CN106834250B

CN106834250B - Esterase EstK1 protein and application thereof

Info

Publication number: CN106834250B
Application number: CN201610878042.4A
Authority: CN
Inventors: 毛相朝; 董浩; 孙建安; 薛长湖
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2016-10-08
Filing date: 2016-10-08
Publication date: 2020-04-28
Anticipated expiration: 2036-10-08
Also published as: CN106834250A

Abstract

The present invention provides an esterase EstK1 protein and an application thereof, which discloses the amino acid sequence and nucleotide sequence of a novel esterase EstK1. Since the enzyme has good tolerance and high transesterification activity in a hydrophobic organic solvent, EstK1 was used to prepare ethyl ester flavors; because EstK1 had the ability to efficiently catalyze the preparation of cinnamyl acetate, most of the cinnamyl alcohol could be converted into cinnamyl acetate within 2 h, and the recovery rate of cinnamyl acetate reached 97.1%. And the study of the substrate spectrum of the present invention shows that EstK1 can be used to prepare n-octanol ethyl ester, 2-phenethyl alcohol ethyl ester, citronellol ethyl ester and geraniol ethyl ester, etc., suggesting that the esterase EstK1 has the ability to prepare ethyl ester fragrances. potential.

Description

Esterase EstK1 protein and application thereof

Technical Field

The invention belongs to the technical field of functional enzyme screening, and particularly relates to esterase EstK1 protein and application thereof.

Background

Esterases (esterases; EC 3.1.1.1) belong to one of the lipolytic enzymes, catalyze the hydrolysis and synthesis of ester bonds, and are widely present in animals, plants and microorganisms. The lipase and esterase belong to the same lipolytic enzymes, have the same mechanism for catalyzing ester bond synthesis and hydrolysis, and are different from the mechanism that lipase catalyzes hydrolysis to synthesize water-insoluble long-chain glyceride (the carbon chain length is more than or equal to 10) and esterase catalyzes hydrolysis to synthesize water-soluble short-chain glyceride (the carbon chain length is less than 10). In the water phase, esterase catalyzes hydrolysis reaction, and in the organic phase, the esterase can catalyze the reactions of esterification, transesterification, ester exchange, aminolysis and the like. Because of its good substrate specificity, stereoselectivity and regioselectivity, esterase becomes an important industrial enzyme and is widely applied to industries such as food, medicine, detergent and chemical industry. However, unlike lipases, only a very small number of esterases are currently used in organic synthesis. Therefore, development of a novel esterase having excellent properties is of great industrial application value.

Acetate is a very important ester, and is often used as a flavoring agent or a perfume in the industries of food, cosmetics, medicine and the like. Wherein cinnamyl acetate belongs to phenylpropanoids and exists in fresh cassia bark under natural conditions. Studies show that the lauryl acetate has good biological activity, particularly oxidation resistance, which is probably due to the structural characteristics of the lauryl acetate. Lauryl acetate has been used to synthesize HIV-protease inhibitors. In addition, the lauryl acetate is widely applied to the perfume industry due to good sweet, fragrant and flowery odor. Natural cinnamyl acetate can be extracted from plants, but the low extraction rate leads to great increase of production cost; the chemical synthesis method is a method for efficiently preparing cinnamyl acetate, but the use of toxic chemicals reduces the safety of the product, and the low specificity of the chemical method causes more byproducts to be generated in the reaction process. The enzyme-catalyzed method is considered to be an ideal alternative, and the enzyme-catalyzed method is receiving increasing attention due to its mild reaction conditions and environmentally friendly process. However, the existing lipase catalysis method mainly used for preparing cinnamyl acetate through enzyme catalysis has the problems of low conversion rate and long reaction time, which may be related to the characteristic that lipase prefers long-chain fatty acid. Esterases favor short chain fatty acids, so the use of esterase catalysis to prepare lauryl acetate is considered a good choice.

Disclosure of Invention

The invention aims to provide esterase EstK1 protein and application thereof, and the esterase is used for efficiently catalyzing the synthesis of acetate such as cinnamyl acetate and the like in a non-aqueous phase by using the transesterification activity of the esterase, so that the defects of the prior art are overcome.

The amino acid sequence of the esterase EstK1 protein is shown in Seq ID NO. 2.

The invention provides an estK1 gene for coding esterase EstK1 protein, and the nucleotide sequence of the gene is shown in Seq ID No. 1.

The invention provides a vector carrying an estK1 gene, which comprises pET21a (+), pET28a (+) and pET32a (+).

