CN112921016A

CN112921016A - Organophosphorus hydrolase mutant and application thereof

Info

Publication number: CN112921016A
Application number: CN202110206457.8A
Authority: CN
Inventors: 刘敏; 顾恰敏; 赵传欣
Original assignee: Insititute Of Nbc Defence
Current assignee: Insititute Of Nbc Defence
Priority date: 2021-02-24
Filing date: 2021-02-24
Publication date: 2021-06-08

Abstract

The invention relates to an organophosphorus hydrolase mutant and application thereof, belonging to the technical field of genetic engineering and biological enzymology. The mutant is formed by mutating histidine at 257 th site of organophosphorus hydrolase into tyrosine, and leucine at 303 th site of organophosphorus hydrolase into threonine, wherein the amino acid sequence of the organophosphorus hydrolase mutant is shown as SEQNO.1. The mutant effectively enhances the activity of organophosphorus hydrolase and the capability of hydrolyzing a substrate, and when the mutant is used for degrading the paraoxon compound, the hydrolysis rate of the paraoxon compound is effectively improved.

Description

Organophosphorus hydrolase mutant and application thereof

Technical Field

The invention relates to an organophosphorus hydrolase mutant and application thereof, belonging to the technical field of genetic engineering and biological enzymology.

Background

The biological enzyme is used as a biological catalyst and can specifically accelerate a series of biochemical reactions. Wherein, the Organophosphorus hydrolase (OPH) is used for biologically degrading Organophosphorus toxicants, and has great significance for decontamination of nerve toxicants, human health protection and ecological environment protection. Organophosphorus hydrolase (OPH) is a typical phosphotriesterase that hydrolyzes many organophosphate triesters, thioesters, and fluorophosphates by hydrolyzing various phosphate bonds (P-O bonds, P-CN bonds, P-F bonds, and P-S bonds) of the ester bond of the cleavage group of the phosphorus atom and the electrophilic atom.

Organophosphorus toxicants such as sarin (GB) contain a typical paraoxon structure, and the mechanism of OPH catalyzed hydrolysis of paraoxon suggests that water molecules are activated at the metal center to bridging hydroxide, which attacks the central phosphorus atom of the substrate, and the catalytic initiation step is independent of the leaving group. The specific reaction process can be divided into four stages: (ii) Zn in the metal center of OPH²⁺Deprotonation of one water molecule, generation of bridging hydroxide and binuclear Zn²⁺And aspartic acid at position 301 (D301); binding the substrate to the active site of the enzyme, bridging hydroxide radicals to attack phosphorus atoms in the substrate molecule, and simultaneously obtaining H protons on the hydroxide radicals by D301; ③ beta-Zn²⁺Polarising phosphoryl oxygen, resulting in beta-Zn²⁺The combination with hydroxyl is weakened, and the electron pushing effect of the phosphorus center is enhanced; cleavage of the substrate P-O bond, release of the leaving group (i.e.the first product P-nitrophenol), forming a complex of the enzyme with the second product, the enzyme-product complex consisting of a metal Zn²⁺Center stabilization; the proton migrates to H254 through D301 and is far away from the active site of the enzyme; the second product is released and the active cavity returns to its original state for the next round of catalysis. Due to the self-property limit, the activity of wild bacteria OPH for degrading the organophosphorus compound cannot meet the requirement of quickly degrading the organophosphorus compound in a high efficiency manner under various conditions.

Disclosure of Invention

In view of the above, the present invention aims to provide an organophosphorus hydrolase mutant and applications thereof, wherein the mutation of histidine at 257 th position to tyrosine (H257Y) and the mutation of leucine at 303 th position to threonine (L303T) in OPH effectively enhances the activity of OPH and the ability to hydrolyze substrates, and when the mutant is used for degrading paraoxon compounds, the mutant effectively increases the hydrolysis rate of paraoxon compounds.

In order to achieve the above object, the technical solution of the present invention is as follows.

