CN114736887A

CN114736887A - Use of carboxylesterase

Info

Publication number: CN114736887A
Application number: CN202210432652.7A
Authority: CN
Inventors: 王萍; 李春明; 殷敬华
Original assignee: Shanghai Weigao Medical Technology Development Co ltd
Current assignee: Shanghai Weigao Medical Technology Development Co ltd
Priority date: 2022-03-25
Filing date: 2022-04-22
Publication date: 2022-07-12

Abstract

The invention relates to the technical field of bioengineering, in particular to application of carboxylesterase. The invention discovers that the nucleotide sequence shown in SEQ ID NO: the EstPS1 enzyme shown in 1 has good activity for degrading ester substances, is prone to degrading short-chain p-nitrophenyl fatty acid esters, and can keep good enzyme activity at high temperature. Research shows that the enzyme has enzyme activity half-lives of 14h, 2h, 31min and 10min at 60, 70, 80, 90 and 100 ℃. The esterase still retains 19% of residual enzyme activity even if the enzyme solution is placed in a boiling water bath for boiling for 10 min.

Description

Use of carboxylesterase

The present application claims priority from the chinese patent application entitled "use of carboxylesterase" filed by the chinese patent office at 25/03/2022 under the application number 202210300169.3, the entire contents of which are incorporated herein by reference.

Technical Field

The invention relates to the technical field of bioengineering, in particular to application of carboxylesterase.

Background

Lipid hydrolases (lipolytics) are a general term for a class of hydrolases having the ability to catalyze the hydrolysis of ester bonds, including carboxylesterase (carboxylesterase) and lipase (lipase), and the esterases commonly referred to as carboxylesterase are compounds used primarily to catalyze fatty acid and aromatic lipids. The lipase has mild reaction condition, stable protein molecular structure and excellent stereoselectivity, and is widely present in animals, plants and microorganisms. Most of esterases used in industry are extracellular enzymes produced by microorganisms, and have more excellent enzymatic properties than esterases derived from animals and plants. Therefore, the esterase derived from microorganisms is widely applied to the fields of agriculture, food brewing, medical and chemical sewage treatment, environmental remediation and the like.

However, in large-scale practical applications, the enzymolysis environment of esterase is usually harsh, and most enzymes cannot maintain good enzyme activity in similar environments.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a carboxylesterase with high temperature resistance and its degradation effect on short-chain substrates at high temperature.

The invention provides a polypeptide with an amino acid sequence shown as SEQ ID NO: 1 in the degradation of esters.

In the following examples, the esters include C2-C18 p-nitrophenyl fatty acid esters.

In the invention, the enzymolysis effect is verified by adopting C2-C18 p-nitrophenyl fatty acid esters, including nitrophenyl acetate (C2), p-nitrophenyl butyrate (C4), p-nitrophenyl hexanoate (C6), p-nitrophenyl octanoate (C8), p-nitrophenyl decanoate (C10), p-nitrophenyl laurate (C12), p-nitrophenyl myristate (C14), p-nitrophenyl palmitate (C16) and p-nitrophenyl stearate (C18).

The invention also provides a method for degrading esters, which uses the amino acid sequence shown as SEQ ID NO: 1 to degrade esters.

In the invention, the esters are p-nitrophenyl fatty acid esters of C2-C18.

In some embodiments, the esters are p-nitrophenyl fatty acid esters from C4 to C12, and C16 to C18.

In some embodiments, the esters include at least one of p-nitrophenyl butyrate, p-nitrophenyl hexanoate, p-nitrophenyl octanoate, p-nitrophenyl laurate, p-nitrophenyl palmitate, and p-nitrophenylstearate.

In the invention, the degradation temperature is 20-100 ℃. In some embodiments, the temperature of the degradation is 80 ℃.

In the invention, the pH value of the degradation is 4.0-10.0. In some embodiments, the pH of the degradation is 8.0.

In the present invention, the degraded buffer comprises Tris-HCl buffer.

The invention discovers that the nucleotide sequence shown in SEQ ID NO: the EstPS1 enzyme shown in 1 has good activity for degrading ester substances, is prone to degrading short-chain p-nitrophenyl fatty acid esters, and can keep good enzyme activity at high temperature. Research shows that the enzyme has enzyme activity half-lives of 14h, 2h, 31min and 10min at 60, 70, 80, 90 and 100 ℃. Even if the enzyme solution is placed in boiling water bath and boiled for 10min, the esterase still retains 19 percent of residual enzyme activity.

