CN107119030B

CN107119030B - Esterase and application thereof

Info

Publication number: CN107119030B
Application number: CN201710359673.XA
Authority: CN
Inventors: 毛相朝; 董浩; 孙建安; 刘振; 薛长湖
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2017-05-20
Filing date: 2017-05-20
Publication date: 2020-05-01
Anticipated expiration: 2037-05-20
Also published as: CN107119030A

Abstract

The invention aims to provide a novel esterase, and the amino acid sequence of the esterase is SEQ ID NO. 1. The esterase of the invention can catalyze the hydrolysis of astaxanthin ester to generate free astaxanthin, after 48 hours of catalytic hydrolysis, most astaxanthin ester can be hydrolyzed, the degree of hydrolysis can reach more than 99 percent, and the potential of the esterase in the preparation of natural free astaxanthin is reflected.

Description

Esterase and application thereof

Technical Field

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

Background

Lipid hydrolases (Liptic enzymes, EC 3.1.1.x) are a member of α/β hydrolase family, can catalyze hydrolysis and synthesis of ester bonds, and are widely present in animals, plants and microorganisms, and can be divided into esterases (EC 3.1.1.1) and lipases (Lipase, EC 3.1.1.3) according to the difference of the length of fatty acid chain of a substrate catalyzed by the lipid hydrolases, the lipid hydrolases can catalyze the hydrolysis reaction in an aqueous phase, and can catalyze the reactions of transesterification, ester synthesis, ester exchange, ammonolysis and the like in an organic system.

The increasing demand for lipid hydrolases for industrialization has also prompted the continued sophistication and development of screening methods. The construction method of the metagenome library is a means for extracting the genome DNA of all microorganisms in a specific environment, and performing gene analysis and cloning. It is reported that about 99% of microorganisms are in an unculturable state in nature, and therefore, constructing a metagenomic library makes it possible to sufficiently utilize the microbial resources in nature, and the metagenomic library becomes an efficient means for obtaining an enzyme catalyst with excellent properties.

Astaxanthin (Astaxanthin), also known as Astaxanthin, Astaxanthin and lobster shell pigment, a carotenoid of formula C₄₀H₅₂O₄Astaxanthin is widely distributed, particularly in aquatic animals such as lobsters, and is a major pigment of such organisms, such as the red flesh color of marine products such as lobsters, i.e., astaxanthin is accumulated in the body, astaxanthin is unstable and is easily oxidized, and the oxidized product is astaxanthin (as-tacene). numerous studies have shown that astaxanthin has good physiological functions, such as oxidation resistance, aging resistance, immunity enhancement, tumor resistance, etc., and the uniqueness of the molecular structure makes astaxanthin the most potent antioxidant in natureOn the other hand, free astaxanthin can be used for the function comparison study with astaxanthin ester. In addition, the prepared high-purity free astaxanthin can be used as a standard substance.

At present, a saponification method is mainly adopted for preparing free astaxanthin by hydrolyzing astaxanthin ester, but a large amount of alkali and organic solvent are used in the saponification process, so that the environment is damaged, the waste is caused, and in addition, the astaxanthin is damaged by using the alkali, and byproducts such as astaxanthin and the like are generated. The other method is hydrolysis by esterase or lipase, which provides a new idea for hydrolysis of astaxanthin ester. However, the existing lipid hydrolase hydrolysis method still has the problems of small treatment capacity, low conversion rate and the like, so that the search for a novel lipid hydrolase to apply the lipid hydrolase to the preparation of free astaxanthin is very important.

Disclosure of Invention

The invention aims to provide a novel esterase and a method for preparing free astaxanthin by efficiently catalyzing and hydrolyzing astaxanthin ester by using the hydrolytic activity of the esterase, thereby overcoming the defects of the prior art.