An organophosphorus hydrolase mutant is characterized in that histidine at the 257 th site of the organophosphorus hydrolase is mutated into tyrosine, and leucine at the 303 th site of the organophosphorus hydrolase is mutated into threonine.

Furthermore, the amino acid sequence of the organophosphorus hydrolase mutant is shown in SEQ NO. 1.

Furthermore, the nucleotide sequence of the gene for coding the organophosphorus hydrolase mutant is shown as SEQ NO. 2.

The application of the mutant of the organophosphorus hydrolase which is used as a biological enzyme for degrading the paraoxon compound.

Further, the paraoxon compound is sarin.

Advantageous effects

1. Compared with wild OPH, the rate of degrading the paraoxon compound by the organophosphorus hydrolase mutated simultaneously in H257Y and L303T is obviously improved.

2. The mutated organophosphorus hydrolase can better promote the hydrolysis of sarin, and the speed of degrading the sarin is increased from 0.452 mg/(min. mg) to 0.879 mg/(min. mg), so that the enzyme has wider prospects in the actual use in the fields of biochemical decontamination, environmental protection and the like.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

The following examples are carried out in the conventional manner in the art unless otherwise specified.

Example 1

Organophosphorus hydrolase point mutation and secretory expression

(I) Point mutation related gene

Based on the data structure of the organophosphorus hydrolase OPH found in Sphingobacterium furiginis (ATCC 27551), the influence of the change of the protein structure on the enzymatic hydrolysis efficiency is analyzed according to the hydrolysis mechanism of the organophosphorus hydrolase OPH, the base pair of the key position is changed by point mutation so as to change the protein sequence and the three-dimensional structure, and the organophosphorus hydrolase with higher hydrolytic activity is obtained, wherein the amino acid sequence of the designed organophosphorus hydrolase mutant is shown as SEQ NO. 1. And obtaining an OPH gene fragment containing H257Y and L303T mutation sites by adopting the conventional overlapped PCR technology in the field, sequencing, wherein the nucleotide sequence of the organophosphorus hydrolase mutant gene is shown as SEQ NO. 2.

The amino acid sequence SEQ NO.1 is:

MQTRRVVLKS AAAAGTLLGG LAGCASVAGS IGTGDRINTV RGPITISEAG FTLTHEHICG SSAGFLRAWP EFFGSRKALA EKAVRGLRRA RAAGVRTIVD VSTFDIGRDV SLLAEVSRAA DVHIVAATGL WFDPPLSMRL RSVEELTQFF LREIQYGIED TGIRAGIIKV ATTGKATPFQ ELVLKAAARA SLATGVPVTT HTAASQRDGE QQAAIFESEG LSPSRVCIGH SDDTDDLSYL TALAARGYLI GLDHIPYSAI GLEDNASASA LLGIRSWQTR ALLIKALIDQ GYMKQILVSN DWTFGFSSYV TNIMDVMDRV NPDGMAFIPL RVIPFLREKG VPQETLAGIT VTNPARFLSP TLRAS

the nucleotide sequence SEQ NO.2 is:

atgcaaacga gaagggttgt gctcaagtct gcggccgccg caggaactct gctcggcggc ctggctgggt gcgcgagcgt ggctggatcg atcggcacag gcgatcggat caataccgtg cgcggtccta tcacaatctc tgaagcgggt ttcacactga ctcacgagca catctgcggc agctcggcag gattcttgcg tgcttggcca gagttcttcg gtagccgcaa agctctagcg gaaaaggctg tgagaggatt gcgccgcgcc agagcggctg gcgtgcgaac gattgtcgat gtgtcgactt tcgatatcgg tcgcgacgtc agtttattgg ccgaggtttc gcgggctgcc gacgttcata tcgtggcggc gaccggcttg tggttcgacc cgccactttc gatgcgattg aggagtgtag aggaactcac acagttcttc ctgcgtgaga ttcaatatgg catcgaagac accggaatta gggcgggcat tatcaaggtc gcgaccacag gcaaggcgac cccctttcag gagttagtgt taaaggcggc cgcccgggcc agcttggcca ccggtgttcc ggtaaccact cacggcagca agtcagcgcg atggtgagca gcaggccgcc atttttgagt ccgaaggcga gcccctcacg ggtttgtatt ggtcacagcg atgatactga cgatttgagc tatctccgcc ctcgctgcgc gcggatacct catcggtcta gaccacatcc cgtatagtgc gatttctaga agataatgcg agtgcatcag ccctcctggg catccgttcg tggcaaacac ggtctcttga tcaaggcgct catcgaccaa ggctacatga aacaaatcct cgtttcgaat ctggactttc gggttttcga gctatgtcac caacatcatg gacgtgatgg atcgcgtgcc ccgacgggat ggccttcatt ccactgagag tgatcccatt cctacgagag aagggcgtcc cacaggaaac gctggcaggc atcactgtga ctaacccggc gcggttcttg tcaccgacct tgcgggcgtc atga