Drawings

FIG. 1 is a SDS-PAGE pattern of the induced expression of the high temperature esterase (EstPS1) and its purification assay;

FIG. 2 substrate specificity analysis of the high temperature esterase (EstPS 1);

wherein Cx represents the length of a fatty acid carbon chain corresponding to p-nitrophenol alkanoate, and the substrate specificity of p-nitrophenol heptanoate is 100 percent;

FIG. 3 is a graph showing the optimum reaction temperature profile of the high temperature esterase (EstPS 1);

FIG. 4 is a graph showing the pH optimum reaction profile of high temperature esterase (EstPS 1);

FIG. 5 is a graph of pH stability of high temperature esterase (EstPS 1).

Detailed Description

The present invention provides the use of carboxylesterases, and those skilled in the art can refer to the disclosure herein and suitably modify the process parameters to achieve this. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The high-temperature esterase is cloned from Pseudomonas chrysogenum (Pseudomonas synxanthomonas PS1) separated from oil well wastewater, and sequencing results show that an esterase gene (EstPS1-1923) is an open reading frame containing 1923 base pairs, and the open reading frame encodes 640 amino acids in total. Thus, the high temperature esterase is a polypeptide containing 640 amino acids. The purified esterase (EstPS1) was characterized and showed a pH optimum of 8.0 and a stable presence of the enzyme in the pH range of 5.0 to 9.0. The optimal enzyme activity temperature of the high-temperature esterase (EstPS1) is 60 ℃, excellent heat resistance is shown, and the activity half-lives at 60 ℃, 70 ℃, 80 ℃, 90 and 100 ℃ are 14 hours, 2 hours, 31 minutes, 10 minutes and 1 minute respectively. The high temperature enzyme (EstPS1) can degrade most of pyrethroid insecticides, the degradation speed is changed along with the change of chemical structures of pyrethroid insecticides, and simultaneously, the high temperature enzyme shows stronger degradation activity to other medium and short carbon chain lipids, thereby indicating that the pyrethroid is high temperature esterase with wide substrate specificity.

The reagents adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples:

example 1: obtaining of high temperature esterase (EstPS1) gene sequence

Extracting and purifying the total DNA of Pseudomonas flavigena (Pseudomonas synxanthomonas PS1), and performing PCR amplification by using the purified genomic DNA as a template, wherein PCR amplification primers are as follows: an upstream primer: 5'-CGGATCCATGCTCAAACCAGCGTTTTTC-3' (containing Bam HI cleavage site) downstream primer: the PCR product obtained 5 '-CCTCGAGTTAGAAATCCAGACTCACCCC 3' (containing Xho I cleavage sites) was sent for sequencing. The sequencing result shows that the nucleic acid fragment obtained by the PCR amplification comprises a restriction enzyme site and a nucleic acid fragment with a nucleotide sequence shown by SEQ ID NO.1 in a sequence table; the nucleotide sequence shown in SEQ ID NO.1 is 1923bp in total, and the amino acid sequence shown in SEQ ID NO.2 in the coding sequence table is 640 amino acid residues in total. The nucleic acid fragment with the nucleotide sequence shown in SEQ ID NO.1 in the sequence table is named EstPS 1-1923. Also can be prepared into nucleic acid fragments with the nucleotide sequence shown in SEQ ID NO.1 in the sequence table through artificial synthesis.

Example 2: obtaining of the high temperature Lipase EstPS1

Construction of recombinant plasmid pET-EstPS1

Plasmid pET28a (purchased from Beijing Quanyujin Biotechnology Co., Ltd.) was double-digested with restriction enzymes Bam HI and Xho I, and the vector backbone was recovered. 3. And (3) connecting the enzyme digestion product in the step (1) with the vector skeleton in the step (2) under the action of T4 DNA ligase to obtain the recombinant plasmid. 4. Sequencing verifies the correctness of the sequence. The sequencing result shows that the obtained plasmid is a nucleic acid fragment with the nucleotide sequence shown by SEQ ID NO.1 in the sequence table and inserted between the BamHI and Xho I enzyme cutting sites of the plasmid pET28a, and the plasmid is named as pET-EstPS 1.

