KR101190631B1 - Novel gene encoding glycosyl hydrolase from cow rumen metagenome - Google Patents

Abstract

The present invention provides a novel glycosyl hydrolase gene derived from bovine rumen metagenome, a recombinant vector comprising the gene, a transformant by the recombinant vector and a glycosyl hydrola produced by the transformant. The present invention relates to an enzyme protein, which provides a novel glycosyl hydrolase gene from ruminant bacteria that is difficult to cultivate by conventional techniques from a bovine rumen metagenomic library, and thereby produces a large amount of recombinant enzymes for the detergent, pulp and paper industries. It can be used for commercial purposes in various fields such as livestock feed, food, and bioenergy.

Description

Novel gene encoding glycosyl hydrolase from cow rumen metagenome derived from cow rumen metagenome

The present invention provides a novel glycosyl hydrolase gene derived from bovine rumen metagenome, a recombinant vector comprising the gene, a transformant by the recombinant vector and a glycosyl hydrolase produced by the transformant. Proteins, more specifically, the rumen metagenomic library of bovine to select a clone having a cellulase activity, a novel glycosyl hydrolase gene identified from the clone, a recombinant vector comprising the gene, said A transformant by a recombinant vector and a glycosyl hydrolase protein produced by the transformant.

It is known that only about 0.1 to 1% of the microorganisms in nature can be cultured. Metagenome is an effective means of discovering useful genes from microorganisms that cannot be cultured. Since 1998, the University of Wisconsin was used to explore new drugs using unknown soil microorganisms, such as marine, desert, river and hot springs. It is applied to the field. When using metagenome, it is difficult to obtain information on the microorganism from which the gene is derived. However, since it has the advantage of simultaneously obtaining the product of the microorganism and the gene, it has become a representative method of discovering useful genes.

Cellulose is a linear polysaccharide of macromolecules in which glucose is linked to beta-1,4-glycosidic bonds, the most abundant organic material in nature and valuable as a renewable energy source. Very high. Cellulose is broken down into its component glucose by a hydrolase called cellulase. Cellulase known to date is largely endo-beta-1,4-glucanase (endo-beta-1,4-glucanase, EC 3.2.1.4), exo-beta-1,4-glucanase (exo-beta- 1,4-glucanase, EC 3.2.1.91), and beta-glucosidase (EC 3.2.1.21). Endo-beta-1,4-glucanase randomly hydrolyzes beta-1,4 bonds of the cellulose inner chain, and exo-beta-1,4-glucanase is endo-beta-1,4-glu The reducing and non-reducing ends of the cellulose polymer produced by the kanase are hydrolyzed sequentially to produce two molecules of glucose-linked cellobiose. The cellobiose is hydrolyzed by beta-glucosidase to finally produce glucose.

Cellulase is widely used in various fields such as detergents, pulp and paper industry, livestock feed and food, and is the next generation energy source to replace fossil fuels. It is the core in the industry that decomposes plant biomass and produces ethanol. It is used as a technique. Cellulase currently forms the third largest market for industrial enzymes around the world. If the biomass-derived bioenergy is commercialized, the market size is expected to be further expanded.

Although much research and development has been conducted to secure excellent cellulase, there is still a need for developing a new cellulase having desirable properties and activities that can be used for sugar conversion of cellulose derived from woody biomass to obtain bioethanol. Therefore, the inventors of the present invention have studied the present invention by discovering excellent novel cellulase and cloned a multifunctional glycosyl hydrolase having a hemicellulase function as well as a cellulase not previously known in the rumen metagenome of cattle. Completed

It is an object of the present invention to provide a novel glycosyl hydrolase gene derived from bovine rumen metagenome and a novel glycosyl hydrolase protein encoded therefrom.

Still another object of the present invention is to provide a recombinant vector comprising the gene and a transformant into which the recombinant vector is introduced.

In order to achieve the above object, the present invention is characterized by providing a base sequence and amino acid sequences of the coding genes of novel glycosyl hydrolase derived from bovine rumen metagenome.

The present invention also provides a recombinant vector comprising the gene and a transformant into which the recombinant vector is introduced.

Hereinafter, the present invention will be described in detail.

