CN113151234A

CN113151234A - Nitrile hydratase lysine mutant HBA-K2H2R, coding gene and application

Info

Publication number: CN113151234A
Application number: CN202110396847.6A
Authority: CN
Inventors: 柳志强; 沈骥冬; 蔡雪; 金利群; 徐建妙; 郑裕国
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2021-04-13
Filing date: 2021-04-13
Publication date: 2021-07-23
Anticipated expiration: 2041-04-13
Also published as: CN113151234B

Abstract

The invention discloses a nitrile hydratase mutant, and particularly relates to a nitrile hydratase lysine mutant HBA-K2H2R and a coding gene thereof, a plasmid and a recombinant bacterium containing the coding gene of the mutant, and application of the nitrile hydratase lysine mutant HBA-K2H2R in preparation of amide compounds by catalyzing organic nitrile compounds. The amino acid sequence of the mutant HBA-K2H2R is shown in SEQ ID NO.1, the optimum pH is 9.0, and the optimum temperature is 35 ℃. Compared with wild enzyme HBA, the mutant nitrile hydratase HBA-K2H2R has improved alkaline activity, and can be applied to the biotechnological fields of biocatalytic production of acrylamide, nicotinamide and other amide compounds with high added values, chemical fiber surface modification, alkaline sewage treatment and the like.

Description

Nitrile hydratase lysine mutant HBA-K2H2R, coding gene and application

(I) technical field

The invention relates to a nitrile hydratase mutant, in particular to a nitrile hydratase lysine mutant HBA-K2H2R and a coding gene thereof, a plasmid and a recombinant bacterium containing the coding gene of the mutant, and application of the nitrile hydratase lysine mutant HBA-K2H2R in preparation of amide compounds by catalyzing organic nitrile compounds.

(II) background of the invention

Compared with a chemical hydrolysis method, the biotransformation method has the advantages of high efficiency, mild conditions, small environmental pollution, realization of chemical, regional and enantiomeric selectivity and the like under the condition of no additional introduction of a protecting/modifying group. In order to industrially produce amide compounds such as acrylamide, nicotinamide and pyrazinamide by catalysis with nitrile hydratase, it is important to reduce the proportion of the production cost of the enzyme to the production cost of the amide compound. In particular, the nitrile hydratase content per weight of enzyme preparation must be increased. With the rapid development of biotechnology, the construction of a recombinant strain having nitrile hydratase activity by means of genetic engineering techniques can be used to overcome the above-mentioned disadvantages. In addition, the gene recombinant bacteria are used for expressing nitrile hydratase, so that the nitrile hydratase can be directionally expressed, side reactions in the catalytic reaction process are avoided, the amide target products are prevented from being partially hydrolyzed in the production process, and the yield and the quality of the amide products are improved.

The basic structural unit of nitrile hydratase is a dimer formed by combining alpha subunit and beta subunit, and the structural units are further combined to form 4-12 polymers (different according to species of origin) to exert activity. The nitrile hydratase needs to take in metal ions (iron ions or cobalt ions) during the expression and translation, and the phenomenon that metal ions coordinate occurs, 2 cysteine residues in the active center region of the nitrile hydratase are subjected to oxidation modification after translation. The process of post-translational maturation of nitrile hydratases requires a specific chaperone to assist the uptake of metal ions by the nitrile hydratase. However, none of the inventions disclose examples of the active post-translational modification of cobalt-type nitrile hydratases without the assistance of a chaperone, nor do they disclose specific methods for artificially regulating the efficiency of post-translational self-modification of nitrile hydratases.

Disclosure of the invention

The invention aims to provide a cobalt-type nitrile hydratase mutant HBA-K2H2R nitrile hydratase lysine mutant HBA-K2H2R with improved posttranslational self-modification efficiency, a plasmid and a recombinant bacterium containing a coding gene of the mutant, and application of the nitrile hydratase lysine mutant HBA-K2H2R in preparation of amide compounds by catalyzing organic nitrile compounds.

The technical scheme adopted by the invention is as follows:

the amino acid sequence of the nitrile hydratase lysine mutant HBA-K2H2R is shown as SEQ ID NO. 1.

