KR20110137761A - Thermostable esterase and use of the same - Google Patents

Abstract

PURPOSE: A thermostable esterase is provided to ensure high activity at high temperature and neutral pH concentration and to be used for a detergent and food industry. CONSTITUTION: An esterase derived from Sulfolobus solfataricus has an amino acid of sequence number 2. The molecular weight of the esterase is 33.5kDa. A method for isolating the esterase gene comprises: a step of collecting chromosomal DNA from a strain containing the esterase gene; a step of partially amplifying the esterase gene by PCR; a step of labeling PCR fragment using a proper reagent; and a step of performing colony hybridization and select esterase gene.

Description

Thermoplastic esterase and use of the same

The present invention relates to a thermophilic esterase and its use, and more particularly to a thermostable esterase derived from Sulfolobus solfataricus.

Esterase is an enzyme that breaks down ester bonds to make low-molecular fats and alcohols, and has activity only against glycerol esters having an acyl chain of 10 carbons or less. Until now, many kinds of esterases have been found in various organisms including plants, including bacteria, yeast, and higher organisms, and studies on biochemical properties and esterase genes themselves have been actively conducted. Esterases are hydrolysis reactions of lipids, acidolysis reactions (replacement of esterified fatty acids with free fatty acids), transesterification, exchange of fatty acids between triglycerides, Used in the synthesis of esters, etc., it is a very important enzyme industrially. In particular, it can be used in the detergent industry as it can decompose fat components in laundry stains into hydrophilic substances that can be easily removed, and it is an anti-aging agent for cheese production and baking such as aging and adding flavor of cheese, or margarine with a natural buttery flavor. It can be used in the food and beverage industry such as manufacturing, the waste treatment industry, and the pharmaceutical industry as a major component of a fire extinguishing agent. In addition, esterases have unique properties such as substrate specificity, optical activity specificity, and site specificity, and thus their use is attracting attention to develop various bioactive chiral drugs.

The production of chiral compounds useful for the production of such optically active drugs is attracting attention as a task that has a very great influence on securing competitiveness of the pharmaceutical industry and promoting public health.

The above important reactions are sometimes carried out effectively at high temperatures and under organic solvents. In particular, beef tallow and palm oil, which are mainly used for the production of free fatty acids in the food industry, remain solid at normal enzymatic reaction temperatures, so a high-temperature reaction environment is required for enzymatic processing of these materials. In addition, even when used in the detergent industry, it must exhibit excellent activity at high temperatures. Therefore, there is an urgent need for heat-resistant esterases that are stable at high temperatures.

The present invention was conceived by the necessity of the above, and an object of the present invention is to provide an esterase having heat resistance.

In order to achieve the above object, the present invention provides an esterase described in SEQ ID NO: 1 or 2 derived from Sulforobus solfataricus.

The term “esterase” as used herein encompasses native sequence polypeptides, polypeptide variants, and fragments of native sequence polypeptides and polypeptide variants that have esterase activity in a manner similar to wild-type esterase. The esterase polypeptides described herein may be isolated from various sources, such as microbial strains or other sources, or prepared by recombinant or synthetic methods. The terms “esterase”, “esterase polypeptide”, “esterase protein” and “esterase molecule” also include variants of the esterase polypeptide as described herein.

“Natural sequence esterase polypeptide” includes a polypeptide having the same amino acid sequence as a corresponding esterase polypeptide derived from nature. In one embodiment, the native sequence esterase polypeptide comprises the protein-coding amino acid sequence of SEQ ID NO: 1 or 2, wherein the first methionine is amino acid position 1. These native sequence esterase polypeptides can be isolated from nature or can be produced by recombinant or synthetic means.