The present invention comprises the steps of extracting a metagenomic DNA from a bovine rumen sample and cloning it into a fosmid vector to produce a metagenomic library; Screening cellulase in said metagenome library; Subcloning the metagenome clone with cellulase activity and analyzing the sequence; Obtaining three open reading frames (ORFs) of different lengths using a polymerase chain reaction (PCR) and introducing them into expression vectors; Introducing and transforming the expression vectors into which the gene is introduced into a host cell; Expressing the recombinant protein using the transformants and confirming the hydrolytic activity of various polysaccharides; Finally, the selected transcriptional detoxification frame is composed of the steps of purifying the recombinant enzyme expressed in the expression vector introduced and the characteristics of the enzyme.

The present invention relates to detergent, pulp and paper industry for mass production of recombinant glycosyl hydrolases using a novel glycosyl hydrolase gene derived from rumen bacteria, which is difficult to cultivate by conventional techniques, from bovine rumen metagenomic library. It can be used for commercial purposes in various fields such as livestock feed, food, and bioenergy.

1 is a diagram for selecting a transformant having a recombinant plasmid with cellulase activity in the metagenome library on a plate medium by Congo red staining method (A: confirming cellulase activity of the metagenome library, B: selected trait) Reconfirm cellulase activity of the convertor).
Figure 2 is a schematic representation of the genetic structure of ORF-E secured by the present invention (GH: glycoside hydrolase).
Figure 3 shows ORF-E and the existing known Prevotella is an illustration showing a homology comparison between the Rana xylene (AAC97596), cellulase (ABB46200) was separated on ruminants of bryontii.
Figure 4 shows the genes encoding the transcriptional decoded frame according to the present invention and the recombinant expression vectors inserted therein.
5 is a schematic diagram of a recombinant vector pEREng13 comprising reng 13, a glycosyl hydrolase coding gene.
6 is a schematic diagram of a recombinant vector pEREng43 comprising reng 43, a glycosyl hydrolase coding gene.
7 is a schematic diagram of a recombinant vector pEREng16 comprising reng 16, a glycosyl hydrolase coding gene.
8 is a meta-genome derived from new articles lycopene E. coli was transformed with the switching chamber hydrolase dehydratase gene E. coli HIT-21 / pEREng13, E. coli HIT-21 / pEREng43, E. coli HIT-21 / pEREng63, E.coli HIT SDS-PAGE analysis of the protein expressed at -21 / pEREng16.
lane M is the molecular weight standard marker;
lane 1, lane 3, lane 6 and lane 8 are cell extracts of the negative control group transformed with pET21a;
lane 2 is a cell extract of E. coli HIT-21 / pEREng13 treated with 0.5 mM IPTG;
lane 4 is cell extract of E. coli HIT-21 / pEREng43 not treated with IPTG;
lane 5 is E. coli HIT-21 / pEREng43 cell extract treated with 0.5 mM IPTG;
lane 7 is E. coli HIT-21 / pEREng63 cell extract treated with 0.5 mM IPTG;
lane 9 is cell extract of E. coli HIT-21 / pEREng16 not treated with IPTG;
Lane 10 shows the cell extract of E. coli HIT-21 / pEREng16 treated with 0.5 mM IPTG.
9 shows the expression of proteins expressed in E. coli HIT-21 / pEREng13, E. coli HIT-21 / pEREng43, E. coli HIT-21 / pEREng63, and E. coli HIT-21 / pEREng16. This figure shows the hydrolytic activity of polysaccharides.
10 is a result of confirming the expression and purification of recombinant proteins REng13 and REng43 by SDS-PAGE,
lane M is the molecular weight standard marker;
lane C is a cell extract of the negative control group transformed with pET21a;
lane CL was E. coli HIT-21 / pEREng13 or E. coli HIT-21 / pEREng43 cell extracts treated with 0.5 mM IPTG;
Lanes E3 to E7 represent the fractions of E. coli HIT-21 / pEREng13 or E. coli HIT-21 / pEREng43 treated with 0.5 mM IPTG, added to the HIS * Bind column, washed several times, and then eluted.
11 is a graph showing the effect of temperature on the activity of recombinant proteins REng13 and REng43.
12 is a graph showing the effect of pH on the activity of recombinant proteins REng13 and REng43 (blue: REng13, red: REng43).
FIG. 13 is a diagram showing the results of TLC analysis of cellooligosaccharide hydrolyzate of recombinant proteins REng13 and REng43.
Lane 1 is cellulose oligosaccharide mixture (G1, glucose; G2, cellobiose; G3, cellulotriose; G4, cellotetraose; G5, cellopentaose);
Lane 2 is hydrolyzate that reacted cellobiose with recombinant REng13 or REng43;
Lane 3 is hydrolyzate that reacted cellulotriose with recombinant REng13 or REng43;
Lane 4 is hydrolyzate that reacted cellotetraose with recombinant REng13 or REng43;
Lane 5 represents the hydrolyzate that reacted cellopentaose with recombinant REng13 or REng43.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are not intended to limit the scope of the present invention, and ordinary changes by those skilled in the art are possible within the scope of the technical idea of the present invention.

