CN112941062B - Nitrile hydratase lysine mutant HBA-K2H1, coding gene and application - Google Patents
Nitrile hydratase lysine mutant HBA-K2H1, coding gene and application Download PDFInfo
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Abstract
The invention discloses a nitrile hydratase mutant, in particular to a nitrile hydratase lysine mutant HBA-K2H1 and a coding gene thereof, a plasmid and recombinant bacterium containing the coding gene of the mutant, and application of the nitrile hydratase lysine mutant HBA-K2H1 in preparing an amide compound by catalyzing an organic nitrile compound. The amino acid sequence of the mutant HBA-K2H1 is shown as SEQ ID NO.1, the optimal pH value is 8.5, and the optimal temperature is 35 ℃. Compared with wild enzyme HBA, the mutant nitrile hydratase HBA-K2H1 has improved alkaline activity, and can be applied to the biotechnological fields such as biocatalysis production of acrylamide, nicotinamide and other amide compounds with high added value, chemical fiber surface modification, alkaline sewage treatment and the like.
Description
Field of the art
The invention relates to a nitrile hydratase mutant, in particular to a nitrile hydratase lysine mutant HBA-K2H1 and a coding gene thereof, a plasmid and recombinant bacterium containing the coding gene of the mutant, and application of the nitrile hydratase lysine mutant HBA-K2H1 in preparing an amide compound by catalyzing an organic nitrile compound.
(II) background art
The nitrile hydratase can convert cyano groups of various organic nitrile compounds into amide groups through water and action, and compared with a chemical hydrolysis method, the biological conversion method has the advantages of high efficiency, mild condition, small environmental pollution, capability of realizing chemical, regional and enantiomer selectivity and the like under the condition of no additional introduced protecting/modifying groups. In order to industrially catalyze the production of an amide compound such as acrylamide, nicotinamide, and pyrazinamide using nitrile hydratase, it is important to reduce the production cost of the enzyme in proportion to the production cost of the amide compound. Specifically, the nitrile hydratase content of the enzyme preparation per unit weight must be increased. With rapid development of biotechnology, construction of a recombinant strain having nitrile hydratase activity by genetic engineering techniques can be used to overcome the above-mentioned drawbacks. In addition, the nitrile hydratase can be directionally expressed by utilizing the gene recombinant bacteria to express the nitrile hydratase, so that side reactions in the catalytic reaction process are avoided, the amide target product is ensured not to be partially hydrolyzed in the production process, and the yield and quality of the amide product are improved.
The basic structural unit of nitrile hydratase is a dimer formed by combining an alpha subunit and a beta subunit, and the structural unit is further combined to form 4-12 polymers (which are different according to the species from which it is derived) to exert activity. The nitrile hydratase needs to take in metal ions (iron ions or cobalt ions) in the process of expressing translation, and is accompanied by the phenomenon that metal ion coordination occurs, and 2 cysteine residues in the active center region are subjected to oxidative modification after translation. The process of posttranslational maturation of nitrile hydratase requires specific molecular chaperones to assist the uptake of metal ions by the nitrile hydratase. However, there has not been any disclosure of examples in which cobalt-type nitrile hydratase can actively accomplish post-translational modification without molecular chaperone assistance, and there has been no disclosure of a specific method in which post-translational self-modification efficiency of nitrile hydratase can be artificially regulated.
(III) summary of the invention
The invention aims to provide a cobalt type nitrile hydratase mutant HBA-K2H1 with improved post-translational self-modification efficiency, a plasmid and recombinant bacterium containing a mutant coding gene, and application of the nitrile hydratase lysine mutant HBA-K2H1 in preparing an amide compound by catalyzing an organic nitrile compound.
The technical scheme adopted by the invention is as follows:
a nitrile hydratase lysine mutant HBA-K2H1 has an amino acid sequence shown in SEQ ID NO. 1.
SEQ ID NO.1 sequence is as follows:
the invention also relates to a gene (HBA-K2H 1) for encoding the nitrile hydratase lysine mutant HBA-K2H 1. Specifically, the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2. The mutant HBA-K2H1 has the optimal pH value of 8.5, the optimal temperature of 35 ℃, the specific activity of 684.5 +/-10.2U/mg and the cobalt ion uptake rate of 0.71+/-0.02.
SEQ ID NO.2 sequence is as follows:
the invention also relates to a plasmid containing the gene for encoding the nitrile hydratase lysine mutant HBA-K2H1 and recombinant bacteria obtained by using the plasmid and containing the gene for encoding the nitrile hydratase lysine mutant HBA-K2H 1.