The invention firstly provides an esterase, which comprises the following components:

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

2) an enzyme derived from 1) by substituting, deleting or adding one or more amino acids in 1) and having the esterase function in 1);

one nucleotide sequence of the gene for coding the esterase is SEQ ID NO. 2;

in still another aspect, the present invention provides a recombinant expression vector carrying a gene encoding the above esterase;

the invention also provides a recombinant host for recombinant expression of the esterase;

the esterase of the invention is used for preparing free astaxanthin by catalyzing and hydrolyzing astaxanthin ester.

One specific preparation method is to use esterase powder to hydrolyze haematococcus pluvialis oil and/or shrimp oil to prepare free astaxanthin.

The esterase of the invention can catalyze the hydrolysis of astaxanthin ester to generate free astaxanthin, after 48 hours of catalytic hydrolysis, most astaxanthin ester can be hydrolyzed, the degree of hydrolysis can reach more than 99 percent, and the potential of the esterase in the preparation of natural free astaxanthin is reflected.

Drawings

FIG. 1: schematic representation of astaxanthin ester hydrolysis catalyzed by different lipid hydrolases;

FIG. 2: the esterase Est3-14 evolutionary tree analysis and multiple sequence alignment schematic diagram is shown;

FIG. 3: SDS-PAGE electrophoresis of esterase Est3-14 of the invention. Lane 0 is a protein Marker, Lane 1 is the supernatant of the disrupted cell solution after expression, Lane 2 is the precipitate of the disrupted cell solution after expression, and Lane 3 is purified Est 3-14;

FIG. 4: est3-14 enzymatic Properties. FIG. a is the preference of carbon chain length of substrate Est3-14, FIG. b is the optimum temperature of Est3-14, FIG. c is the optimum pH of Est3-14, and FIG. d is the effect of surfactant on Est3-14 esterase activity;

FIG. 5: est3-14 catalyzes the HPLC results of astaxanthin ester hydrolysis.

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.

Example 1: heterologous expression of novel esterase and screening of astaxanthin ester hydrolase

After sequencing the constructed marine sludge metagenome library, through sequence function analysis, we find some target fragments with assumed lipid hydrolase activity. Partial lipid hydrolase fragments (est3-14, X1, X2, X3, X4, X5 and X6) are selected for heterologous expression, pET-28a (+) is used as a vector, and BL21(DE3) is used as a host.

And (2) carrying out induction fermentation on the lipid hydrolase engineering bacteria from the metagenome, centrifuging 50mL of fermentation liquor after fermentation is finished to remove a supernatant, washing the bacteria by using 0.9% NaCl solution, then re-dissolving by using 6 mM Tris-HCl buffer solution (100mM, pH 7.5), and carrying out ultrasonic crushing in ice bath. The supernatant of the crushing liquid is used for verifying the function of hydrolyzing astaxanthin ester, and a hydrolysis reaction system comprises the following steps: 0.5% haematococcus pluvialis oil, 0.5mL of absolute ethyl alcohol and 6mL of supernatant of the crushing liquid. After the reaction mixture was incubated in a 37 ℃ water bath shaker for 48 hours with shaking, the extraction was repeated using an extract (isopropanol/dichloromethane ═ 1:1), and the produced free astaxanthin was detected by TLC method. TLC results (FIG. 1) showed that lipid hydrolase Est3-14 had the ability to hydrolyze astaxanthin ester to produce free astaxanthin, whereas 6 lipid hydrolases X1, X2, X3, X4, X5, X6 were unable to hydrolyze astaxanthin ester.

Example 2: sequence analysis of esterase Est3-14 Gene

The classification of the family of lipid hydrolases Est3-14 was carried out using literature reported methods, using MEGA 6.0 software to construct a phylogenetic tree of Est3-14 and other families of lipid hydrolases, Clustal X software for multiple sequence alignments of lipid hydrolases, and ESPrip 3.0(http:// ESPript. ibcp. fr/ESPrip /) for the export of aligned sequences. As shown in FIG. 2a, Est3-14 belongs to the V family of lipid hydrolyzing enzymes and has the typical catalytic triad Ser (position 115), Asp (position 237), His (position 363) (FIG. 2 b). The amino acid similarity of the enzyme with the reported protein in GenBank is only 51 percent at most (Acidimicrobium sp.).