≪ Example 1 >

Construction of a Metagenome Library of Bovine Rumen Samples

1-1. Isolation of Metagenome DNA from Bovine Rumen Samples

Rumen samples were obtained using an autosampler cannula. The rumen samples were spontaneously precipitated at 4 ° C. to remove large impurities, and then centrifuged at a low speed of less than 1,000 rpm to further remove extracellular bacteria to collect microorganisms. The microorganism is extracted from the DNA using a metagenomic DNA isolation kit (Metagenomic DNA isolation kit for water, Epicentre, USA) and then blunt end the DNA end extracted with a DNA end-repair enzyme mixture (Epicentre, USA). Repaired.

1-2. Building the rumen metagenome library

DNA repaired to the blunt end of 1-1 was electrophoresed with 0.4% agarose gel to purify only about 40 kb of DNA with a gel extraction kit (QIAEX II gel extraction kit, Qiagen, Germany) Linked to a phosmid pCC1FOS vector using a Copy Control Fosmid Library Production Kit (Epicentre, USA), packaged in a λDNA packaging kit and transduced into E. coli EPI300-T1 to the rumen metagenomic library Secured.

<Example 2>

Screening and Isolation of Recombinant Plasmids with Cellulase Activity

In order to find a recombinant plasmid with cellulase activity in the metagenome library, the transformants were inoculated into LB plate medium containing 0.5% CM-cellulose (carboxymethyl-cellulose) and then incubated at 37 ° C for 24 hours. The plate was then stained with 0.5% (w / v) Congo red solution for 15 minutes and decolorized with 1M NaCl for 15 minutes. The cellulase enzyme activity can be seen as a transparent circumference formed around the colony, where it was confirmed that one transformant had cellulase activity (FIG. 1). The transformant showing the cellulase activity was named R1-4-G9.

<Example 3>

Determination of Cellulase Gene Sequences from Recombinant Plasmids with Cellulase Activity

3-1. Shotgun Library Construction of Recombinant Plasmids with Cellulase Activity

Recombinant plasmid pC1R14G9 was isolated from plasmid midi kit (QIAGEN, Germany) from transformant R1-4-G9 with cellulase activity of Example 2. The isolated recombinant plasmid was fragmented using a hydroshear device, and the DNA end was repaired with a blunt end. The repaired DNA fragments were electrophoresed to remove only the gel corresponding to the 2-5kb position, purified using a gel extraction kit, and then subcloned into a pC31A21 vector to prepare a shotgun library.

3-2. Sequencing of Recombinant Plasmids with Cellulase Activity

The sequence analysis of the shotgun library prepared in 3-1 was performed using an automatic base sequence analyzer (ABI 3730 DNA analyzer). Combining the results of sequence analysis of the shotgun library, it was found that the recombinant plasmid pC1R14G9 had a DNA fragment consisting of 32,747 bp. The base sequence of the DNA fragment was analyzed by the ORF finder of the National Center for Biotechnology Information (NCBI). As a result, an open reading frame of 2,760bp estimated to be endo-beta-1,4-glucanase, one of cellulase ORF). This transcription decoding frame was named ORF-E.