(II) designing expression system

The bacillus subtilis selected by the secretion expression bacteria has strong protein secretion capacity, and can directly secrete exogenous proteins into a culture medium, so that the method has important significance on later-stage purification and activity maintenance of the proteins. In addition, the genetic background of the bacillus subtilis is clear, so that the modification of genetic engineering is facilitated; has no preference of the codon, has simple requirements on the culture medium, and can grow in high density in the culture medium with simpler nutrition.

The vector plasmid selects an E.coli-B.subtilis shuttle pMA0911, and the vector can carry a target gene segment to realize secretion expression in a bacillus subtilis cell. The base sequence of the pMA0911 shuttle was analyzed and NdeI and EcoRI sites were chosen for cleavage. NdeI is positioned at the 10 th site, EcoRI is positioned at the 111 th site, and both are unique enzyme cutting sites on a pMA0911 plasmid ring, are close to each other, do not contain key base sequences in the range, and are suitable for being used as sites for inserting OPH genes.

1. Extraction of plasmid and Gene

Extraction of pMA0911 shuttle and OPH (OPH-YT) gene plasmid containing the H257Y & L303T mutation site was performed with reference to the plasmid miniprep extraction kit (Tiangen Biochemical technology Co., Ltd., DP 105).

2. Double digestion of plasmid and Gene

Carrying out double enzyme digestion on OPH (OPH-YT) gene plasmid containing H257Y & L303T mutation sites by NdeI and EcoRI to obtain an enzyme-digested OPH-YT gene fragment;

and (3) cutting the shuttle pMA0911 by NdeI and EcoRI double enzymes to obtain an enzyme-cut shuttle pMA0911 fragment.

3. Construction of expression vectors

The OPH-YT gene fragment and the E.coli-B.subtilis shuttle pMA0911 fragment were recovered and purified using agarose gel recovery kit (Tiangen Biochemical technology Co., Ltd., DP 219). The target OPH-YT gene was inserted into shuttle pMA0911 to construct expression vector pMA-YT 1.

4. Transformation of competent e

The expression vector pMA-YT1 was transferred to competent E.coli, spread on LB solid medium containing ampicillin, and cultured overnight in a 37 ℃ incubator. And selecting resistant transformants, and extracting a target pMA-YT1 plasmid after strain amplification.

5. Preparation and transformation of the competent cells of subtilis

The Spizizen method for preparing and transforming the subtilis competent cells comprises the following specific operation steps:

(1) preparing a culture medium:

10 × lowest salt solution: k₂HPO₄ 14g，KH₂PO₄ 6g，(NH₄)₂SO₄2g, sodium citrate (Na)₃C₆H₅O₇·2H₂O)1g，MgSO₄·7H₂0.2g of O is dissolved in deionized water in sequence, and finally the volume is fixed to 100 mL.

Amino acid (L-trp) solution: the concentration is 2mg/mL, and the mixture is filtered and sterilized by a sterile 0.22um filter, stored in a brown bottle, wrapped by black paper and stored in dark.

