KR101999782B1 - A novel endo-β-1,4-glucanases from metagenomic resources and method for preparing the same - Google Patents

Abstract

The present invention relates to a new endo-beta-1,4-glucanase derived from a metagenome and a method for producing the same, and more particularly to a novel endo-beta-1-glucanase derived from a metagenome library extracted from a black goat rumen , 4-glucanase, a polynucleotide encoding the same, and a method for producing the same.
Since the peptide of SEQ ID NO: 1 of the present invention exhibits excellent endo-beta-1,4-glucanase activity, it can be used for microbial industrial waste treatment, ink removal of pulp and paper, and fiber biopolishing, Can be usefully used to improve substantially the production of better animal feed, improved beer brewing, reduced viscosity of the beta -glucan solution, and biofinishing in the textile industry.

Description

A novel endo-β-1,4-glucanases derived from a metagenome and a method for preparing the same.

The present invention relates to a new endo-beta-1,4-glucanase derived from a metagenome and a method for producing the same, and more particularly to a novel endo-beta-1-glucanase derived from a metagenome library extracted from a black goat rumen , 4-glucanase, a polynucleotide encoding the same, and a method for producing the same.

Cellulose is the main polysaccharide compound in plant cell walls and is composed of repeating units of cellobiose linked via β-1,4 glycosidic bonds. Cellulose is considered the most abundant renewable biomass in nature (Bhat and Bhat 1997). Cellulases can be classified into three types based on catalysis: endo-beta-1,4-glucanases (EC 3.2.1.4), cellobiose hydro- lase (cellobiohydrolases, EC 3.2.1.91) and beta-glucosidases (EC 3.2.1.21). Of these, endo-beta-l, 4-glucanase optionally attacks the amorphous sites inside the cellulosic polymer and thus generates new chain ends. Cellobiose hydrolyzate removes cellobiose without reducing or reducing the ends of the cellulose polymer. Beta-glucosidase hydrolyzes cyclodextrin and cellobiose to glucose. Cellulase has attracted many commercial interests in a variety of industries covering food, food, energy, pulp and textiles. Of the three cellulases, endo-beta-1,4-glucanase can release small pieces of cellulose of any length. Therefore, endo-beta-1,4-glucanase has biotechnological potential in various industrial fields. The enzyme can be used for microbial industrial waste treatment, pulp and paper ink removal, and fiber biopolishing. Enzymes can also be used to substantially improve the production of better animal feed, improved beer brewing, reduced viscosity of the beta -glucan solution, and biofinishing in the textile industry.

Cellulase can be prepared by various microbial plants and animals. In symbiotic microbes in herbivore rumen, cellulosic polymers can be hydrolyzed and fermented, thus allowing the host to obtain energy from polymers that are difficult to break down. Thus, the rumen ecosystem of ruminants is considered to be an attractive source of cellulase because this ecosystem has adapted to the utilization of lignocellulose plant biomass.

Korean black goats are classified as herbivores such as leaves, stems, and so forth. The small rumen of a black goat is consumed by many herbs and is comparable to other larger herbivores, and the particles of food can quickly move through the digestive tract. Therefore, although various environmental systems are expected to exist in the rumen rumen, most of the rumen microorganisms can not be cultured even if the rumen microbial environment system is very diverse. Therefore, efforts are needed to find the enzymes of the majority of microorganisms that have never actually been cultured and identified, which is believed to be possible through the expression of molecular biologic genetic recombination.

Metagenomes are defined as collecting genomes of all microorganisms that exist in the natural world. Generally, the metagenomic research consists of separating the metagenome without culturing the microorganism from the natural sample, and then preparing them as a library and introducing them into culturable Escherichia coli. This is a method for securing useful products from microorganisms that could not be cultured. It is difficult to obtain information on the microorganisms originating from the gene, but it has an advantage of securing the products and genes of microorganisms at the same time.

As a result, the present inventors made intensive efforts to select a cellulolytic enzyme derived from a meta genome library from Korean black goat rumen. As a result, the present inventors have found that a novel endo-beta-1,4-glucanase endo-beta-1,4-glucanases), and their excellent enzymatic activity was confirmed, thereby completing the present invention.