<Example 4>

Analysis of New Cellulase Sequences

The transcription-translation frame ORF-E, estimated to be endo-beta-1,4-glucanase found in Example 3, was composed of 920 amino acids, had a transmembrane domain at the N-terminal region, and two glyco Seed hydrolase had a catalytic domain (FIG. 2). Glycoside hydrolase is an enzyme that hydrolyzes glycosidic bonds between two or more carbohydrates or between carbohydrates and non-carbohydrates, based on the amino acid homology and structural properties of each enzyme. It is divided into two groups. The glycoside hydrolase family 26 present in ORF-E has beta-mannanase and beta-1,3-xylanase activities. In the glycoside hydrolase family 5, chitosanase, beta-mannosidase, and cellulase have been shown to exhibit a wide range of activities. It is known. In addition, amino acid homology analysis using the ORF-E sequence confirmed that 54% and 62% of Xylanase of the previously known Prevotella bryantii or cellulase isolated from ruminant animals, respectively (Fig. 3).

<Example 5>

Functional Expression of Novel Cellulase in Escherichia Coli

5-1. Preparation of Transformant

DNA fragments encoding four types of transcriptional deciphering frames depending on the position of the start codon (ATG) and the catalytic domain included in the base sequence of the ORF-E of Example 3 to express the novel cellulase of the present invention Were amplified by PCR to include Bam HI and Xho I restriction sites. DNA fragments amplified by the above PCR were reng 13 (base sequence: SEQ ID NO: 1, amino acid sequence: SEQ ID NO: 2), reng 43 (base sequence: SEQ ID NO: 3, amino acid sequence: SEQ ID NO: 4), reng 63 (base sequence) : SEQ ID NO: 5, amino acid sequence: SEQ ID NO: 6), reng 16 (base sequence: SEQ ID NO: 7, amino acid sequence: SEQ ID NO: 8). These DNA fragments were cleaved with the restriction enzymes and then introduced into the pET21a (Novagen, USA) vector digested with the same enzymes, which were named pEREng13, pEREng43, pEREng63, and pEREng16, respectively (Figure 4). The cleavage map for the recombinant vector pEREng13 is shown in FIG. 5, the cleavage map for the recombinant vector pEREng43 is shown in FIG. 6, and the cleavage map for the recombinant vector pEREng16 is shown in FIG. 7. The vectors were transformed into E. coli HIT-21 (RBC, USA), which was used as an expression host. They were named E. coli HIT-21 / pEREng13, E. coli HIT-21 / pEREng43, E. coli HIT-21 / pEREng63, and E. coli HIT-21 / pEREng16, respectively.

5-2. Expression of Cellulase Enzyme

The transformants were suspended in culture so that the OD value was 0.4-0.6 in a liquid LB medium and 0.5 mM IPTG was added to the medium to induce protein expression at 30 ° C. and 200 rpm for 4 hours. The cultures were centrifuged at 3,000 rpm for 15 minutes to collect only the cells and then added to the Bugs Buster Master Mix solution (Novagen, USA) to destroy the cells and centrifuged to remove insoluble residues. The cell extracts obtained here were used to confirm the enzyme activity. Cell extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transformants E. coli HIT-21 / pEREng13, E. coli HIT-21 / pEREng43, and E.coli HIT-21 / pEREng63. , E. coli HIT-21 / pEREng16 It was confirmed that the recombinant proteins of about 103, 63, 54, 40 kDa size were expressed (Fig. 8). Recombinant proteins expressed from genes reng 13, reng 43, and reng 16 were named REng13, REng43, and REng16, respectively.

5-3. Confirmation of Hydrolytic Activity of Recombinant Glycosyl Hydrolases

The hydrolyzate activity of the polysaccharides was confirmed using the 5-2 cell extracts. The enzyme reaction was performed at 37 ° C. for 1 hour by adding 100 mM Sodium Acetate buffer pH 5.5, 1% (w / v) polysaccharide and 10% (v / v) cell extract. The activity of the enzyme was determined by quantifying the reducing sugar from which the polysaccharide was decomposed, and a known DNS method (Miller GL. Anal. Chem., (1959) 31: 426-428) was used to quantify the reducing sugar. As a result, the recombinant protein REng13 hydrolyzed all CM-cellulose, xylan, beta-glucan, and beta-mannan. Glucan could be hydrolyzed. Recombinant protein REng16 had activity to degrade beta-mannan only, and recombinant protein REng63 showed no activity (FIG. 9).