Expression and purification of recombinant proteins

The plasmid pET-EstPS1 obtained in the step (I) is transformed into Escherichia coli BL21(DE3) by a chemical transformation method (purchased from Beijing Quanji Biotechnology Co., Ltd.), and transformants are screened on LB agar plates containing 50mg/mL kanamycin, so as to obtain a recombinant bacterium pET-EstPS1/E.coli BL21(DE 3). The recombinant strain obtained by transforming Escherichia coli BL21(DE3) with plasmid pET28a was used as a control strain. The recombinant bacterium pET-EstPS1/E.coli BL21(DE3) was inoculated into 100mL of LB liquid medium (containing 50mg/mL of kanamycin), cultured with shaking at 37 ℃ until OD600 reached about 0.6, added with 0.5mM IPTG to the final concentration, and induced overnight at 25 ℃. The cells were collected by centrifugation at 6000rpm for 10 minutes, and the ratio of the collected cells was 1: 5 Add 100mM Tris-HCl buffer (pH8.0) and sonicate the cells for 10min (200w, sonicate for 2s, stop for 3 s). Cell debris was removed by centrifugation at 12000rpm for 10 minutes to obtain a crude enzyme solution. The crude enzyme solution was purified by ProteinIso Ni-NTA Resin (available from Kyoto gold Biotech Co., Ltd.). After the crude enzyme solution completely flows through the chromatographic column, 5mL of washing solution is used for washing the non-combined hybrid protein in the nickel column, and then 10mL of washing solution is used for washing the weak-combined protein in the nickel column. Finally eluting the target protein by using 5mL of eluent, collecting the eluent, adding 25mL of 100mM Tris-HCl buffer (pH8.0), using a 10000Da protein concentration tube, centrifuging for 30 minutes at 63000rpm, removing redundant imidazole to obtain concentrated eluent, and detecting the obtained high-temperature lipase EstPS1 by SDS-PAGE electrophoresis. The results of the experiment are shown in FIG. 1.

EXAMPLE 3 study of the enzymatic Properties of the high temperature esterase (EstPS1)

Substrate specificity analysis of (I) high temperature esterase (EstPS1)

The activity of the lipase is defined as 1g of solid enzyme powder (or 1mL of liquid enzyme, under the conditions of optimal temperature and pH value, 1 mu mol of titratable fatty acid generated by hydrolyzing a substrate for 1 minute is 1 enzyme activity unit, and is expressed by mu mol (or mu mol). The substrate specificity and the enzyme activity of the esterase are measured by adopting p-nitrophenol alkanoate with different carbon chain lengths, the p-nitrophenol alkanoate with different carbon chain lengths is diluted to 100mmol/L by using acetonitrile, the substrate solution is prepared by the following steps of 1: 4: 95 by using acetonitrile (containing the substrate)/isopropanol/50 mmol/L Tris-HCl buffer solution, the concentration of the p-nitrophenol alkanoate is ensured to be 1mmol/L, 3 mu L of diluted enzyme solution is added, after the incubation is carried out for 15min at 60 ℃, the absorbance of the reaction solution at 405nm is measured, and the experimental result is shown in figure 2.

Experimental results show that the hydrolysis activity of esterase EstPS1 is related to the length of a substrate fatty acid carbon chain of p-nitrophenyl fatty acid ester, EstPS1 is more prone to hydrolyzing p-nitrophenyl fatty acid ester substrates of C4-C18, and the hydrolysis rate of C4 and C8 can reach more than 80%.

Determination of optimum reaction temperature of (II) high temperature esterase (EstPS1)

The optimum reaction temperature of the esterase is determined within the range of 20 ℃ to 70 ℃. Using the reaction system used above, the reaction is carried out for 15min at 20, 30, 40, 50, 60 and 70 ℃, and the absorbance at 405nm is measured, and the measurement result shows that: SEQ ID NO: 1 (EstPS1) has an optimum reaction temperature of 50 ℃ and still has 80 percent of relative activity at 70 ℃.