An “esterase polypeptide variant” or a variable thereof is an esterase polypeptide as defined herein, generally an active esterase, having an amino acid sequence identity of at least about 80% with the original sequence esterase polypeptide sequence as described herein. I mean the first polypeptide. Such esterase polypeptide variants include, for example, esterase polypeptides in which one or more amino acid residues are added or deleted at the N- or C-terminus of the original amino acid sequence. Typically, esterase polypeptide variants have an amino acid sequence identity of at least about 80% with the original sequence esterase polypeptide sequence as described herein, and on the other hand about 81%, 82%, 83%, 84%, 85. %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more. Typically, esterase polypeptide variants are about 10 or more amino acids in length, and on the other hand about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or more amino acids. Optionally, the esterase polypeptide variant is one or less conservative amino acid substitutions compared to the original esterase polypeptide sequence, on the other hand 2, 3, 4, 5, 6, 7, 8, 9, or 10 It will have the following conservative amino acid substitutions.

"Amino acid sequence identity (%)" with respect to a peptide or polypeptide sequence aligns the sequences and, if necessary, introduces gaps to achieve maximum sequence identity, and after no conservative substitutions are considered as part of the sequence identity, specific It is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the peptide or polypeptide sequence. Alignment for the purpose of determining% amino acid sequence identity can be performed using, for example, publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megaline (DNASTAR) software. It can be achieved in various ways within the skill level of the field. One of skill in the art can determine suitable parameters for measuring alignment, including all algorithms necessary to achieve maximum alignment across the full length sequences to be compared. However, for the purposes of the present application, amino acid sequence identity (%) values are generated using the computer program ALIGN-2 for sequence comparison as described in US Pat. No. 6,828,146.

In one embodiment of the present invention, the esterase preferably has a molecular weight of 33.5kDa and 35kDa, respectively, but is not limited thereto.

In one embodiment of the present invention, the esterase preferably has an optimum activity at a pH of 6 to 8 and a temperature of 70 to 95°C, but is not limited thereto.

The esterase gene of the present invention is isolated from sulfolobus solfataricus cells. First, chromosomal DNA is obtained from a strain having an esterase gene. Next, polymerase chain reaction (PCR) is performed using the designed oligonucleotide as a primer, and the chromosomal DNA of the Sulfolobus solfataricus strain as a template, and the esterase gene is partially amplified. The PCR amplified fragment thus obtained is a fragment having near 100% homology to the esterase gene of the sulfolobus sulfataricus strain, and a high S/N ratio can be expected as a probe during colony hybridization. At the same time, it facilitates the stringency control of hybridization. The PCR amplified fragment is labeled with an appropriate reagent, colony hybridization is performed on the chromosomal DNA library, and an esterase gene is selected (Current Protocols in Molecular Biology, Vol. 1, p. 603, 1994). .

A DNA fragment containing an esterase gene can be obtained by recovering a plasmid from E. coli selected by the above method using an alkaline method (Current Protocols in Molecular Biology, Vol. 1, p. 161, 1994). Further, after determining the nucleotide sequence by the above method, it is possible to obtain the entire gene of the present invention by hybridizing the DNA fragment prepared by digestion of the DNA fragment having the nucleotide sequence with a restriction enzyme as a probe.

The transformed microorganism of the present invention is obtained by introducing the recombinant vector of the present invention into a host suitable for the expression vector used when constructing the recombinant vector. For example, when bacteria such as Escherichia coli are used as a host, the recombinant vector according to the present invention can be autonomously replicated in the host, and at the same time, DNA containing a promoter, an esterase gene, and a transcription termination sequence are used. It is preferable that it has a constitution necessary for expression. As the expression vector used in the present invention, any expression vector that satisfies the above requirements can be used.

In the production of an esterase enzyme according to the present invention, a transformant obtained by transforming a host with a recombinant vector having a gene encoding it is cultivated, and the gene product, Estera, is placed in a culture (cultured cell or culture supernatant). This is carried out by generating and accumulating the first enzyme and obtaining the enzyme from the culture.

Acquisition and purification of the esterase enzyme can be performed by centrifugal recovery of the cells or the supernatant from the obtained culture, lysis of cells, affinity chromatography, cation or anion exchange chromatography, etc., alone or in combination.

Hereinafter, the present invention will be described in detail.

Archaea, an archaea, is a creature living in a very extreme environment. Among them, Sulfolobus solfataricus strain belonging to thermoacidophiles is a strain that grows at a pH of 4 or less and 75℃ or more. It aims to investigate the possibility of a detergent by mass-producing it from .coli (E. coli).