The invention also relates to application of the nitrile hydratase lysine mutant HBA-K2H1 in catalyzing organic nitrile compounds to prepare amide compounds.
Specifically, the catalytic reaction is carried out at 35-45 ℃ and pH 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-K2H1 with improved alkaline activity can be carried out according to the following steps:
1) Connecting hba-k2h1 with an expression vector pET-28a, and transforming the connection product into escherichia coli B121 (DE 3) to obtain a recombinant strain containing hba-k2h1;
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-K2H1;
4) And (3) activity measurement.
The beneficial technical effects of the invention are mainly as follows: compared with wild enzyme, the post-translational self-modification efficiency of the mutant nitrile hydratase HBA-K2H1 is improved by more than 90%, the specific activity is improved by more than 200%, the optimal pH of the purified mutant HBA-K2H1 is 8.5, and the optimal temperature is 35 ℃. Under the optimal condition, the specific activity of the mutant HBA-K2H1 purified enzyme is improved by 232% compared with that of the wild type HBA, and the post-translational self-modification efficiency is improved by 97% compared with that of the wild type HBA. The mutant nitrile hydratase HBA-K2H1 can be applied to the biotechnologies such as amide compound production, chemical fiber surface modification, sewage treatment and the like.
(IV) description of the drawings
FIG. 1 shows the specific activity and cobalt ion relative content of recombinant nitrile hydratase HBA and its mutant HBA-K2H1 of the invention.
(fifth) detailed description of the invention
The present invention will be described in further detail with reference to the following specific examples, which are only for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.
Test materials and reagents:
1. strains and vectors: recombinant E.coli HBA containing recombinant plasmid pET-28-HBA was provided by the full Gene synthetic cloning service of Kirschner Biotech Co.
2. Enzymes and other biochemical reagents: plasmid miniprep kit was purchased from ai si biotechnology (hangzhou) limited and Fast Mutagenesis System kit was purchased from beijing holo biotechnology limited. Other reagents were analytically pure reagents purchased from national pharmaceutical systems chemical reagent limited.
3. Culture medium:
LB medium: peptone 10g,Yeast extract 5g,NaCl 10g, distilled water was added to 1000mL and the pH was natural (about 7). The solid medium was supplemented with 20g/L agar. Description: molecular biology experimental methods not specifically described in the examples below are referred to
The specific procedures set forth in the guidelines for molecular cloning experiments (third edition) J.Sam Brookfield, or in accordance with 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 E.coli according to the instructions of the plasmid miniprep kit of Axygen company. The amino acid sequence of the wild nitrile hydratase HBA is shown as SEQ ID NO. 3.
2) Site-directed mutagenesis primers 5'-CAAACATGGTTTCGGCAAGATCATTCGTGAGAAAAACGAGCCGCTGTTCC-3' (SEQ ID NO. 5) and 5'-GATCTTGCCGAAACCATGTTTGCCACCCAGG TCGTGG-3' (SEQ ID NO. 6) were designed based on the nucleotide sequence of the wild-type nitrile hydratase HBA (SEQ ID NO. 4).
3) According to the specification of Beijing full gold biotechnology Co., ltd Fast Mutagenesis System kit, the recombinant plasmid pET-28-HBA obtained in the step 1 is used as a template, and the primer synthesized in the step 2 is used for single-point fixed-point mutation PCR.
4) The site-directed mutagenesis PCR product is transformed into escherichia coli BL21 (DE 3), after being cultured at night, single colony is selected from a screening plate containing Kan resistance and placed in LB culture solution containing 50Kan, the strain is transferred into glycerol with a final concentration of 15% (v/v) after being rapidly cultured at 37 ℃ for about 16 hours in a shaking way, and the strain is preserved at-80 ℃.
Example 2: enzyme preparation of nitrile hydratase mutant HBA-K2H1 and wild type HBA
1) Recombinant strains containing mutant HBA-K2H1 and wild-type HBA were inoculated in LB (containing 50. Mu.g/mL Kan) medium at an inoculum size of 0.1%, respectively, and cultured at 37℃with shaking at 180rpm for 16 hours.
2) Inoculating the overnight culture activated bacterial liquid into fresh LB (containing 50 mug/mL Kan) culture liquid at 1% inoculum size, and shaking culturing at 37deg.C and 180rpm3h(OD 600nm Reaching 0.6-1.0).
3) IPTG (isopropyl thiogalactoside) was added to a final concentration of 0.5mM for induction, and the culture was carried out at 20℃and 150rpm with shaking for about 20 hours.