The sequence of SEQ ID NO.1 is as follows:

the invention also relates to a gene for coding the nitrile hydratase lysine mutant HBA-K2H 2R. Specifically, the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2. The optimum pH of the mutant HBA-K2H2R is 8.5, the optimum temperature is 35 ℃, and the specific activity is 267.4 +/-1.4U/mg.

The sequence of SEQ ID NO.2 is as follows:

the invention also relates to a plasmid containing a gene coding the nitrile hydratase lysine mutant HBA-K2H2R and a recombinant bacterium obtained by using the plasmid and containing the gene coding the nitrile hydratase lysine mutant HBA-K2H 2R.

The invention also relates to application of the nitrile hydratase lysine mutant HBA-K2H2R in preparation of amide compounds by catalyzing organic nitrile compounds.

Specifically, the catalytic reaction is carried out at 35-45 ℃ and pH of 8.0-9.0.

Preferably, the organic nitrile compound is 3-cyanopyridine, and the amide compound is 3-pyridinecarboxamide.

The method for preparing the cobalt-type nitrile hydratase lysine mutant HBA-K2H2R with improved alkaline activity can be carried out according to the following steps:

1) hba-k2h2r and an expression vector pET-28a are connected, and the connection product is transformed into Escherichia coli B121(DE3) to obtain a recombinant strain containing hba-k2h2 r;

2) culturing the recombinant strain, and inducing the expression of recombinant mutant cobalt-type nitrile hydratase;

3) recovering and purifying the expressed mutant nitrile hydratase HBA-K2H 2R;

4) and (4) measuring the activity.

The beneficial technical effects of the invention are mainly reflected in that: compared with wild enzyme, the posttranslational self-modification efficiency of the mutant nitrile hydratase HBA-K2H2R is improved by more than 90%, the specific activity is improved by more than 20%, the optimum pH value of the purified mutant HBA-K2H2R is 8.5, and the optimum temperature is 35 ℃. Under the optimal conditions, the maximum reaction rate and the catalytic constant of the purified enzyme of the mutant HBA-K2H2R are improved by 142 percent compared with the wild type HBA, the specific activity is improved by 29 percent compared with the wild type HBA, and the self-modification efficiency after translation is improved by 100 percent compared with the wild type HBA. The mutant nitrile hydratase HBA-K2H2R can be applied to the biotechnological fields of amide compound production, chemical fiber surface modification, sewage treatment and the like.

(IV) description of the drawings

FIG. 1 shows the specific activity and the relative content of cobalt ions of the recombinant nitrile hydratase HBA and its mutant HBA-K2H2R of the invention.

(V) detailed description of the preferred embodiments

For the purpose of enhancing understanding of the present invention, the present invention will be described in further detail with reference to specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention.

Test materials and reagents:

1. strains and vectors: the recombinant Escherichia coli HBA containing the recombinant plasmid pET-28-HBA is provided by the complete gene synthesis cloning service of the Kingsler Biotechnology Ltd.

2. Enzymes and other biochemical reagents: the plasmid miniprep kit was purchased from Aisijin Biotechnology (Hangzhou) Inc., and the Fast Mutagenesis System kit was purchased from Beijing Quanjin Biotechnology Inc. All other reagents were analytically pure reagents purchased from chemical reagents of national drug group, ltd.

3. Culture medium:

LB culture medium: peptone 10g, Yeast extract 5g, NaCl 10g, distilled water to 1000mL, natural pH (about 7). On the basis of the solid medium, 20g/L agar was added.

Description of the drawings: the molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or according to the kit and product instructions.

Example 1: site-directed mutagenesis of nitrile hydratase HBA

1) The recombinant plasmid pET-28-HBA containing the nitrile hydratase HBA gene was extracted from recombinant Escherichia coli HBA according to the instructions of the plasmid minikit of Axygen. The amino acid sequence of the wild-type nitrile hydratase HBA is shown in SEQ ID NO. 3.

2) The multi-site-directed mutagenesis primers 5'-CAAACATGGTTTCGGCAAGATCATTCGTGAGAAACATGAGCCGCTGTTCC-3' (SEQ ID NO.5), 5'-GATCTTGCCGAAACCATGTTTGCCACCCAGGTCGTGG-3' (SEQ ID NO.6), 5'-CAGCAGACCAAACGCGCGACGTTCCC AGTC-3' (SEQ ID NO.7) and 5'-CGCGCGTTTGGTCTGCTGATTGGCACCGCGGGTC-3' (SEQ ID NO.8) were designed based on the nucleotide sequence of the wild-type nitrile hydratase HBA (SEQ ID NO. 4).