The Sulfolobus solfataricus P1 strain (DSM1616) of the present invention was purchased from American Type Culture Collection (ATTC), and cultured and used.

The present invention was transformed into E. coli and mass-produced, and the properties (molecular weight, temperature, pH, thermal stability) of these enzymes were confirmed through purification.

As can be seen from the specification of the present invention, it was confirmed that the esterase of the present invention exhibited high activity between 70 and 95 °C and showed high activity between 7 and 8 similar to the pH of the water we use, Therefore, it can be used in the detergent or food industry, etc., which is stable at high temperatures and has high activity.

1 is a diagram showing the molecular weight and activity confirmation of arylesterase. In Figure 1, (B) shows the molecular weight measurement through MALDI-TOF, and (A) and (C) show the results of silver staining and active staining (fast blue + substrate (alpha-naphthyl acetate)), respectively. )
Figure 2 shows the activity of arylesterase according to the pH,
3 shows the activity of arylesterase according to the temperature,
4 shows the activity of arylesterase according to the thermal stability. In FIG. 4, the residual activity was determined after incubation for the time indicated on the x-axis at 50°C for diamonds, 70°C for squares, and 90°C for triangles. Activity measurements were performed using standard enzyme assays, and error bars represent standard deviations.
5 is a diagram showing molecular weight and activity confirmation of carboxylesterase. In Figure 1, (B) shows the molecular weight measurement through MALDI-TOF, and (A) and (C) show the results of silver staining and active staining (fast blue + substrate (alpha-naphthyl acetate)), respectively. kDa)
6 shows the activity of carboxylesterase according to pH,
7 shows the activity of carboxylesterase according to the temperature,
8 shows the activity of carboxylesterase according to the thermal stability. In Fig. 8, the residual activity was determined after incubation at 50°C for the square, 70°C for the black circle, and 80°C for the white circle for the time indicated on the x-axis. Activity measurements were performed using standard enzyme assays, and error bars represent standard deviations.

Hereinafter, the present invention will be described in more detail through non-limiting examples. However, the following examples are described for the purpose of illustrating the present invention, and the scope of the present invention is not to be construed as being limited by the following examples.

Example One: Esterase Separation

Aryl esterase

Sulfolobus solfataricus P1 (ATCC 35091) was stirred in a 5L fermentor at 75° C. and pH 4 and cultured in yeast medium. The cells were collected by centrifugation, and 8g of the cells were dissolved in 40ml of Sodium Phosphate Buffer (pH 7), and the cells were lysed with a high-pressure crusher.

After that, the cells were collected by centrifugation at 3000 rpm to collect the cells that were not lysed, and the supernatant (lysis cells) were collected to remove the membrain at 100000 x g.

After that, the nucleic acid is removed using protamin sulfate and purified by putting it in DEAE-sepharose (0~0.15M). Fractions with esterase activity are collected and corrected with 2M NaCl, and a step gradient is applied to butyl-sepharose up to 30%, 40%, 50%, 60%, and 70% to collect fration (using ethylene glycol). After removal, the elution was performed by using a 0~0.2M NaCl linear gradient using Q-sepharose, and the enzyme was purified and sequenced using a 10mM~100mM sodium phosphate buffer using a hydroxyapatite column (SEQ ID NO: 1).

Caboxylesterase

Sulfolobus solfataricus P1 (ATCC 35091) was stirred in a 5L fermentor at 75° C. and pH 4 and cultured in yeast medium. The cells were collected by centrifugation, and 8g of the cells were dissolved in 40ml of Sodium Phosphate Buffer (pH 7), and the cells were lysed with a high-pressure crusher.

After that, the cells were collected by centrifugation at 3000 rpm to collect the cells that were not lysed, and the supernatant (lysis cells) were collected to remove the membrain at 100000 x g.

After that, remove the nucleic acid using protamin sulfate, apply butyl-sepharose (40%, 50%, 60% step gradient, ethylene glycol for elution), and apply 0~0.2M NaCl gradient using Q-sepharose. After elution, a step gradient of 30%, 40%, 50%, and 60% 70% was applied using Phenyl-sepharose to purify and sequence the enzyme at 5-60% (SEQ ID NO: 2).