4) Centrifugation was performed at 8000rpm for 5min, and after the cells were collected, the cells were suspended in PBS buffer.
5) The bacterial cells were broken by ultrasonic waves in a low-temperature ice water bath. The ultrasonic crushing conditions are that the power is 300W,5s is on, 10s is off, the temperature is 4 ℃, and the effective crushing time is 30min.
6) After the cell suspension subjected to ultrasonic disruption was centrifuged at 15000rpm for 15min, the supernatant was aspirated to obtain a crude enzyme solution containing the objective nitrile hydratase.
7) The crude enzyme solution obtained was purified by using NuviaTMIMAC Cartridges pre-packed column according to BIO-RAD official instructions, and the purified product was desalted by dialysis to obtain the target protein.
Example 3: determination of the Properties of the purified enzyme of the nitrile hydratase mutant HBA-K2H1 and wild-type HBA
1) Activity analysis of mutant HBA-K2H1 and wild-type HBA purified enzyme
Dissolving substrate 3-cyanopyridine in 50mM boric acid-borax buffer solution (pH 8.0) to obtain 10mM reaction solution, preheating at 40deg.C for 5min, adding enzyme solution to obtain 3.2 μg/mL, reacting at 40deg.C for 10min, and adding 5M concentrated HCl
20. Mu.L of the reaction was terminated. After removing impurities by filtration through a 0.22 μm filter, the amount of 3-pyridinecarboxamide produced was measured by HPLC. HPLC detection conditions were using Agilent InfinityLab Poroshell EC-C18 column (4.6X105 mm,4 μm) at 36℃with mobile phase 10% (v/v) acetonitrile, flow rate 0.5mL/min, detection wavelength 215nm;1 enzyme activity unit (U) is defined as the amount of enzyme required to hydrolyze a substrate under the given conditions to produce 1. Mu. Mol of product per minute.
2) Measurement of the pH Activity of mutant HBA-K2H1 and wild-type HBA purified enzyme the mutant HBA-K2H1 and wild-type HBA purified enzyme solutions were subjected to enzymatic reactions in boric acid-borax buffers at pH7.5,8.0,8.5 and 9.0 at 37 ℃. 3-cyanopyridine is used as a substrate and reacts for 10min, and the enzymatic properties of the purified nitrile hydratase are measured. The results (Table 1) show that: the mutant enzyme HBA-K2H1 and the wild-type HBA purified enzyme had an optimum pH of 8.5.
Table 1: PH Activity of mutant HBA-K2H1 and wild-type HBA purified enzyme
3) Thermal activity assay of mutant HBA-K2H1 and wild-type HBA purified enzyme
The mutant HBA-K2H1 and the wild-type HBA purified enzyme were enzymatically reacted in boric acid-borax buffer at pH8.0 at a temperature gradient of 35, 40, 45, 50 and 60 ℃. 3-cyanopyridine is used as a substrate and reacts for 10min, and the enzymatic properties of the purified nitrile hydratase are measured. The results (Table 2) show that: the optimum temperatures of mutant HBA-K2H1 and wild-type HBA purified enzyme were 35℃and 40℃respectively.
Table 2: thermal Activity of mutant HBA-K2H1 and wild-type HBA purified enzyme
4) Enzymatic reaction kinetic parameter determination of mutant HBA-K2H1 and wild-type HBA purified enzyme
The mutant HBA-K2H1 and the wild-type HBA purified enzyme were subjected to enzymatic reaction in a reaction system having different substrate concentrations (3-cyanopyridine concentration gradient of 1-10 mM) at pH8.0 and 40℃for a first reaction time of 5min, and the enzymatic properties of the purified nitrile hydratase were determined. Mie constant (Km), maximum reaction rate (Vmax) and catalytic constant (kcat) were determined using the Lineweaver-Burk double number plot method. The results show that: the enzymatic reaction kinetic parameters Km, vmax and kcat of mutant enzyme HBA-K2H1 purified enzyme were 4.1+ -0.2 mM, 465.3+ -1.3U/mg and 391.9 + -1.8 s, respectively -1 The method comprises the steps of carrying out a first treatment on the surface of the The enzymatic kinetic parameters Km, vmax and kcat of the wild-type HBA purified enzyme were 1.5.+ -. 0.1mM, 141.7.+ -. 1.6U/mg and 119.4.+ -. 1.3s, respectively -1 The maximum reaction rate and the catalytic constant of the mutant enzyme HBA-K2H1 are improved by 228% compared with those of the wild type HBA.