Example 3: purification of esterase Est3-14

And (3) carrying out induction fermentation on esterase Est3-14 again, collecting thalli by centrifugation after the fermentation is finished, and washing the thalli once by using sterilized normal saline. After the completion of washing, the cells were reconstituted with Tris-HCl buffer (pH 8.0,100mM) containing 10mM imidazole and sonicated in an ultrasonic cell disruptor, and the supernatant of the disruption solution was used for purification of esterase Est 3-14. The purification was carried out using a nickel column (1mL, Qiagen, Hilden, Germany) and gradient elution of the proteins was carried out using Tris-HCl buffers (pH 8.0,100mM) containing different concentrations of imidazole (20mM-500 mM). And respectively collecting the eluted solutions, concentrating and desalting by using an ultrafiltration concentration tube (10 kDa), and carrying out protein electrophoresis analysis and lipase/esterase enzyme activity detection.

The results of protein gel electrophoresis of the esterase Est3-14 are shown in FIG. 3, wherein a band 0 is a protein marker, a band 1 is a supernatant of a cell disruption solution, a band 2 is a precipitate of the cell disruption solution, and a band 3 is purified Est 3-14.

Example 4: esterase Est3-14 enzymological Properties

Esterase Est3-14 purified in example 3 was used for the enzymatic characterization.

(1) Esterase Est3-14 substrate specificity study

The optimal substrate of Est3-14 was explored by selecting pNP esters of different carbon chain lengths (C2, C4, C8, C10, C12, C14, C16): pNP acetate (pNPA), pNP butyl (pNPB), pNP captylate (pNPC), pNPdecanoate (pNPD), pNP laurate (pNPL), pNP myrtate (pNPM) and pNP palmate (pNPP). The activity at the optimum carbon chain length is defined as 100% and the activities at other carbon chain lengths are expressed as a percentage of the highest activity. As shown in FIG. 4a, the substrate spectrum of Est3-14 is broad, and the hydrolysis activity is good for pNP ester of C2-C16, wherein the hydrolysis activity of Est3-14 to C4(pNPB) is highest, and the hydrolysis activity to pNP ester with carbon chain length >10 is weaker, which indicates that Est3-14 is esterase.

(2) Esterase Est3-14 optimum temperature

Optimum temperature of esterase Est3-14 by incubating the reaction at different temperatures (20, 25, 30, 35, 40, 45, 50 ℃) and detecting the absorbance A₄₀₅To be determined. The activity at the optimum temperature is defined as 100%, and the activities at other temperatures are expressed as a percentage of the maximum activity. From the results (FIG. 4b), it can be seen that the temperature<At 40 deg.C, esterase activity gradually increases with increasing temperature, and temperature>After 40 ℃, the esterase activity gradually decreases with increasing temperature, and the optimum temperature of Est3-14 is 40 ℃.

(3) Esterase Est3-14 optimum pH

The effect of pH on esterase Est3-14 activity was examined using different buffers at different pH. The buffers used in this process included: 100mM citric acid buffer (pH 4.0-6.0), 100mM sodium phosphate buffer (pH 6.0-8.0), 100mM Tris-HCl buffer (pH 8.0-9.0), and 100mM Na₂CO₃-NaHCO₃Buffer (pH 9.0-10.0). The activity at the optimum pH is defined as 100%Activity at other pH is expressed as a percentage. The results (FIG. 4c) show that Est3-14 was Na at pH 9₂CO₃-NaHCO₃The highest hydrolytic activity was shown in the buffer, and Est3-14 showed higher hydrolytic activity under alkaline conditions than under acidic conditions, indicating that Est3-14 is a typical alkaline esterase. (4) Effect of surfactants on esterase Est3-14 Activity