③ GMI solution: 95mL of 1 Xminimum salt solution, 1mL of 50% glucose, 0.4mL of 5% hydrolyzed casein, 1mL of 10% yeast juice and 2.5mL of L-trp solution.

GM II solution: 97.5mL of 1 Xminimum salt solution, 1mL of 50% glucose, 0.08mL of 5% hydrolyzed casein, 0.04mL of 10% yeast juice, 0.5M MgCl₂ 0.5mL，0.1M CaCl₂0.5mL, and 0.5mL of L-trp solution.

(2) Inoculating Bacillus subtilis WB800 monoclonal in 2mL GMI culture medium, and culturing overnight in a constant temperature shaking table at 37 ℃;

(3) mu.L of overnight culture was inoculated into 5mL of SPI medium, and the absorbance (OD600) at a wavelength of 600nm was measured after shaking culture at 37 ℃ for 4 hours. When OD600 is 1.0, 200. mu.L of the bacterial suspension is transferred to 2mL of GMII medium, and shake culture is carried out at 37 ℃ and 100r/min for 1.5 h.

(4) 20 μ L of 100 XEGTA solution was added to the tube, and after precise culture in a shaker at 37 ℃ and 100r/min for 10min, 600 μ L of the culture medium was dispensed into each 1.5mL centrifuge tube.

(5) pMA-YT1 plasmid is added into the tube, and after being evenly sucked, the tube is placed in a shaking table with the temperature of 37 ℃ and the speed of 100r/min for cultivation for 2 hours.

(6) After the culture is finished, collecting bacterial liquid by centrifugation at 4000r/min, discarding part of supernatant, leaving 150 mu L of resuspended thallus, coating the thallus on a selective plate containing kanamycin, and culturing overnight at 37 ℃ and 180r/min to obtain the target recombinant strain carrying pMA-YT1 plasmid.

6. Preservation of recombinant bacteria

The resulting recombinant strain was inoculated into 10mL of LB medium (containing 50. mu.g. multidot.mL)^-1Kanamycin) is added into a proper amount of glycerol for storing bacteria after overnight culture at 37 ℃ and 180 r/min.

Example 2

The mutant organophosphorous hydrolase was mutated as described in example 1 to tyrosine (H257Y) from histidine at position 257 of the organophosphorous hydrolase, and was designated as OPH-Y.

Example 3

The organophosphorus hydrolase mutant was obtained by mutating leucine at position 303 to threonine (L303T) as OPH-T in the same manner as in example 1.

Example 4

Activity assay of recombinant organophosphorus hydrolase:

the recombinant strain is inoculated into 30mL of TB expression medium and is subjected to constant temperature shaking culture at 37 ℃ and 180r/min for 24 h. Centrifuging the fermentation liquor, taking a precipitate part, washing the precipitate part by 0.1M phosphate buffer solution (PBS, pH7.4), suspending the precipitate part in 5mLPBS, setting ultrasonic bacteria breaking conditions to be 300w, ultrasonic for 5s and interval of 5s, centrifuging the precipitate part for 10min at 4000r/min after ultrasonic bacteria breaking for 15min, taking the supernatant of the bacteria breaking, performing BCA protein concentration determination and a peroxide-benzidine quantitative method to detect the GB degrading ability of enzyme, and detecting the hydrolysis ability of the genetic engineering recombinant strain.

BCA protein concentration assay: the BCA protein concentration assay kit (Beijing Soilebao Tech Co., Ltd.) was used. Under alkaline conditions, the protein converts Cu into²⁺Reduction to Cu⁺，Cu⁺A purple blue complex was formed with the BCA reagent, and its absorbance at 562nm was measured to calculate the protein concentration.

Enzyme activity detection by a perbenzidine quantitative method: sodium perborate decomposes hydrogen peroxide in alkaline water, hydrogen peroxide reacts with G type toxin agent to generate peroxophosphonic acid, the peroxophosphonic acid has strong oxidizing power, can oxidize benzidine to generate colored azo fuel, has specific absorption peak at the position of 406nm of wavelength, detects the light absorption value (A406) of a sample at the position of 406nm, and further calculates the content of residual sarin (GB) and the activity of enzyme.

