KR20110118002A - Thermostable esterase and use of the same - Google Patents

Abstract

FIELD OF THE INVENTION The present invention relates to pyrogenic esterases and their use, and more particularly to thermostable esterases derived from Sulphobus solfataricus.

Description

Thermophilic esterase and its use {Thermostable esterase and use of the same}

The present invention relates to pyrogenic esterases and their use, and more particularly to thermostable esterases derived from Sulphobus solfataricus.

Esterases are enzymes that break down ester bonds to produce low-molecular fats and alcohols. They are only active against glycerolesters with up to 10 carbonyl acyl chains. To date, many kinds of esterases have been found in various organisms including plants, including bacteria, yeasts and higher organisms, and research on biochemical properties and the esterase genes themselves has been actively conducted. Esterases are hydrolysis of lipids, acidolysis (replacement of esterified fatty acids with free fatty acids), transesterification, exchange of triglyceride fatty acids, Used in the synthesis of esters, etc., it is an enzyme of great industrial importance. In particular, it can be used in the detergent industry because it can decompose fat components in laundry stains into easily removable hydrophilic substances, and margarine with anti-aging or natural butter flavors for cheese production and baking, such as the addition and flavor of cheese. It can be used in the food and beverage industry, such as manufacturing, in the waste disposal industry, and in the pharmaceutical industry as a major component of the extinguishing agent. In addition, esterases have unique characteristics such as substrate specificity, optical activity specificity, and site specificity, and thus, their use has been attracting attention in developing 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 great influence on securing the competitiveness of the pharmaceutical industry and promoting the health of the public.

This important reaction is sometimes carried out effectively under high temperatures and organic solvents. In particular, bee 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 should show excellent activity at high temperatures. Therefore, there is an urgent need for stable heat resistant esterases at high temperatures.

The present invention has been made in view of the above necessity, 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 as set forth in SEQ ID NO: 1 or 2 derived from Sulfobus solfataricus.

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

A “natural sequence esterase polypeptide” includes a polypeptide having the same amino acid sequence as the corresponding esterase polypeptide derived from nature. In one embodiment, the original 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. Such native sequence esterase polypeptides can be isolated from nature or can be produced by recombinant or synthetic means.

An “esterase polypeptide variant” or variables thereof may be an esterase polypeptide, generally an active ester, as defined herein, having an amino acid sequence identity of at least about 80% with the original sequence esterase polypeptide sequence as described herein. Means a second 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 at least about 80% amino acid sequence identity 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 may have one or less conservative amino acid substitutions compared to the original esterase polypeptide sequence, and 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 (%)” in relation to a peptide or polypeptide sequence achieves the maximum sequence identity by aligning the sequences and introducing gaps if necessary, and conservative substitutions as part of sequence identity are not considered at all, It is defined as the percentage of amino acid residues in the candidate sequence that are identical to amino acid residues in the peptide or polypeptide sequence. Alignments 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 Megaalign (DNASTAR) software. It can be achieved in various ways within the skill level of the art. Those skilled in the art can determine appropriate parameters for measuring alignment, including all algorithms necessary to achieve maximum alignment over the full length sequence to be compared. However, for purposes herein,% amino acid sequence identity rate 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 pH 6-8, temperature 70-95 ℃ but is not limited thereto.

The esterase gene of the present invention is isolated from Sulphobus solfataricus cells. First, chromosomal DNA is obtained from a strain having an esterase gene. Next, a polymerase chain reaction (PCR) is carried out using a designed oligonucleotide as a primer, and a chromosomal DNA of a Sulfolobus solfataricus strain as a template to partially amplify the esterase gene. The PCR amplified fragment thus obtained is a fragment having a homology close to 100% of the esterase gene of the sulfoverse sulfatariscus strain, and a high S / N ratio can be expected as a probe for colony hybridization. At the same time, it facilitates stringency control of hybridization. The PCR amplification fragments are labeled with appropriate reagents, and colony hybridization is performed on the chromosomal DNA library to select esterase genes (Current Protocols in Molecular Biology, Vol. 1, p. 603, 1994). .

DNA fragments containing an esterase gene can be obtained by recovering a plasmid from the E. coli selected by the above method using an alkaline method (Current Protocols in Molecular Biology, Vol. 1, p. 161, 1994). 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 by restriction enzymes of the DNA fragment having the nucleotide sequence 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 producing the recombinant vector. For example, when a bacterium such as Escherichia coli is used as a host, the recombinant vector according to the present invention can autonomously replicate in the host, and at the same time, a DNA containing a promoter, an esterase gene, a transcription termination sequence, or the like. It is preferable to have what is necessary for expression. As the expression vector used in the present invention, any expression vector satisfying the above requirements can be used.

Production of the esterase enzyme according to the present invention involves culturing a transformant obtained by transforming a host with a recombinant vector having a gene encoding the same, and an ester product which is a gene product in a culture (cultured cell or culture supernatant). It is performed by generating and accumulating the first enzyme and acquiring the enzyme from the culture.

Acquisition and purification of the esterase enzyme can be carried out by centrifuging the cells or supernatant from the obtained culture, followed by cell disruption, affinity chromatography, cation or anion exchange chromatography alone or in combination.