Accordingly, an object of the present invention is to provide endo-beta-1,4-glucanases comprising the amino acid sequence of SEQ ID NO: 1.

Another object of the present invention is to provide a polynucleotide encoding said endo-beta-1,4-glucanase.

Another object of the present invention is to provide a vector comprising the polynucleotide.

Another object of the present invention is to provide a recombinant microorganism transformed with said vector.

Another object of the present invention is to provide a method for producing endo-beta-1,4-glucanases by (a) culturing the recombinant microorganism of claim 5 to produce endo-beta-1,4-glucanases; And

beta-1, 4-glucanase of claim 1, comprising the step of (b) obtaining the endo-beta-1,4- endo -? - 1,4-glucanases.

In order to accomplish the object of the present invention, the present invention provides endo-β-1,4-glucanases comprising the amino acid sequence of SEQ ID NO: 1.

Another object of the present invention is to provide a polynucleotide encoding said endo-beta-1,4-glucanase.

According to another aspect of the present invention, there is provided a vector comprising the polynucleotide.

In order to accomplish another object of the present invention, there is provided a recombinant microorganism transformed with said vector.

According to another aspect of the present invention, there is provided a method for producing endo-beta-1,4-glucanases, comprising: (a) culturing the recombinant microorganism of claim 5 to produce endo-beta-1,4-glucanases; And

beta-1, 4-glucanase of claim 1, comprising the step of (b) obtaining the endo-beta-1,4- endo -? - 1,4-glucanases.

Hereinafter, the present invention will be described in detail.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

The present invention provides endo-beta-1,4-glucanases comprising the amino acid sequence of SEQ ID NO: 1.

In the present invention, a metagenomic library was constructed using a molecular biological method (Sambrook et al., Molecular Cloning, 1989) to select a novel cellulase (exocellulase) In order to extract the DNA for production and construct a fosmid library, it was ligated to a phosphide vector and packed in a phosphide library construction kit and transfected into E. coli.

The 'metagenome' of the present invention is defined as "a genome set of all microorganisms existing in a specific natural environment", and collectively refers to a genome or a genome-containing clone extracted from environmental samples (Handelsman, J. et al., Chem Biol , 5: R245,1998). The metagenomic library is usually cloned by inserting the genome directly into the vector, extracting the genome from tens of billions of microorganisms that are mixed in various environments such as soil, seawater, tidal flats, rivers, and animals. Although commonly used plasmids are used as vectors, BAC, YAC, Fosmid, Cosmid, etc., which can clone genes or gene clusters of a larger size, have been used , 'Fosmid' used in the present invention is known to be able to insert a gene or a dielectric of approximately 37 to 52 kb in size and has an advantage of exhibiting high transformation efficiency (Alduina, R. et al., FEMS Microbiol Lett., 218: 181, 2003).

SEQ ID NO: 1 of the present invention is a peptide consisting of the following amino acid sequence. In addition, the peptide of the present invention includes a peptide comprising the specific sequence of SEQ ID NO: 1 and further comprising 1 to 50 amino acids at the N-terminus and / or the C-terminus. Most preferably, the endo-beta-1,4-glucanases comprise the amino acid sequence of SEQ ID NO: 1.

SEQ ID NO: 1:

MNKNDERKGEATMNTCFDRHGALRVSGTQLLDEAERRVQLRGVSTLGLAWYPEYVNEAAF

RTLRDGWGANTVRLAMYTCESGGYMTGGDRAALEALIDRGVKLCAALGLYVIIDWHILSD

GDPNTHADAAEDFFGRISARYAGQPHVLYEICNEPNGPAATWPAVKRYARRIIPVIRANA

PDAVILCGTPNWSQDVDLAAADPLDDGNLMYALHFYAATHKEALREKAERALAAGAPLFI

TEFSICDASGDGAIDYDSAAAWRALIAKHGLSYVAWSLSNRDESAALIRADCPRTSDWTD

DDLTDTGRWLKAALNEDRLQ

According to one embodiment of the present invention, the peptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention exhibits excellent endo-beta-1,4-glucanases activity . It was also confirmed that the endo-beta-1,4-glucanase exhibits the best activity at pH 5.0 to 7.0 and 30 to 50 ° C.