To examine the effect of surfactants on esterase Est3-14, we measured by adding different surfactants (final concentration 0.5%) to the reaction solution, using reagents including: SDS, TritonX-100, Tween 20, Tween60 and Tween 80. The activity measured in the reaction solution without the addition of a surfactant was defined as 100%, and the activity with the addition of a surfactant was expressed as a percentage. The result is shown in FIG. 4d, and through comparison with a sample without surfactant, Triton X-100, Tween 20, Tween60 and Tween 80 are found to have a promoting effect on the improvement of the enzyme activity of esterase, wherein the improvement of the hydrolysis activity of esterase Est3-14 by Tween 20 is the largest and is increased by 22.7%, and SDS has a remarkable inhibiting effect on the activity of Est 3-14.

(5) Effect of Metal ions on enzyme Activity

Effect of Metal ions on esterase Est3-14 Activity by adding Metal ions (CoCl) to the reaction System at final concentrations of 1mM and 10mM₂，KCl，LiCl，FeSO₄，FeCl₃，MnCl₂，CaCl₂，MgCl₂，ZnCl₂And NiCl₂) And Na₂EDTA. Without addition of metal ions and Na₂EDTA as a control, the activity of which is defined as 100%, the results are shown in the following table.

Metal ions are added externally, the improvement of Est3-14 hydrolytic activity is not remarkably promoted, and under the condition of the addition of 1mM metal ions, Co²⁺And K⁺Has slight promotion effect on the improvement of enzyme activity, the enzyme activity is respectively improved to 107.9 percent and 105.6 percent, and other metal ions and Na₂EDTA has the inhibition effect on the enzyme activity of Est 3-14. At 10mM metal ion and Na₂The hydrolytic activity of the esterase Est3-14 was varied with the addition of EDTADegree of inhibition of Zn²⁺The inhibition effect on the enzyme activity is strongest, and the activity is only 23.2 percent of that of a control group.

(6) Influence of organic solvent on enzyme Activity

The effect of organic solvents on esterase Est3-14 activity was examined by adding organic solvents to the reaction system at final concentrations of 25%, 50% and 100%, the organic solvents selected included: methanol, ethanol, acetonitrile, n-hexane, chloroform, dimethyl sulfoxide, acetone, n-propanol, isopropanol, isooctane and cyclohexane. Before the determination, the enzyme solution and the organic solvent are mixed and then placed at 30 ℃ for shaking incubation for 3h, and the enzyme activity of the residual esterase is determined by a colorimetric method.

For a hydrophobic organic solvent, after removing the organic solvent from the incubated mixed solution through centrifugation, performing enzyme activity determination on the residual enzyme liquid water phase; for hydrophilic organic solvent, the incubated mixed solution is diluted by buffer solution until the concentration of the organic solvent is 5%, and then the measurement is carried out, so that the influence of the organic solvent on the enzyme activity measurement is eliminated.

As shown in the following table, Est3-14 maintains better activity in a low concentration (25%) of hydrophilic organic solvent, and the enzyme activity shows a remarkable descending trend as the concentration is increased to 50%, but when the concentration of the hydrophilic organic solvent is increased to 100%, the activity of Est3-14 in a part of hydrophilic organic solvent is remarkably improved compared with that of 50%, for example, the residual activity of Est3-14 in 50% acetonitrile is 20.0%, and the activity in 100% acetonitrile is 90.2%. Est3-14, a residual activity in hydrophobic organic solvents, showed an irregular change. The activity of Est3-14 in anhydrous n-hexane is high, and the residual activity can still reach 88.2% after incubation, which suggests the potential of non-aqueous phase catalysis of Est3-14 in n-hexane.