Hereinafter, the present invention will be described in detail.

Archaea, archaea, is a living organism in very extreme environments. Among them, the Sulfolobus solfataricus strain belonging to thermoacidophiles is a strain that grows at pH below 75 ℃. In this study, the genes of esterases (arylesterase, carboxylesterase), which are ester-binding compounds (particularly fat-soluble substances), are identified. It aims to investigate the potential as a detergent by mass production from .coli.

Sulfolobus solfataricus P1 strain (DSM1616) of the present invention was purchased from the American Type Culture Collection (ATTC) was used to culture.

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

As can be seen in the specification of the present invention, the esterase of the present invention was found to show high activity between 70 ~ 95 ℃ and 7 ~ 8 similar to the pH of the water we use, Therefore, it can be used in detergents or food industries having good effect at high temperature and having high activity.

1 is a diagram showing the molecular weight and activity of the arylesterase. In Figure 1 (B) shows the molecular weight measurement through MALDI-TOF, (A) and (C) shows the results of silver staining and active staining (fast blue + substrate (alpha-naphthyl acetate), respectively). )
Figure 2 shows the activity according to the pH of the arylesterase,
3 shows the activity according to the temperature of the arylesterase,
Figure 4 shows the activity according to the thermal stability of the arylesterase. In FIG. 4, after incubating the diamond at 50 ° C., the square at 70 ° C., and the triangle at 90 ° C., the residual activity is determined. Activity measurements were performed using standard enzyme assays and error bars indicate standard deviation.
5 is a diagram showing the molecular weight and activity of the carboxylesterase. (B) in Fig. 1 shows the molecular weight measurement through MALDI-TOF, (A) and (C) shows the results of silver staining and active staining (fast blue + substrate (alpha-naphthyl acetate), respectively) carboxylesterase molecular weight (33.5 kDa)
Figure 6 shows the activity according to the pH of the carboxylesterase,
Figure 7 shows the activity according to the temperature of the carboxylesterase,
8 shows the activity according to the thermal stability of the carboxylesterase. In Figure 8, the square is 50 ℃, the black circle is 70 ℃, the white circle is incubated for 80 hours at the time described on the x-axis residual activity is determined. Activity measurements were performed using standard enzyme assays and error bars indicate standard deviation.

The present invention will now be described in more detail by way of 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 limited by the following examples.

Example  One: Esterase  detach

Aryl esterase

Sulfolobus solfataricus P1 (ATCC 35091) was incubated in yeast medium by stirring in a 5L fermentor at 75 ℃, pH 4 conditions. The cells were collected by centrifugation, and 8 g of cells were dissolved in 40 ml of sodium phosphate buffer (pH 7), followed by cell lysis using a high pressure crusher.

Afterwards, the cells were collected by centrifugation at 3000 rpm, and the supernatant (lysis) was collected to remove the membrain portion at 100000 x g.

Subsequently, the nucleic acid is removed using protamin sulfate and purified by adding it to DEAE-sepharose (0 ~ 0.15M). Fractions with esterase activity are collected and corrected with 2M NaCl, followed by step gradients up to 30%, 40%, 50%, 60%, and 70% on Butyl-Sepharose to collect fration (using ethylene glycol). Elution was carried out by using 0-0.2M NaCl linear gradient using Q-sepharose, and the enzyme was purified and sequenced using 10mM-100mM sodium phosphate buffer using hydroxyapatite column (SEQ ID NO: 1).

Caboxylesterase

Sulfolobus solfataricus P1 (ATCC 35091) was incubated in yeast medium by stirring in a 5L fermentor at 75 ℃, pH 4 conditions. The cells were collected by centrifugation, and 8 g of cells were dissolved in 40 ml of sodium phosphate buffer (pH 7), followed by cell lysis using a high pressure crusher.

Afterwards, the cells were collected by centrifugation at 3000 rpm, and the supernatant (lysis) was collected to remove the membrain portion at 100000 x g.

Subsequently, the nucleic acid was removed using protamin sulfate, butyl sepharose (step gradient of 40%, 50%, 60%, and ethylene glycol was used for elution), followed by 0-0.2M NaCl gradient using Q-sepharose. After elution, a step gradient of 30%, 40%, 50%, and 60% 70% was used to purify enzymes from 5 to 60% using phenyl sepharose (SEQ ID NO: 2).

Example  2: transformation

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

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

Also, 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 gas extracts at 70 ℃ for 30 minutes, the cells were down by the same method as the above centrifugation, and only the supernatant was collected for active staining to confirm the presence of esterase.

Chromatography was followed by purification through a butyl sepharose column followed by Q-sepharose. Silver staining and active staining were performed simultaneously (FIGS. 1 and 5).

Example  3: pH  And thermal stability experiments

The activity and thermal stability of pH and temperature of the arylesterase obtained in Example 2 were tested using ELISA at 405 nm absorbance using p-nitrophenyl caprylate as substrate. (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 one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 2 derived from Sulphobus solfataricus. The esterase according to claim 1, wherein the esterase has a molecular weight of 35 kDa and 33.5 kDa, respectively. 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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