The results of the experiment are shown in FIG. 3. The temperature tolerance of EstPS1 was determined using 50mM Tris-HCl buffer (pH8.0) and the enzyme solutions were stored in water baths (60, 70, 80, 90 and 100 ℃) at different temperatures for a period of time. The residual enzyme activity was determined under standard experimental conditions.

The optimum temperature of purified EstPS1 is 60 ℃, and the enzyme activity is 2226U/mg at most. And for the temperature tolerance of the enzyme, the temperature is kept for 14h at 60 ℃, and the residual enzyme activity is more than 50%.

EstPS1 is very stable at low temperature of 20-50 ℃, and the half lives of the enzyme at 60, 70, 80, 90 and 100 ℃ are 14h, 2h, 31min and 10min respectively. The esterase still retains 19% of residual enzyme activity even if the enzyme solution is placed in a boiling water bath for boiling for 10 min.

The results of the experiments show that the esterase EstPS1 has very good thermal stability, which is better than that of most esterases reported in the literature. Esterases EstPS1 and EstPS1 delta AT were incubated AT different temperatures for 10min, and the results of comparison of the residual enzyme activities are shown in Table 1:

comparison of thermal stability of esterases reported in the literature of Table 1

Determination of optimum reaction pH of (III) high temperature esterase (EstPS1)

The optimum reaction pH of the high temperature esterase (EstPS1) is determined in the range of 4.0-10.0. The buffer solutions used were: citric acid-sodium citrate buffer system (pH4.0-5.0), phosphate buffer system (pH6.0-7.0), 50mmol/L Tris-HCl (pH8.0), and glycine-sodium hydroxide system (pH 9.0-1.0). The results of the experiment are shown in FIG. 4.

The results show that the effect of pH on esterase EstPS1 activity was determined using p-NPB as the hydrolysis substrate in buffers of different pH values. Purified EstPS1 showed higher esterase activity at pH values ranging from 6.0 to 9.0, with the esterase having the highest hydrolytic activity at pH 8.0. The activity of EstPS1 is obviously reduced when the pH value of the acidic solution is less than 5.0, and only about 30 percent of the maximum enzyme activity is obtained.

(IV) determination of the pH stability of the high temperature esterase (EstPS1)

The pH stability of the high temperature esterase (EstPS1) was determined in the range of 4.0-10.0. And (3) placing the purified enzyme solution into buffer solutions with different pH values, incubating at 37 ℃, and taking out the bacteria solution every 5 hours to determine the enzyme activity. The results show that the lipase (EstPS1) has better pH stability, and the relative enzyme activity is higher than 60% under each pH condition at 20 h. The results of the experiment are shown in FIG. 5.

In conclusion, the invention clones and obtains a new thermostable esterase gene from Pseudomonas flavigena (Pseudomonas synxanthomonas PS1), and discovers that an enzyme protein encoded by the gene has excellent enzymological properties and can be applied to related production processes of ester catalytic reaction. Meanwhile, the engineering bacteria capable of expressing a large amount of esterase are successfully cloned, the large-scale production of the esterase can be realized, and the method becomes the basis of subsequent industrial application.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Sequence listing

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Claims

1. The amino acid sequence is shown as SEQ ID NO: 1 in the degradation of esters.

2. The use of claim 1, wherein the esters comprise p-nitrophenyl fatty acid esters from C2 to C18.

3. A method for degrading esters, characterized in that the degradation method is characterized in that the degradation method comprises the following steps of: the carboxylesterase shown in 1 degrades esters.

4. The degradation method according to claim 3, wherein the ester is a p-nitrophenyl fatty acid ester having a carbon number of from 2 to 18.

5. The degradation method according to claim 4, wherein the esters are C4-C12 and C16-C18 p-nitrophenyl fatty acid esters.

6. The degradation method according to claim 5, wherein the ester comprises at least one of p-nitrophenyl butyrate, p-nitrophenyl hexanoate, p-nitrophenyl octanoate, p-nitrophenyl laurate, p-nitrophenyl palmitate, and p-nitrophenylstearate.

7. The degradation method according to claim 3, wherein the temperature of the degradation is 20-100 ℃.

8. The degradation method according to claim 7, wherein the temperature of degradation is 80 ℃.

9. The degradation method according to claim 3, wherein the pH value of the degradation is 4.0 to 10.0.

10. A degradation method according to claim 3, characterized in that the pH value of the degradation is 8.0.