* Example 2: Transformation

E. coli containing recombinant DNA was treated with Amp+ in LB medium, and then treated with IPTG when the O.D value was 0.5 at an absorbance of 600 nm to overexpress the protein, followed by centrifugation at 3000 rpm for 40 minutes. After the cells were down, they were dissolved in sodium phosphate buffer, and then lysed in a high-pressure crusher at a pressure of 1280 psi.

In the case of the primer used for the recombinant DNA of the present invention, Arylesterase used a Primer sequence: (forward 5'-AATCCATGGAAATGCCGTTGGATCCTGA-3'; SEQ ID NO: 3), (reverse 5'-CGCCCATGGTTACTTAAAAATGTCCTTAAGTA-3'; SEQ ID NO: 4), For carboxylesterase, Primer sequences: (forward 5'-CGCGGATCCCATGCCTTTAGATCCAACC-3'; SEQ ID NO: 5) and (reverse 5'-CGCGGATCCTCAAAAACTGTTG-3'; SEQ ID NO: 6) were used.

In addition, pET-11d (New England Biolabs) was used as a vector, and E.coli BL21 (DE3) was put into a Competent cell through heat shock.

After heat-treating the above cell extract at 70° C. for 30 minutes, the cells were down in the same manner as in the above centrifugation, and then only the supernatant was collected and active stained, and the presence or absence of esterase was checked.

The chromatography was purified through a Q-Sepharose process followed by a butyl Sepharose column. Silver staining and Active staining were performed at the same time (Figs. 1 and 5).

Example 3: pH And thermal stability experiment

The activity and thermal stability of the arylesterase obtained in Example 2 with respect to pH and temperature were tested using p-nitrophenyl caprylate as a substrate and using ELISA at an absorbance of 405 nm (Figs. 2, 3, 4). , 6, 7 and 8)

