL
T4Polynucletide Kinase	1μL
		ddH₂O	4μL

After mixing gently, the mixture was reacted at 16 ℃ for 1 hour.

(4) Taking 10 mu L of the circularized plasmid in (3), transferring the circularized plasmid into E.coli JM109 through transformation, picking a transformant, and carrying out sequencing verification on the circularized plasmid as shown in FIG. 1. Sequencing, wherein mutation sites are mutated according to a preset target to obtain a mutant coding gene mLEH shown in SEQ ID NO.4, and the amino acid sequence of the corresponding mutant is shown in SEQ ID NO. 2; the plasmid and related bacteria were stored at-80 ℃.

Example 2 expression of leucine-5-hydroxylase

The wild type coding gene LEH and the mutant coding gene mLEH are respectively constructed on a pET28a (+) expression vector to obtain recombinant vectors LEH-pET28a and mLEH-pET28a, and the recombinant vectors are respectively transferred into escherichia coli BL21(DE3) to obtain BL21/LEH and BL21/V77A recombinant bacteria.

Inoculating the 2 recombinant bacteria to 5mL LB culture medium (containing 50 ug/mL kanamycin), and culturing at 37 deg.C for 12 hr at 220 r/min; inoculated in 100mL LB medium (containing 50. mu.g/mL kanamycin) at an inoculum size of 1%, to be inoculated at OD₆₀₀0.6-0.8 IPTG was added to a final concentration of 0.75mM and induced at 16 ℃ at 180r/min for 20 h.

Example 3 purification and purification of leucine-5-hydroxylase

The bacterial suspension obtained in example 2 was centrifuged at 5000r/min and 15min to collect the cells, resuspended in solution A (20mM Tris-HCl, pH 8.0,300mM NaCl,20mM imidazole, 1.5mM DTT), added with lysozyme (final solubility of 200. mu.g/mL) and protease inhibitor (final solubility of 1mM) and placed on ice for 30min, sonicated on ice (3s on, 5s off, 350W power), and centrifuged at low temperature and high speed (4 ℃ C., 18000r/min) to remove cell debris to obtain a supernatant.

Ni affinity chromatography: 4 Open columns (Open-Column) were taken and 1mL of Ni-NTA resin (QIAGEN) was added to each Column. The resin was equilibrated with 20mL of solution A. The high speed centrifugation supernatant was combined in 1mL of resin at 4 ℃ for 40-60 min. The mixture is passed through an open column and the protein-bound resin is retained. The resin was rinsed with 20mL of solution A. Finally, the protein was eluted with 15mL of solution B (20mM Tris-HCl, pH 8.0,300mM NaCl,300mM imidazole, 2mM DTT).

Gel filtration chromatography: the GE-AKTA Pure 25L chromatographic system and GE-Superdex 200 Increate 10/300 GL^TMAnd (4) realizing gel filtration chromatography purification, and further purifying the protein. The above 15mL eluate was concentrated to 500. mu.L with an ultrafiltration tube (Millipore-Amicon-Ultra-15-MWCO-10kD) and loaded by a pump. Finally, the target protein, i.e., the wild-type enzyme solution and the mutant enzyme solution, was eluted in solution C (20mM Tris-HCl, pH 8.0,150mM NaCl,1mM DTT) containing 150mM NaCl.

Protein size and purity were identified by 12% native polyacrylamide gel electrophoresis (SDS-PAGE) to obtain a protein with a molecular weight of about 31kDa, which was consistent with DNAMAN predictions and a protein purity of over 95%, as shown in FIG. 2.

Example 4 measurement of leucine-5-hydroxylase Activity

1. Determination of enzyme concentration

Protein concentration was determined by BCA method and the kit was purchased from solibao corporation.

(1) Preparing a working solution: according to the number of the standard products and the number of the samples, 50 volumes of BCA reagent and 1 volume of Cu reagent (50:1) are prepared into BCA working solution, and the BCA working solution is fully mixed.

(2) Diluting the standard substance: mu.L of BSA standard was diluted to 100. mu.L with PBS (samples can be diluted with PBS in general) to a final concentration of 0.5 mg/ml. The standard was added to the protein standard wells of a 96-well plate at 0,2,4,6,8,12,16, 20. mu.L, and PBS was added to make up to 20. mu.L.