Reaction system 1.25 mL: the disrupted supernatant was diluted with 0.01M PBS solution to make the protein concentration uniform. 250 mu L of diluted enzyme solution is added into 250 mu L of sarin with the concentration of 100 mu g/mL, after 5min of room temperature reaction, 125 mu L of acetone, 125 mu L of 0.5 wt% benzidine aqueous solution and 500 mu L of 0.25 wt% sodium perborate aqueous solution are sequentially added. A406 was measured and the residual GB content and the enzyme activity were calculated.

Drawing a Shalin standard curve: GB standard solutions 0, 50, 100, 150, 200 and 250 μ L of 100 μ g/mL are weighed, 0.01M PBS solution is added to 500 μ L respectively, 125 μ L acetone, 125 μ L0.5 wt% benzidine aqueous solution and 500 μ L0.25 wt% sodium perborate aqueous solution are sequentially added, and A406 is determined.

The experimental results are shown in Table 1, compared with the non-mutated wild-type OPH, the hydrolysis rate of the sarin is slightly improved by the OPH-Y (mutation point H257Y), reduced by the OPH-T (mutation point L303T), and remarkably improved by increasing the hydrolysis rate of the sarin from 0.452 mg/(min. mg) to 0.879 mg/(min. mg) by the OPH-YT (mutation points H257Y and L303T).

TABLE 1 Activity of OPH mutants to hydrolyze sarin

In summary, the invention includes but is not limited to the above embodiments, and any equivalent replacement or local modification made under the spirit and principle of the invention should be considered as being within the protection scope of the invention.

Sequence listing

<110> China civil liberation army chemical defense institute

<120> organophosphorus hydrolase mutant and application thereof

<160> 2

<210> 1

<211> 365

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Gln Thr Arg Arg Val Val Leu Lys Ser Ala Ala Ala Ala Gly Thr