3) According to the specification of a Fast MultiSite Mutagenesis System kit of Beijing Quanji Biotechnology Limited, the multi-site-directed Mutagenesis PCR is carried out by using the recombinant plasmid pET-28-HBA obtained in the step 1 as a template and the primer synthesized in the step 2.

4) The site-directed mutagenesis PCR product is transformed into escherichia coli BL21(DE3), a single colony is selected from a screening plate containing Kan resistance after overnight culture in LB culture solution containing 50Kan, after rapid shaking culture at 37 ℃ for about 16h, the bacterial solution is transferred into glycerol with the final concentration of 15% (v/v), and the mixture is uniformly mixed and stored at-80 ℃.

Example 2: enzyme preparation of nitrile hydratase mutant HBA-K2H2R and wild type HBA

1) The recombinant strain containing the mutant HBA-K2H2R and the wild type HBA was inoculated in LB (containing 50. mu.g/mL Kan) culture medium at an inoculum size of 0.1%, respectively, and cultured at 37 ℃ for 16 hours with shaking at 180 rpm.

2) The overnight culture activated bacterial suspension was inoculated into fresh LB (containing 50. mu.g/mLKan) culture medium at an inoculum size of 1%, and cultured at 37 ℃ for 3 hours (OD) with shaking at 180rpm_600nmTo 0.6-1.0).

3) Induction was carried out by adding IPTG (isopropyl thiogalactoside) at a final concentration of 0.5mM, and shaking culture was carried out at 150rpm for about 20h at 20 ℃.

4) The cells were centrifuged at 8000rpm for 5min, collected and suspended in PBS.

5) And breaking the thalli by ultrasonic waves in a low-temperature ice-water bath. The ultrasonic crushing conditions are that the power is 300W, the time is 5s on, the time is 10s off, the temperature is 4 ℃, and the effective crushing time is 30 min.

6) The ultrasonically disrupted cell suspension was centrifuged at 15000rpm for 15min, and the supernatant was aspirated to obtain a crude enzyme solution containing the objective nitrile hydratase.

7) Nuvia according to BIO-RAD official notes^TMAnd purifying the obtained crude enzyme solution by using an IMAC Cartridges pre-packed column, and dialyzing and desalting the purified product to obtain the target protein.

Example 3: determination of Properties of purified enzymes of nitrile hydratase mutant HBA-K2H2R and wild-type HBA

1) Activity analysis of mutant HBA-K2H2R and wild type HBA purified enzyme

The substrate 3-cyanopyridine was dissolved in 50mM boric acid-borax buffer (pH 8.0) to give a final concentration of 10mM, the reaction solution was preheated at 40 ℃ for 5min, then pure enzyme solution was added thereto to give a final concentration of 7.2. mu.g/mL, the reaction was carried out at 40 ℃ for 10min, and then 5M concentrated HCl was added thereto in an amount of 20. mu.L to terminate the reaction. After removing impurities by filtration through a 0.22 μm filter, the amount of the produced 3-pyridinecarboxamide was measured by HPLC.

HPLC detection conditions are that an Agilent Infinity Lab Poroshell 120 EC-C18 column (4.6X 150mm,4 μm) chromatographic column is used, the column temperature is 36 ℃, the mobile phase is 10% (v/v) acetonitrile, the flow rate is 0.5mL/min, and the detection wavelength is 215 nm; 1 enzyme activity unit (U) is defined as the amount of enzyme required to hydrolyze a substrate to produce 1. mu. mol of product per minute under given conditions.

2) Determination of pH Activity of mutant HBA-K2H2R and wild type HBA purified enzyme

The mutant HBA-K2H2R and the wild type HBA purified enzyme solution are put in boric acid-borax buffer solution with pH of 7.5, 8.0, 8.5 and 9.0 at 37 ℃ for enzymatic reaction. 3-cyanopyridine was used as a substrate, and the enzymatic properties of the purified nitrile hydratase were measured after 10min of reaction. The results (table 1) show that: the optimum pH of the mutant HBA-K2H2R and the wild-type HBA purified enzyme was 8.5.