The invention provides a recombinant expression vector carrying the vector, which is Escherichia coli BL21(DE 3).

The invention provides a transesterification reaction, which utilizes esterase EstK1 protein to produce cinnamyl acetate in a non-aqueous phase.

Further, the transesterification reaction was catalyzed using EstK1 lyophilized whole cells.

Further, the acyl donor of the transesterification reaction is vinyl acetate.

Further, the organic solvent of the transesterification reaction is isooctane.

Further, the molar ratio of the substrate cinnamyl alcohol and vinyl acetate of the transesterification reaction is 1: 4.

Further, the reaction temperature of the transesterification reaction was 40 ℃.

The esterase EstK1 protein is used for producing n-octanol ethyl ester, 2-phenethyl alcohol ethyl ester, citronellol ethyl ester or geraniol ethyl ester in a non-aqueous phase with a good effect.

Has the advantages that:

the esterase can efficiently catalyze the transesterification reaction of cinnamyl alcohol and vinyl acetate to generate cinnamyl acetate. The recovery rate of the cinnamyl acetate reaches 94.1% in 1 hour, most of the substrate cinnamyl alcohol is converted in 2 hours, the recovery rate of the ethyl laurate reaches 97.1%, and the preparation time of the cinnamyl acetate is greatly shortened.

The esterase can catalyze the synthesis of other acetates, such as n-octanol ethyl ester, 2-phenethyl alcohol ethyl ester, citronellol ethyl ester and geraniol ethyl ester, and embodies the potential of the esterase in the preparation of acetate perfumes.

Drawings

FIG. 1: the esterase EstK1 evolutionary tree analysis and multiple sequence alignment schematic diagram is shown;

FIG. 2: SDS-PAGE electrophoresis of the esterase EstK1 of the invention; wherein, Lane 1 is purified EstK1, Lane 2 is protein Marker, Lane 3 is the supernatant of the expressed thallus crushing liquid, Lane 4 is the sediment of the expressed thallus crushing liquid;

FIG. 3: EstK1 enzymatic property study; wherein, the graph a is the preference of EstK1 carbon chain length, the graph b is the optimum pH of EstK1, the graph c is the optimum temperature of EstK1, and the graph d is the influence of detergent on the activity of EstK1 esterase;

FIG. 4: optimizing conditions for preparing cinnamyl acetate from EstK1 non-aqueous phase; wherein, panel a is the effect of acyl donor on transesterification, panel b is the effect of organic solvent on transesterification, panel c is the effect of vinyl acetate molar ratio of cinnamyl alcohol on transesterification, panel d is the effect of temperature on transesterification;

FIG. 5: the synthesis time curve of cinnamyl acetate under different addition amounts of thalli;

FIG. 6: transesterification to generate lauryl acetate 2h HPLC result chart;

FIG. 7: the substrate spectrum of acetate synthesized by EstK 1.

Detailed Description

The present invention is described in detail below with reference to examples and figures, but the scope of protection is not limited thereto.

The invention obtains a novel IV family esterase with good properties by gene library construction and specificity screening methods, and the esterase comprises:

1) an enzyme having the amino acid sequence of SEQ ID NO. 1;

2) the gene of the esterase has a nucleotide sequence of SEQ ID NO. 2;

the esterase EstK1 has the capability of catalyzing transesterification in a non-aqueous phase, and can catalyze specific alcohols and short-chain acetate to carry out transesterification so as to generate corresponding acetate perfume.

As shown in FIGS. 1 to 7, the esterase EstK1 protein of the present invention and its use are illustrated in detail by the following five examples.

Example 1: construction of Gene libraries and analysis of lipolytic enzyme sequences

In previous studies, this laboratory screened a strain (OUC _ Estk) that produced esterase in high yield and identified it as Acinetobacter hemolyticus.genomic DNA was extracted, incompletely digested with Sau3AI restriction enzyme and 2-7kb DNA fragments were recovered.the recovered fragments were ligated to pBluescript IISK (+) vector that was completely digested with BamHI and dephosphorylated with alkaline phosphatase.A recombinant vector was introduced into DH5 α competent cells and spread on LB ampicillin resistant solid plates containing tributyrin, after 1-2d static culture in 37 ℃ incubator, positive clones with distinct clearing circles were picked up for culture, plasmids were extracted and sent to sequencing, after sequencing was completed, a lipolytic enzyme full-length gene evolution tree analysis (FIG. 1a) with 1071bp size was obtained, EstK1 belongs to the family of lipolytic enzymes IV, catalytic triads (FIG. 1b) were Ser (201), Asp (299), His (325), respectively.