From the above results, it was found that the recombinant protein REng13 according to the present invention is a multifunctional glycosyl hydrolase capable of decomposing not only cellulose but also hemicelluloses such as xylan and beta-mannan.

Meanwhile, through the difference in substrate specificity between the recombinant proteins REng43 and REng16, glycoside hydrolase family 26 of the two active sites exhibit beta-mannanase activity and glycoside hydrolase family 5 The family was found to exhibit cellulase and xylanase activity.

The transformant E. coli HIT-21 / pEREng43 was deposited on September 16, 2009 at the Korea Institute of Biotechnology and Biotechnology Center, the transformant E. coli HIT-21 / pEREng13 and E.coli HIT-21 / pEREng16 were deposited on March 15, 2010 at the Korea Institute of Bioscience and Biotechnology. The deposit number of the transformant E. coli HIT-21 / pEREng43 is KCTC 11556BP, the deposit number of the transformant E. coli HIT-21 / pEREng13 is KCTC 11655BP, and the transformant E. coli HIT-21 The deposit number for / pEREng16 is KCTC 11656BP.

<Example 6>

Characterization of Recombinant Proteins

6-1. Expression and Purification of Recombinant Proteins REng13 and REng43

The recombinant proteins REng13 and REng43 according to the present invention were expressed and purified, respectively, to examine their properties. After expressing proteins from transformants E. coli HIT-21 / pEREng13 and E. coli HIT-21 / pEREng43 in the same manner as 5-2, HIS * Bind column (Novagen, USA) and HIS * Bind buffer kit (Novagen, USA) was used to purify the recombinant proteins (FIG. 10).

6-2. Characterization of temperature and pH

In order to investigate the effect of temperature on the activity of the recombinant proteins REng13 and REng43 according to the present invention, the CM-cellulose hydrolysis reaction was performed between 30 and 80 ° C. Both the recombinant proteins REng13 and REng43 showed optimal activity at 43 ° C. (FIG. 11).

In order to investigate the effect of pH on the activity of recombinant proteins, CM-cellulose hydrolysis was performed in buffers of various pHs. Both recombinant proteins REng13 and REng43 of the present invention showed optimal activity at pH 3.5. (FIG. 12).

6-3. Confirmation of Cellooligosaccharide Degradation of Recombinant Proteins

Cell oligosaccharide degradation of recombinant proteins according to the present invention was confirmed. 5 μL of enzyme solution is mixed with 20 μL of 100 mM sodium acetate buffer solution (pH 5.3) containing 5 mg / mL of cellopentaose, cellotetraose, cellotriose, and cellobiose, respectively. After reacting at 43 ° C. for 1 hour, the hydrolyzate was confirmed by TLC. As a mobile phase, 1-butanol: acetic acid: water (v / v / v, 2: 1: 1) was used. After the development, a methanol solution containing 5% sulfuric acid was sprayed and heated to 100 ° C. to confirm the reaction. TLC development showed no difference between the recombinant proteins REng13 and REng43, where cellopentaose was almost completely degraded while cellotetraose was partially degraded (FIG. 13). Cellulotriose or cellobiose hardly decomposes, so the recombinant proteins according to the present invention seem to be able to use four or more bound celloligosaccharides as substrates.

Korea Biotechnology Research Institute KCTC11556BP 20090916 Korea Biotechnology Research Institute KCTC11655BP 20100225 Korea Biotechnology Research Institute KCTC11656BP 20100225

Attach an electronic file to a sequence list

Claims (12)

Glycosyl hydrolase gene represented by the nucleotide sequence of SEQ ID NO: 1.
Glycosyl hydrolase protein represented by the amino acid sequence of SEQ ID NO.
delete delete delete delete Recombinant expression vector pEREng13 having a cleavage map of FIG. 5 comprising the glycosyl hydrolase gene of SEQ ID NO: 1.
E. coli HIT-21 / pEREng13 transformants transformed with the recombinant expression vector of claim 7 (Accession No .: KCTC 11655BP).
delete delete delete delete

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