(3) Samples were diluted appropriately and 20. mu.L was added to the sample wells of a 96-well plate.

(4) 200 μ L of BCA working solution was added to each well, and left at 37 ℃ for 15-30 min. Determination of 562nm light absorption OD by enzyme-labeling instrument₅₆₂And calculating the protein concentration according to the standard curve.

2. Method for measuring enzyme activity

The protein concentrations of the wild-type enzyme solution and the mutant enzyme solution obtained in example 3 were respectively diluted to 0.2mg/mL, and the reaction system for measuring 1mL of enzyme activity was as follows:

the blank control group is the reaction system containing the inactivated enzyme solution;

the reaction system was incubated at 30 ℃ for 10min, and the change in absorbance of the reaction solution at 340nm within 10min was measured using a succinic acid kit (Megazyme, Irland).

Definition of enzyme activity: 1U is defined as the consumption of 5mM leucine or methionine (i.e., the amount of succinic acid produced) in mM min per minute^-1The enzyme activity calculation formula is as follows:

in the formula: a0 and a1 each represent the absorbance at the start and at the end of the reaction;

v is the reaction volume and is 1 mL;

t is reaction time, and is 10 min;

ξ₆₀₀is extinction coefficient, xi₆₀₀＝18.7×10³cm^-1·M^-1。

3. Comparison of the specific activities of the wild type and the mutant

The measured enzyme activity is divided by the enzyme concentration in the reaction system to obtain the specific enzyme activity, and the specific enzyme activities of the wild type and the mutant are shown in the following table. The specific enzyme activity of the V77A mutant is higher than that of a wild type, and the specific enzyme activity is respectively improved by 69.53 percent and 23.26 percent for leucine and methionine serving as substrates.

Example 5 HPLC identification of leucine and methionine catalytic products by leucine-5-hydroxylase

Leucine-5-hydroxylase specifically catalyzes the far-end carbon number 5 of leucine to generate 5-hydroxy-leucine, and can specifically catalyze methionine to generate oxidative sulfonation reaction to generate methionine sulfoxide. The catalytic process is schematically shown in FIG. 3. The mutant enzyme solution prepared in example 3 was used to establish an enzyme-catalyzed reaction system (1mL) as shown in the following table (wherein ferrous sulfate and alpha-ketoglutarate are cofactors of the enzyme solution-catalyzed reaction):

the control group (with the inactivated protein) and 1mL of the reaction were left to react overnight at 25 ℃. Boiling at high temperature to inactivate protein, and centrifuging to remove protein. Taking 300 mu L of supernatant, and adopting 2, 4-dinitro-fluorobenzene to carry out pre-column derivatization. The product was detected by HPLC as shown in fig. 4 and 5.

The HPLC detection conditions were as follows:

a chromatographic column:

C18 Column(5μm,4.6×250mm,Waters,Ireland)；

mobile phase: 50% methanol, 50mM sodium acetate (pH 6.4);

flow rate: 1 mL/min;

sample introduction amount: 10 mu L of the solution;

ultraviolet detection wavelength: 360 nm.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

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Claims

1. A leucine-5-hydroxylase mutant is characterized in that the amino acid sequence of the mutant is shown as a sequence table SEQ ID No. 2.

2. A gene encoding the leucine-5-hydroxylase mutant according to claim 1.

3. The gene encoding the leucine-5-hydroxylase mutant according to claim 2, which is represented by the sequence table SEQ ID No. 4.

4. A recombinant vector or recombinant strain comprising the gene of claim 2.

5. The recombinant vector or recombinant strain of claim 4, wherein the expression vector is pET28a (+), and the host cell is E.coli BL21(DE 3).

6. Use of the recombinant vector or the recombinant strain according to claim 4 for producing the leucine-5-hydroxylase according to claim 1.

7. Use of the mutant leucine-5-hydroxylase of claim 1 in hydroxylation reactions with leucine as a substrate, wherein the site of hydroxylation is at the 5 th carbon position of leucine.