<110> UNIVERSITY-INDUSTRY COOPERATION FOUNDATION KANGWON NATIONAL UNIVERSITY <120> Thermostable esterase and use of the same <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 306 <212> PRT <213> Sulfolobus solfataricus <400> 1 Met Pro Leu Asp Pro Glu Val Arg Asn Phe Leu Gln Val Tyr Tyr Lys 1 5 10 15 Ala Asn Ile Ile Asp Phe Thr Lys Tyr Gln Phe Gln Glu Ile Arg Gln 20 25 30 Lys Val Asn Glu Leu Leu Ala Lys Ala Val Pro Lys Asp Pro Val Gly 35 40 45 Glu Thr Arg Asp Met Lys Ile Lys Leu Glu Asp Tyr Glu Leu Pro Ile 50 55 60 Arg Ile Tyr Ser Pro Ile Lys Arg Thr Asn Asn Gly Leu Val Met His 65 70 75 80 Phe His Gly Gly Ala Trp Ile Leu Gly Ser Ile Glu Thr Glu Asp Ala 85 90 95 Ile Ser Arg Ile Leu Ser Asn Ser Cys Glu Cys Thr Val Ile Ser Val 100 105 110 Asp Tyr Arg Leu Ala Pro Glu Tyr Lys Phe Pro Thr Ala Val Tyr Asp 115 120 125 Cys Phe Asn Ala Ile Val Trp Ala Arg Asp Asn Ala Gly Glu Leu Gly 130 135 140 Ile Asp Lys Asp Lys Ile Ala Thr Phe Gly Ile Ser Ala Gly Gly Asn 145 150 155 160 Leu Val Ala Ala Thr Ser Leu Leu Ala Arg Asp Asn Lys Leu Lys Leu 165 170 175 Thr Ala Gln Val Pro Val Val Pro Phe Val Tyr Leu Asp Leu Ala Ser 180 185 190 Lys Ser Met Asn Arg Tyr Arg Lys Gly Tyr Phe Leu Asp Ile Asn Leu 195 200 205 Pro Val Asp Tyr Gly Val Lys Met Tyr Ile Arg Asp Glu Lys Asp Leu 210 215 220 Tyr Asn Pro Leu Phe Ser Pro Leu Ile Ala Glu Asp Leu Ser Asn Leu 225 230 235 240 Pro Gln Ala Ile Val Val Thr Ala Glu Tyr Asp Pro Leu Arg Asp Gln 245 250 255 Gly Glu Ala Tyr Ala Tyr Arg Leu Met Glu Ser Gly Val Pro Thr Leu 260 265 270 Ser Phe Arg Val Asn Gly Asn Val His Ala Phe Leu Gly Ser Pro Arg 275 280 285 Thr Ser Arg Gln Val Thr Val Met Ile Gly Ala Leu Leu Lys Asp Ile 290 295 300 Phe Lys 305 <210> 2 <211> 305 <212> PRT <213> Sulfolobus solfataricus <400> 2 Met Pro Leu Asp Pro Thr Ile Lys Lys Leu Leu Glu Ser Gly Phe Val 1 5 10 15 Ile Pro Ile Gly Lys Val Pro Val Asp Glu Val Arg Lys Val Phe Arg 20 25 30 Gln Ile Ser Ser Ala Ala Pro Lys Met Asp Val Gly Asn Ile Glu Asp 35 40 45 Ile Lys Ile Pro Gly Thr Glu Thr Met Ile Pro Ala Arg Val Tyr Tyr 50 55 60 Pro Lys Thr Lys Gly Pro Tyr Gly Ile Leu Val Tyr Leu His Gly Gly 65 70 75 80 Gly Phe Val Ile Gly Asp Ile Glu Ser Tyr Asp Pro Val Cys Arg Ala 85 90 95 Ile Thr Asn Ala Cys Asn Cys Val Val Ala Ser Ile Asp Tyr Arg Leu 100 105 110 Ala Pro Glu Asn Lys Phe Pro Ser Ala Val Val Asp Ser Val Asp Ala 115 120 125 Thr Lys Trp Ile Tyr Glu Asn Ala Glu Ser Leu Glu Gly Lys Gln Gly 130 135 140 Val Ala Val Gly Gly Asp Ser Ala Gly Gly Asn Leu Ser Ala Val Val 145 150 155 160 Ser Ile Leu Thr Lys Gly Lys Ile Asn Leu Lys His Gln Val Leu Ile 165 170 175 Tyr Pro Ala Thr Gly Ala Asp Cys Thr Ser Arg Ser Met Val Glu Tyr 180 185 190 Tyr Asp Gly Tyr Phe Leu Thr Arg Glu Gln Ile Glu Trp Phe Gly Ser 195 200 205 Gln Tyr Leu Arg Ser Tyr Met Asp Leu Leu Asp Ile Lys Phe Ser Pro 210 215 220 Ile Leu Asn Gln Asp Leu Thr Gly Leu Pro Pro Ala Leu Ile Ile Thr 225 230 235 240 Ala Glu Tyr Asp Pro Leu Arg Asp Gln Gly Glu Ala Tyr Ala Asn Lys 245 250 255 Leu Leu Met Ala Gly Ile Pro Val Thr Ser Val Arg Phe Asn Asn Val 260 265 270 Ile His Gly Phe Val Ser Phe Phe Pro Leu Ile Pro Gln Gly Met Asp 275 280 285 Ala Ile Gly Leu Ile Gly Ala Thr Leu Arg Arg Val Phe Tyr Asn Ser 290 295 300 Phe 305 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 aatccatgga aatgccgttg gatcctga 28 <210> 4 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 cgcccatggt tacttaaaaa tgtccttaag ta 32 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 cgcggatccc atgcctttag atccaacc 28 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 cgcggatcct caaaaactgt tg 22

Claims (3)

Esterase having the amino acid sequence of SEQ ID NO: 2 derived from Sulphobus solfataricus. The esterase according to claim 1, wherein the esterase has a molecular weight of 33.5 kDa. The esterase according to claim 1, wherein the esterase has an optimum activity at a temperature of 70 to 95 ° C.

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