Example 4: esterase Est3-14 for catalyzing and hydrolyzing astaxanthin ester in haematococcus pluvialis oil

And (3) fermenting esterase Est3-14 again, centrifuging the collected escherichia coli, redissolving the escherichia coli by using ultrapure water, and placing the escherichia coli in an ultrasonic crusher for crushing treatment. After the precipitate of the crushing liquid is removed by centrifugation, the supernatant of the crushing liquid is frozen and dried, and the prepared Est3-14 enzyme powder is used for research on hydrolysis of astaxanthin ester. The reaction system is as follows: 0.5% Haematococcus pluvialis oil, 0.5mL absolute ethanol, 6mL Tris-HCl buffer, 1700U esterase Est3-14 (defined as hydrolytic activity on pNPB). Placing the reaction mixed solution in a water bath shaker at 37 ℃ for shaking reaction, sampling at 48h and 72h respectively, and detecting the content of the generated free astaxanthin by using an HPLC method.

As shown in FIG. 5, in the HPLC chart, peak 1 was the free astaxanthin formed, and the substance which showed a peak after 20min was astaxanthin ester. Compared with the result of 0h, the astaxanthin ester can be hydrolyzed completely in 48h after 1700U esterase Est3-14 is added externally, the hydrolysis rate reaches more than 99%, and the result shows that the screened Est3-14 has very good potential in hydrolyzing the astaxanthin ester.

Example 5: esterase Est3-14 for catalyzing hydrolysis of astaxanthin ester in shrimp oil

Est3-14 enzyme powder was prepared by fermentation and used in the hydrolysis study of astaxanthin ester in shrimp oil as in example 4. The hydrolysis system is as follows: 1.0% shrimp oil, 0.5mL absolute ethanol, 6mL Tris-HCl buffer, 2000U esterase Est3-14 (defined as hydrolytic activity towards pNPB). Placing the reaction mixed solution in a water bath shaking table at 37 ℃ for shaking reaction for 48 hours, and detecting the content of the generated free astaxanthin by using an HPLC method.

The hydrolysis result shows that under the condition of adding 2000U esterase Est3-14, astaxanthin ester in the shrimp sauce is basically and completely hydrolyzed within 48 hours, the hydrolysis rate can reach more than 99 percent, and the generation of byproducts such as astaxanthin and the like is greatly reduced.