The endo-beta-1,4-glucanases of the present invention are understood to include functional equivalents of peptides having the amino acid sequence of SEQ ID NO: 1. &Quot; Functional equivalent " means a peptide exhibiting substantially the same physiological activity as the peptide of SEQ ID NO: 1. &Quot; Homologous physiological activity " refers to an isomerism capable of having an amino acid sequence homology of at least 60%, preferably 70%, more preferably 90% or more. "Functional equivalents" include amino acid sequence variants in which some or all of the native protein amino acids are substituted, or in which some of the amino acids are deleted or added. Substitution of amino acids is preferably conservative substitution. Examples of conservative substitutions of amino acids present in nature are as follows: (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met).

The peptides of the present invention can be prepared by methods known to those skilled in the art. Such peptides can be produced in prokaryotic or eukaryotic cells by expressing polynucleotides encoding the peptide sequences of the invention, often as part of larger polypeptides. Alternatively, such peptides can be synthesized by chemical methods. Methods for expression of heterologous proteins in recombinant hosts, chemical synthesis of polypeptides and in vitro transcription are well known in the art and are described in Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd Ed Merrifield, J. (1969) J. Am., J. Am. Chem. Soc., Vol. Rev. Biochem. 57: 957; and Offord, RE (1980) Semisynthetic < RTI ID = 0.0 > Proteins, Wiley Publishing).

The present invention provides a polynucleotide encoding said peptide.

The " polynucleotide " is a polymer of deoxyribonucleotides or ribonucleotides present in single-stranded or double-stranded form. RNA genomic sequences, DNA (gDNA and cDNA) and RNA sequences transcribed therefrom, and include analogs of natural polynucleotides unless otherwise specified.

The polynucleotide includes not only a nucleotide sequence encoding the peptide but also a sequence complementary to the sequence. The complementary sequence includes not only perfectly complementary sequences but also substantially complementary sequences. It is known that under stringent conditions known in the art, a nucleotide sequence encoding endo-beta-1,4-glucanases comprising the amino acid sequence of SEQ ID NO: 1 ≪ RTI ID = 0.0 > and / or < / RTI >

The polynucleotide may also be modified. Such modifications include addition, deletion or non-conservative substitution or conservative substitution of nucleotides. The polynucleotide encoding the amino acid sequence is also interpreted to include a nucleotide sequence that exhibits substantial identity to the nucleotide sequence. The above substantial identity is determined by aligning the nucleotide sequence with any other sequence as closely as possible, and analyzing the aligned sequence using algorithms commonly used in the art to obtain a sequence having at least 80% homology, At least 90% homology or at least 95% homology.

Preferably, the polynucleotide encoding endo-beta-1,4-glucanase comprising the amino acid sequence of SEQ ID NO: 1 of the present invention is characterized by being represented by the nucleotide sequence of SEQ ID NO:

The present invention provides a vector comprising the polynucleotide.

The term "vector" means means for expressing a gene of interest in a host cell. For example, viral vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors, adenovirus vectors, retroviral vectors, and adeno-associated viral vectors. The vector that can be used as the recombinant vector may be a plasmid (for example, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14 , pGEX series, pET series, and pUC19), phage (e.g.,? gt4? B,? -charon,?? z1 and M13) or viruses (e.g., CMV, SV40 and the like).

The polynucleotide encoding the amino acid sequence of SEQ ID NO: 1 in the recombinant vector may be operatively linked to a promoter. The term "operatively linked" refers to the functional linkage between a nucleotide expression control regulatory sequence (e.g., a promoter sequence) and another nucleotide sequence. Thus, the regulatory sequence may thereby regulate transcription and / or translation of the other nucleotide sequence.

The recombinant vector can typically be constructed as a vector for cloning or as a vector for expression. The expression vector may be any conventional vector used in the art to express foreign proteins in plants, animals or microorganisms. The recombinant vector may be constructed by a variety of methods known in the art.