1 5 10 15

Leu Leu Gly Gly Leu Ala Gly Cys Ala Ser Val Ala Gly Ser Ile Gly

20 25 30

Thr Gly Asp Arg Ile Asn Thr Val Arg Gly Pro Ile Thr Ile Ser Glu

35 40 45

Ala Gly Phe Thr Leu Thr His Glu His Ile Cys Gly Ser Ser Ala Gly

50 55 60

Phe Leu Arg Ala Trp Pro Glu Phe Phe Gly Ser Arg Lys Ala Leu Ala

65 70 75 80

Glu Lys Ala Val Arg Gly Leu Arg Arg Ala Arg Ala Ala Gly Val Arg

85 90 95

Thr Ile Val Asp Val Ser Thr Phe Asp Ile Gly Arg Asp Val Ser Leu

100 105 110

Leu Ala Glu Val Ser Arg Ala Ala Asp Val His Ile Val Ala Ala Thr

115 120 125

Gly Leu Trp Phe Asp Pro Pro Leu Ser Met Arg Leu Arg Ser Val Glu

130 135 140

Glu Leu Thr Gln Phe Phe Leu Arg Glu Ile Gln Tyr Gly Ile Glu Asp

145 150 155 160

Thr Gly Ile Arg Ala Gly Ile Ile Lys Val Ala Thr Thr Gly Lys Ala

165 170 175

Thr Pro Phe Gln Glu Leu Val Leu Lys Ala Ala Ala Arg Ala Ser Leu

180 185 190

Ala Thr Gly Val Pro Val Thr Thr His Thr Ala Ala Ser Gln Arg Asp

195 200 205

Gly Glu Gln Gln Ala Ala Ile Phe Glu Ser Glu Gly Leu Ser Pro Ser

210 215 220

Arg Val Cys Ile Gly His Ser Asp Asp Thr Asp Asp Leu Ser Tyr Leu

225 230 235 240

Thr Ala Leu Ala Ala Arg Gly Tyr Leu Ile Gly Leu Asp His Ile Pro

245 250 255

Tyr Ser Ala Ile Gly Leu Glu Asp Asn Ala Ser Ala Ser Ala Leu Leu

260 265 270

Gly Ile Arg Ser Trp Gln Thr Arg Ala Leu Leu Ile Lys Ala Leu Ile

275 280 285

Asp Gln Gly Tyr Met Lys Gln Ile Leu Val Ser Asn Asp Trp Thr Phe

290 295 300

Gly Phe Ser Ser Tyr Val Thr Asn Ile Met Asp Val Met Asp Arg Val

305 310 315 320

Asn Pro Asp Gly Met Ala Phe Ile Pro Leu Arg Val Ile Pro Phe Leu

325 330 335

Arg Glu Lys Gly Val Pro Gln Glu Thr Leu Ala Gly Ile Thr Val Thr

340 345 350

Asn Pro Ala Arg Phe Leu Ser Pro Thr Leu Arg Ala Ser

355 360 365

<210> 2

<211> 1084

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgcaaacga gaagggttgt gctcaagtct gcggccgccg caggaactct gctcggcggc 60

ctggctgggt gcgcgagcgt ggctggatcg atcggcacag gcgatcggat caataccgtg 120

cgcggtccta tcacaatctc tgaagcgggt ttcacactga ctcacgagca catctgcggc 180

agctcggcag gattcttgcg tgcttggcca gagttcttcg gtagccgcaa agctctagcg 240

gaaaaggctg tgagaggatt gcgccgcgcc agagcggctg gcgtgcgaac gattgtcgat 300

gtgtcgactt tcgatatcgg tcgcgacgtc agtttattgg ccgaggtttc gcgggctgcc 360

gacgttcata tcgtggcggc gaccggcttg tggttcgacc cgccactttc gatgcgattg 420

aggagtgtag aggaactcac acagttcttc ctgcgtgaga ttcaatatgg catcgaagac 480

accggaatta gggcgggcat tatcaaggtc gcgaccacag gcaaggcgac cccctttcag 540

gagttagtgt taaaggcggc cgcccgggcc agcttggcca ccggtgttcc ggtaaccact 600

cacggcagca agtcagcgcg atggtgagca gcaggccgcc atttttgagt ccgaaggcga 660

gcccctcacg ggtttgtatt ggtcacagcg atgatactga cgatttgagc tatctccgcc 720

ctcgctgcgc gcggatacct catcggtcta gaccacatcc cgtatagtgc gatttctaga 780

agataatgcg agtgcatcag ccctcctggg catccgttcg tggcaaacac ggtctcttga 840

tcaaggcgct catcgaccaa ggctacatga aacaaatcct cgtttcgaat ctggactttc 900

gggttttcga gctatgtcac caacatcatg gacgtgatgg atcgcgtgcc ccgacgggat 960

ggccttcatt ccactgagag tgatcccatt cctacgagag aagggcgtcc cacaggaaac 1020

gctggcaggc atcactgtga ctaacccggc gcggttcttg tcaccgacct tgcgggcgtc 1080

atga 1084

Claims

1. An organophosphorus hydrolase mutant, which is characterized in that: the mutant is formed by mutating histidine at 257 th site of organophosphorus hydrolase into tyrosine, and mutating leucine at 303 th site into threonine.

2. The mutant organophosphorous hydrolase according to claim 1, wherein: the amino acid sequence of the organophosphorus hydrolase mutant is shown in SEQ NO. 1.

3. The mutant organophosphorous hydrolase according to claim 1, wherein: the nucleotide sequence of the organophosphorus hydrolase mutant gene is shown in SEQ NO. 2.

4. Use of an organophosphorus hydrolase mutant according to claim 1, wherein: the organophosphorus hydrolase mutant is used as a biological enzyme for degrading paraoxon compounds.

5. The use of an organophosphorus hydrolase mutant according to claim 3, wherein: the paraoxon compound is sarin.