Example 2: cloning, expression and purification of esterase EstK1

According to the obtained EstK1 full-length gene, a full-length primer of the gene is designed, wherein an upstream primer: CGCGGATCCATGTTTGCATTGAATGAATCACTTA (BamHI cleavage site), downstream primer: CCCAAGCTTTTAACTACGTTTATCCCAAAATTTACG (HindIII cleavage site). The digested target fragments were ligated to pET21a (+), pET28a (+), and pET32a (+) vectors which were similarly digested, respectively, and the recombinant plasmids were finally introduced into BL21(DE3) for inducible expression. After induction expression, thalli are broken, enzyme activity detection shows that active esterase can be expressed by using three vectors, and pET32a (+) is selected as a final expression vector because the protein content expressed by using pET32a (+) is higher. The recombinant protein EstK1 was purified.

The gel electrophoresis of the esterase EstK1 is shown in FIG. 2, wherein a band 1 is purified EstK1, a band M is a protein marker, a band 2 is a supernatant of a crushing liquid, and a band 3 is a precipitate of the crushing liquid.

Example 3: esterase EstK1 enzymological Properties

(1) Esterase EstK1 substrate specificity study

The preference of esterase EstK1 for carbon chain length was examined in 100mM Tris-HCl buffer pH 8.0 using pNP esters of different carbon chain lengths (C2, C4, C8, C10, C12, C14, C16). As shown in FIG. 3a, the esterase EstK1 showed the highest activity for C4 and very low activity for pNP > 10 carbons, indicating that esterase EstK1 is an esterase.

(2) Optimum pH

The pH optimum of EstK1 was determined for the purified esterase EstK1 using different pH buffers. The reaction process takes pNPB (C4) as a substrate, and different pH buffers are respectively as follows: sodium phosphate buffer (

pH

6, 7, 8), Tris-HCl buffer (pH 8, 9) and Na₂CO₃-NaHCO₃Buffer (pH 9, 10). As shown in fig. 3bThe optimum pH of EstK1 was 8.0, and EstK1 showed higher activity in a more alkaline environment, indicating that EstK1 is an alkaline esterase.

(3) Optimum temperature

The process takes pNPB (C4) as a substrate, and detects the influence of different temperatures (30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 60 ℃) on the enzyme activity of EstK1 in 100mM Tris-HCl buffer solution with the pH value of 8.0. As shown in FIG. 3c, the optimum temperature of EstK1 was 40 ℃ and the enzyme activity at 35 ℃ was close to that at 40 ℃.

(4) Effect of detergents

The process is to study the change of enzyme activity by adding 0.5% of detergent (Tween 20, Tween 60, Tween 80, Triton X-100, SDS) into the reaction system, wherein the enzyme activity is defined as 100% when no detergent is added as a control. As shown in FIG. 3d, after Tween 60, Tween 80 and Triton X-100 are added, the enzyme activity of esterase is improved to different degrees, wherein the enzyme activity of Tween 60 is improved to the maximum extent, the enzyme activity is improved to 119.47%, Tween 20 has a slight reduction effect on the enzyme activity, and SDS significantly inhibits the activity of esterase EstK 1.

(5) Effect of Metal ions on enzyme Activity

Enzyme activity detection is carried out by adding different metal ions, and different effects are shown as shown in table 1. Wherein Fe²⁺The esterase activity is obviously improved, and 10mM Fe is added²⁺And the esterase activity is improved to 234.97%. Zn²⁺Has the maximum inhibition effect on the enzyme activity, and the enzyme activity is reduced to 31.79 percent.

TABLE 1

(6) Influence of organic solvent on enzyme Activity

Adding different organic solvents into the enzyme solution to enable the organic solvents to reach final concentrations of 25% and 50% respectively, placing the mixture in a temperature of 37 ℃, shaking and incubating the mixture for 3 hours, and then detecting the enzyme activity of the residual esterase. The water-soluble organic solvent is diluted to 5% for detection, and the water-insoluble organic solvent is removed by centrifugation, and the esterase activity is detected. As can be seen from the results in Table 2, hydrophobic organic solvents such as alkylhydrins (cyclohexane, n-hexane and isooctane) had the least effect on the esterase EstK 1.