SEQUENCE LISTING

<110> China oceanic university

<120> esterase and application thereof

<130>

<160>2

<170>PatentIn version 3.5

<210>1

<211>290

<212>PRT

<213>1

<400>1

Met Ser Val Lys Pro Thr Ser Val Met Asp Ile Pro Pro Leu Leu Pro

1 5 10 15

Gly Arg Leu Ile Ser Leu Pro Gly Arg Gly Glu Ile Phe Val Arg His

20 25 30

His Gln His Val Asn Pro Asp Ala Pro Thr Leu Leu Leu Leu His Gly

35 40 45

Trp Thr Ala Ser Ser Asp Leu Gln Phe Phe Thr Ala Tyr Glu Glu Leu

50 55 60

Ser Arg Asn Tyr Ser Ile Val Gly Val Asp His Arg Gly His Gly Arg

65 70 75 80

Gly Leu Arg Pro Asn His Thr Phe Ser Leu Glu Asp Cys Ala Asp Asp

85 90 95

Ala Ala Ala Val Val Arg Ala Leu Gly Ile Arg Asn Val Ile Thr Val

100 105 110

Gly Tyr Ser Met Gly Gly Pro Ile Ser Leu Leu Val Trp Gln Arg His

115 120 125

Ser Asp Leu Val Thr Gly Met Val Leu Gln Ala Thr Ala Leu Glu Trp

130 135140

Ser Gly Thr Arg Gln Glu Arg Asn Lys Trp Arg Val Met His Val Ile

145 150 155 160

Asp Pro Leu Phe Arg Arg Ile Asn Ser Pro Arg Leu Thr Arg Trp Tyr

165 170 175

Val Arg Arg Leu Ile Pro Arg Gly His Glu Ile Asn Arg Tyr Leu Pro

180 185 190

Trp Ile Thr Gly Glu Leu Arg Arg Asn Asp Ser Trp Met Ile Ser Glu

195 200 205

Ala Gly Arg Ala Ile Ser Arg Phe Asp Ala Arg Gly Phe Ala His Thr

210 215 220

Val Asn Val Pro Thr Ser Phe Val Leu Thr Thr Leu Asp Lys Leu Val

225 230 235 240

Leu Pro His Lys Gln Gln Ala Leu Ala Asp Ala Val Arg Ala Glu Val

245 250 255

Val Glu Leu Glu Gly Asp His Leu Ala Pro Met Gln Gln Pro Arg Glu

260 265 270

Phe Ser Trp Ala Thr Ala Arg Ala Val Glu Ile Val Val Arg Gln Thr

275 280 285

Asn Gln

290

<210>2

<211>873

<212>DNA

<213>2

<400>2

atgtcagtaa aaccaacgag cgtgatggac attccaccgc ttttgcccgg ccgtttgatt 60

tcactgcctg gtcgaggcga gatttttgtg cgccatcatc aacatgtaaa ccccgacgcg 120

ccaacgttgc tattgcttca cgggtggaca gcatcatctg atttgcagtt tttcaccgcg 180

tatgaagaac tctcacgtaa ttattcaatt gttggcgtcg accatcgcgg tcatggtcga 240

ggcttacgcc cgaaccacac gttctcgctt gaagattgcg ccgatgatgc agcagcagtt 300

gtgcgagcgc ttggcattcg taacgtcatt acggttggtt actcaatggg tggccccatc 360

agcttgttgg tgtggcaacg acacagtgat ctcgtgaccg gaatggttct gcaagcaaca 420

gcacttgagt ggagcggcac acgtcaagaa cgcaacaagt ggcgcgtcat gcacgtcatt 480

gacccgttat tccgtcgcat caactcacct cgtctgacgc gttggtatgt ccggcgactc 540

attccgcgcg gtcatgaaat caaccgttac ctgccgtgga tcacaggcga gttgcgacgt 600

aatgattcgt ggatgatcag cgaagcaggt cgtgccatct cgcgttttga tgctcgcggg 660

tttgcgcaca ccgtgaatgt gccaacaagt tttgtgttga cgacgctcga caaacttgtc 720

ttgccgcaca aacaacaagc gctcgctgat gctgttcgcg cagaagtggt ggaacttgag 780

ggcgatcact tggcgcccat gcagcaacca cgcgaatttt cgtgggctac tgctcgcgct 840

gtggagattg ttgttcgcca gacgaatcaa tga 873

Claims

1. An esterase, characterized in that the esterase is an enzyme with an amino acid sequence of SEQ ID NO. 1.

2. A gene encoding the esterase of claim 1.

3. The gene of claim 2, wherein the nucleotide sequence of the gene is SEQ ID NO 2.

4. A recombinant expression vector carrying the gene of claim 2.

5. A recombinant host microorganism transformed/transfected with the recombinant expression vector of claim 4.

6. Use of a recombinant host microorganism according to claim 5 for the recombinant expression of an esterase according to claim 1.

7. Use of the esterase of claim 1 for catalyzing the hydrolysis of astaxanthin ester to produce free astaxanthin.

8. The use according to claim 7, wherein the astaxanthin ester is provided by Haematococcus pluvialis oil and/or shrimp oil.

9. A method for producing free astaxanthin, comprising hydrolyzing an astaxanthin ester with the esterase of claim 1 to produce free astaxanthin.

10. The method of claim 9, wherein the astaxanthin ester is provided by Haematococcus pluvialis oil and/or shrimp oil.