The recombinant vector may be constructed with prokaryotic or eukaryotic cells as hosts. For example, when the vector used is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (for example, pL? Promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.) , A ribosome binding site for initiation of translation, and a transcription / translation termination sequence. When the eukaryotic cell is used as a host, the origin of replication that functions in the eukaryotic cells contained in the vector is f1 replication origin, SV40 replication origin, pMB1 replication origin, adeno replication origin, AAV replication origin, CMV replication origin, and BBV replication origin But is not limited thereto. Also, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, The cytomegalovirus (CMV) promoter and the tk promoter of HSV) can be used, and generally have a polyadenylation sequence as a transcription termination sequence.

The present invention provides a recombinant microorganism transformed with said vector.

The recombinant microorganism of the present invention may be any host cell known in the art and includes, for example, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E Bacillus strains such as E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis and Bacillus tulifene, and enterobacteria and strains such as Salmonella typhimurium, Serratia marcesensis and various Pseudomonas species Saccharomyce cerevisiae, insect cells, plant cells and animal cells such as SP2 / 0, CHO (Chinese hamster ovary) K1, CHO DG44, PER, and the like are used as host cells when transforming eukaryotic cells. C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN and MDCK cell lines.

The insertion of the polynucleotide or a recombinant vector containing the polynucleotide into a recombinant microorganism can be carried out using an insertion method well known in the art. For example, when the recombinant microorganism is a prokaryotic cell, the CaCl2 method or the electroporation method may be used. When the recombinant microorganism is a eukaryotic cell, the microinjection method, the calcium phosphate precipitation method, the electroporation method, the liposome -Mediated transfection, and gene bombardment, but are not limited thereto. When E. coli and other microorganisms are used, the productivity is higher than that of animal cells, but it is not suitable for intact Ig-type antibody production due to glycosylation problems. However, production of antigen-binding fragments such as Fab and Fv Can be used.

The method of selecting the transformed host cells can be easily carried out according to a method widely known in the art by using a phenotype expressed by a selection marker. For example, when the selection mark is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic.

The present invention also provides a method for producing endo-beta-1,4-glucanases, comprising: (a) culturing the recombinant microorganism to produce endo-beta-1,4-glucanases; Beta-1, 4-glucanases of claim 1, comprising the steps of: (a) obtaining endo-beta-1,4-glucanases; (endo -? - 1,4-glucanases).

Since the peptide of SEQ ID NO: 1 of the present invention exhibits excellent endo-beta-1,4-glucanase activity, it can be used for microbial industrial waste treatment, ink removal of pulp and paper, and fiber biopolishing, Can be usefully used to improve substantially the production of better animal feed, improved beer brewing, reduced viscosity of the beta -glucan solution, and biofinishing in the textile industry.