Table 1: pH Activity of mutant HBA-K2H2R and wild-type HBA purified enzyme

3) Determination of thermal Activity of mutant HBA-K2H2R and wild-type HBA purified enzyme

The mutant HBA-K2H2R and the wild type HBA purified enzyme solution are subjected to enzymatic reaction in a boric acid-borax buffer solution with the pH value of 8.0 in a temperature gradient of 35, 40, 45, 50 and 60 ℃.

3-cyanopyridine was used as a substrate, and the enzymatic properties of the purified nitrile hydratase were measured after 10min of reaction.

The results (table 2) show that: the optimum temperatures of the mutant enzyme HBA-K2H2R and the wild type HBA purified enzyme are 35 ℃ and 40 ℃ respectively.

Table 2: thermal activity of mutant HBA-K2H2R and wild type HBA purified enzyme

Determination of enzymatic reaction kinetics parameters of mutant HBA-K2H2R and wild type HBA purified enzyme mutant HBA-K2H2R and wild type HBA purified enzyme were subjected to enzymatic reaction in reaction systems of different substrate concentrations (concentration gradient of 3-cyanopyridine of 1-10 mM) at pH8.0 and 40 ℃ for 5min for the first-order reaction time, and the enzymatic properties of the purified nitrile hydratase were determined. The Michaelis constant (Km), maximum reaction rate (Vmax) and catalytic constant (kcat) were determined using a Lineweaver-Burk double-iteration plot. The results show that: the enzymatic reaction kinetic parameters Km, Vmax and kcat of the purified enzyme of the mutant enzyme HBA-K2H2R are 2.3 +/-0.8 mM, 343.6 +/-2.1U/mg and 289.4 +/-2.8 s respectively^-1(ii) a The enzymatic reaction kinetics parameters Km, Vmax and kcat of the wild type HBA purified enzyme are 1.5 +/-0.1 mM, 141.7 +/-1.6U/mg and 119.4 +/-1.3 s respectively^-1. The maximum reaction rate and catalytic constant of the mutant enzyme HBA-K2H2R are improved by 142% compared with wild-type HBA.

Example 4: determination of cobalt ion content of nitrile hydratase mutant HBA-K2H2R and wild type HBA purified enzyme

The nitrile hydratase mutant HBA-K2H2R and the wild type HBA pure enzyme 1mg are precisely measured, 1mL of concentrated nitric acid is added, the temperature is kept at 70 ℃ for 1H, protein samples are sufficiently digested, the mixture is cooled to room temperature, and mass spectrum deionized water is added to dilute the mixture until the final concentration of the nitric acid is 5%. Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the concentration of cobalt ions in the samples, which was repeated three times for each sample, using the same treated mass-fraction deionized water without protein as a negative control. The results (fig. 1) show that: the specific activities of the mutant HBA-K2H2R and the wild type HBA purified enzyme are 267.4 +/-1.4 and 206.1 +/-14.6U/mg respectively, and the relative cobalt ion content of a unit catalytic center is 0.61 +/-0.02 and 0.30 +/-0.01 mol/mol respectively. The posttranslational self-modification efficiency of the mutant HBA-K2H2R is improved by 100 percent compared with that of the wild HBA; under the optimal conditions, the specific activity of the purified enzyme of the mutant HBA-K2H2R is improved by 29 percent compared with that of the wild HBA.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Sequence listing

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Claims

1. The amino acid sequence of the nitrile hydratase lysine mutant HBA-K2H2R is shown as SEQ ID NO. 1.

2. A gene encoding the nitrile hydratase lysine mutant HBA-K2H2R according to claim 1.

3. The encoding gene of claim 2, wherein the nucleotide sequence of the encoding gene is shown as SEQ ID No. 2.

4. A plasmid containing a gene encoding the nitrile hydratase lysine mutant HBA-K2H2R according to claim 1.

5. A recombinant bacterium containing a gene encoding the nitrile hydratase lysine mutant HBA-K2H2R according to claim 1.

6. Use of the nitrile hydratase lysine mutant HBA-K2H2R according to claim 1 for catalyzing organic nitrile compounds to produce amide compounds.

7. The use of claim 6, wherein the catalytic reaction is carried out at a temperature of 30-45 ℃ and a pH of 8.0-9.0.

8. The use according to claim 6, wherein the organic nitrile compound is 3-cyanopyridine and the amide compound is 3-pyridinecarboxamide.