TABLE 2

Example 4: non-aqueous preparation of cinnamyl acetate by esterase EstK1

The acetic ester is prepared by adopting non-aqueous phase catalysis, so that the subsequent purification and other steps can be simplified, and the method has obvious advantages. In the process, EstK1 freeze-dried whole cells are used as a catalyst, and cinnamyl acetate is prepared in an organic phase in a catalytic manner.

(1) Effect of acyl donors on transesterification

As shown in fig. 4a, the effect of different acyl donors (vinyl acetate, isopropenyl acetate, isoamyl acetate, methyl acetate, ethyl acetate and butyl acetate) on the transesterification effect was investigated. Among them, vinyl acetate is the most active as an acyl donor, isopropenyl acetate is the next to vinyl acetate, and other acyl donors are less active. This is probably because when vinyl acetate and isopropenyl acetate are used as acyl donors, the product enol is unstable and is converted into aldehyde, and the reaction from the enol to the aldehyde is an irreversible process, thereby promoting efficient progress of the transesterification reaction.

(2) Effect of organic solvents on transesterification

Their effect on EstK1 transesterification activity was investigated by using different hydrophobic organic solvents, and the results are shown in fig. 4 b. From the results, it can be seen that when isooctane is used as an organic solvent, EstK1 is most effective in transesterification, and therefore isooctane is selected as the best organic solvent.

(3) Effect of the cinnamyl alcohol vinyl acetate molar ratio on the transesterification

The process examined the effect of the molar ratio of cinnamyl alcohol to vinyl acetate on the transesterification reaction, setting the molar ratio at 1:1,1:2,1:3,1:4,1:5 and 1:6, and the results are shown in fig. 4 c. It can be seen from the results that the transesterification effect becomes gradually significant as the molar ratio increases, but the difference between the transesterification effects is no longer significant after the molar ratio is 1: 4. For economic reasons, 1:4 was taken as the optimum molar ratio of cinnamyl alcohol vinyl acetate.

(4) Influence of temperature on transesterification

The temperature affects not only the activity and stability of the enzyme but also the viscosity of the medium and the reaction equilibrium, etc. By carrying out the transesterification reaction at different temperatures (25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃), it was found that different temperatures have a significant influence on the transesterification reaction (FIG. 4 d). Of these, EstK1 is most effective in transesterification at 40 ℃ and is therefore selected as the optimum reaction temperature at 40 ℃.

(5) Curve of transesterification at different addition levels of bacteria

In this process, under the optimum reaction conditions (vinyl acetate as acyl donor, isooctane as organic solvent, molar ratio 1:4, reaction at 40 ℃), transesterification curves were examined under different cell addition amounts, and the experimental results are shown in fig. 5 with time as abscissa and recovery rate of lauryl acetate as ordinate. When the addition amount of the bacterial cells is 10mg/mL, the recovery rate of the cinnamyl acetate reaches 94.1% after 1 hour, and the recovery rate gradually increases along with the prolonging of time. After 2h of reaction, the substrate cinnamyl alcohol is mostly consumed, and the recovery rate of cinnamyl acetate reaches 97.1%, wherein the liquid phase result is shown in figure 6.

Example 5: non-aqueous preparation of other acetates by esterase EstK1

In order to verify the ability of esterase EstK1 to prepare other acetates, different alcohols (n-butanol, n-pentanol, isoamyl alcohol, n-octanol, terpineol, phenol, 3-chlorophenol, 2-phenylethyl alcohol, cinnamyl alcohol, citronellol, geraniol and linalool) were used as substrates, and after the same reaction time, the amount of the product produced was detected, wherein cinnamyl alcohol was used as a positive control. The experimental result shows that esterase EstK1 can utilize n-octanol, 2-phenethyl alcohol, citronellol and geraniol as substrates to prepare corresponding acetate, wherein the transesterification efficiency of esterase EstK1 to 2-phenethyl alcohol is highest, and the result is shown in FIG. 7.

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.

Claims

1. A transesterification method, comprising: producing cinnamyl acetate by using esterase EstK1 protein in a non-aqueous phase; the amino acid sequence of the esterase EstK1 protein is shown in Seq ID NO. 2.

2. A transesterification method according to claim 1, wherein: it uses esterase EstK1 protein freeze-dried whole cells for catalytic reaction.

3. A transesterification method according to claim 1, wherein: the acyl donor is vinyl acetate.

4. A transesterification method according to claim 1, wherein: the organic solvent is isooctane.

5. A transesterification method according to claim 3, wherein: the molar ratio of the substrates cinnamyl alcohol and vinyl acetate is 1: 4.

6. A transesterification method according to claim 1, wherein: the reaction temperature was 40 ℃.