Example 4: determination of purified enzyme cobalt ion content of nitrile hydratase mutant HBA-K2H1 and wild type HBA
Accurately measuring 1mg of nitrile hydratase mutant HBA-K2H1 and wild type HBA pure enzyme, adding 1mL of concentrated nitric acid, preserving heat for 1H at 70 ℃ to fully digest a protein sample, cooling to room temperature, and adding mass spectrometry grade deionized water to dilute until the final concentration of nitric acid is 5%. The concentration of cobalt ions in the samples was determined by inductively coupled plasma mass spectrometry (ICP-MS), and each sample was repeated three times with the same treated mass spectrometry grade deionized water containing no protein as a negative control. The results (fig. 1) demonstrate that: the specific activities of mutant HBA-K2H1 and wild type HBA purified enzyme are 684.5 +/-10.2 and 206.1+/-14.6U/mg respectively, the relative cobalt ion content of unit catalytic center is 0.71+/-0.03 and 0.36+/-0.01 mol/mol respectively, the post-translational self-modification efficiency of mutant nitrile hydratase HBA-K2H1 is improved by 97% compared with wild type HBA, and the specific activity is improved by 232% compared with wild type HBA.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to 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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caccaccacc acccgcaccc ggagagcttc tggagcgcgc gtgcgaaggc gctggaaagc 780
ctgctgatcg agaaaggcat tctgagcagc gacgcgatcg atcgtgtggt tcagcactac 840
gaacacgagc tgggcccgat gaacggtgcg aaggtggttg cgaaagcgtg gaccgacccg 900
gcgtttaagc agcgtctgct ggaagatccg gagaccgttc tgcgtgagct gggttactat 960
ggcctgcaag gtgaacacat tcgtgtggtt gagaacaccg ataccgtgca caacgtggtt 1020
gtgtgcaccc tgtgcagctg ctatccgtgg ccgctgctgg gtctgccgcc ggcgtggtac 1080
aaagaaccga cctatcgtag ccgtatcgtt aaggagccgc gtaaagtgct gcgtgaggag 1140
ttcggtctgg acctgccgga taccgttgaa attcgtgtgt gggacagcag cagcgagatg 1200
cgttatatgg tgctgccgca gcgtccggaa ggtaccgagg gtatgaccga ggaagagctg 1260
gcgaagatcg tgacccgtga tagcatgatt ggtgttgcga aagtgcaacc gagcagcgtt 1320
accgtgcgtt ga 1332
<210> 5
<211> 50
<212> DNA
<213> Unknown (Unknown)
<400> 5
caaacatggt ttcggcaaga tcattcgtga gaaaaacgag ccgctgttcc 50
<210> 6
<211> 37
<212> DNA
<213> Unknown (Unknown)
<400> 6
gatcttgccg aaaccatgtt tgccacccag gtcgtgg 37
Claims (8)
1. A nitrile hydratase lysine mutant HBA-K2H1 has an amino acid sequence shown in SEQ ID NO. 1.
2. A gene encoding the nitrile hydratase lysine mutant HBA-K2H1 of claim 1.
3. The coding gene according to claim 2, wherein the nucleotide sequence of the coding gene is shown in SEQ ID NO. 2.
4. A plasmid comprising a gene encoding the nitrile hydratase lysine mutant HBA-K2H1 of claim 1.
5. A recombinant bacterium comprising a gene encoding the nitrile hydratase lysine mutant HBA-K2H1 according to claim 1.
6. Use of the nitrile hydratase lysine mutant HBA-K2H1 according to claim 1 for catalyzing the preparation of amide compounds from organic nitrile compounds.
7. The use according to claim 6, wherein the catalytic reaction is carried out at a temperature of 30 to 45℃and a pH of 8.0 to 9.0.
8. The use according to claim 6, wherein the organic nitrile compound is 3-cyanopyridine and the amide compound is 3-pyridinecarboxamide.
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| CN109251882A (en) * | 2018-11-28 | 2019-01-22 | 江南大学 | The Escherichia coli recombinant strain and its application of one plant of heat-resisting nitrile hydratase of heterogenous expression |
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Non-Patent Citations (2)
| Title |
|---|
| Ji-Dong Shen 等.Structural insights into the thermostability mechanism of a nitrile hydratase from Caldalkalibacillus thermarum by comparative molecular dynamics simulation.Proteins.2021,第1-10页. * |
| 史悦 等.有效增强重组腈代谢酶系表达活性的策略.现代化工.2003,第23卷(第9期),第23-28页. * |
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