Figure 1 shows multiple alignments of the KG35 endo-beta-1,4-glucanase GH5 domain according to the invention, along with other homologous GH5 protein domains. More specifically, the amino acid sequence of the GH5 lineage domain in KG35 endo-beta-1,4-glucanase was aligned with the GH5 lineage domain of the homologous enzyme from the following microorganisms (the signature sequence of the GH5 domain is shown in gray The Glu residues under the asterisks indicate catalytic sites): Eubacterium cellulosolvens (WP_051527028.1), Agathobacter rectalis (WP_055222932.1), Roseburia inulinivorans (WP_055169535.1), Agathobacter rectalis (WP_012741522.1), Ruminococcus torques (WP_055163516.1), Eubacterium rectale CAG : 36 (CDC71888.1), Roseburia inulinivorans (WP_055040325.1), and Lachnospiraceae bacterium C6A11 (WP_051646390.1).
FIG. 2 shows PAGE analysis of recombinant KG35 endo-beta-1,4-glucanase overexpressed in E. coli Rosetta-gami (Lane M: protein molecular weight marker, Lane 1: cell-free pre- 2: soluble fractions after induction, Lane 3: KG35 endo-beta-1,4-glucanase in polyacrylamide gels containing purified KG35 endo-beta-1,4-glucanase, Lane 4: Analysis of the presenter anemogram).
Figure 3 shows the results of analysis of the effect of pH (A) or temperature (B) on the activity of recombinant KG35 endo-beta-1,4-glucanase. More specifically,
FIG. 3A shows the results of measurement of the enzyme activity at 50 ° C. for 15 minutes in the indicated pH range (4.0 to 10.0). The buffer used at each pH was as follows: 0.1 M sodium acetate (pH 4.0 to 6.0, 0.1 M sodium phosphate (pH 6.0 to 8.0,?), 0.1 M Tris-HCl (pH 8.0 to 9.0,?), And 0.1 M glycine-NaOH (pH 9.0 to 10.0,?).
Fig. 3B shows the results of measurement of the enzyme activity in the indicated temperature range (20-70 DEG C) for 15 minutes in 100 mM sodium phosphate buffer (pH 7.0).
Figure 4 shows the results of analysis of the effect of pH (A) or temperature (B) on the stability of recombinant KG35 endo-beta-1,4-glucanase. More specifically,
Figure 3A shows the stability of the enzyme measured at 50 ° C. for 15 minutes in the indicated pH range (4.0-10.0). The buffers used at each pH are as follows: 0.1 M sodium acetate (pH 4.0-6.0, , 0.1 M sodium phosphate (pH 6.0 to 8.0, ∘), 0.1 M Tris-HCl (pH 8.0 to 9.0, ▼), and 0.1 M glycine-NaOH (pH 9.0 to 10.0, Δ).
FIG. 3B shows the stability of the enzyme measured in 100 mM sodium phosphate buffer (pH 7.0) for 15 minutes in the indicated temperature range (20-70 DEG C).

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

(1) Treatment of experimental animals and group genomics Fosmid  Building the Library

All animal research and operation procedures were reviewed and approved by the Animal Science Research Institute of Korea, Suwon, and Rural Development Administration (No. 2009-007, D-grade, surgery). Three Korean black goats consisting of two males and one female of 18 months old were obtained from the National Institute of Animal Science and were supplied with rice straw and mineral supplements for one month in order to maximize the fibrin adaptation of the rumen microorganisms. The rumen contents of the three goats were collected after slaughter, and then the fiber-adherent microorganisms and liquid fraction-related microorganisms were isolated. Each genomic DNA was extracted from each fraction using hexadecyl trimethyl ammonium bromide and sodium dodecyl sulfate (CTAB-SDS) (Zhou et al. 1996). A DNA fragment of about 40 kb obtained by partially digesting with HindIII was amplified using a copy control Fosmid library preparation kit based on pCC1FOS vector according to the manufacturer's instructions and a group genomic phosmid library . The traits of E. coli DH5TM (Life Technologies, Carlsbad, Calif., USA) carrying other recombinant fosmids were transferred to 384-well plates and stored at -70 ° C. A portion of the library contains chloramphenicol (15 g / mL) and 0.2% carboxymethyl cellulose (CMC; Sigma-Aldrich, Germany) to select the activity of endo-beta-1,4- (LB) agar plate fed with a buffer solution (LB, St. Louis, Mo., USA). After incubation at 37 ° C for 18 hours, all plates were stained with 0.2% Congo red for 30 min and washed with 1 M NaCl solution. A fosmid clone with fibrin activity was detected to form a clear region of the colonies representing the hydrolysis of CMC on a red background.

A group genomic phosmid library containing 115,200 clones was constructed of a black goat rumen microorganism. The NotI restriction analysis showed that the average insertion size was typically 31kb (no data). A total of 155 independent phosmid clones expressing carboxymethylcellulase (CMCase) activity were identified in two libraries. One of the phosphide clones (Ad125D08) formed the largest specific region on the screening plate containing CMC and was selected for further analysis.

(2) Enzyme positive Fosmid  Clone's shotgun  Sequencing

Shotgun sequencing and molecular analysis were performed to localize the gene for cellulase within the enzyme-positive phospho-clone and to subclone the gene for effective biochemical properties. The fibrin phosphide clone Ad125D08 carrying bacteria was cultured in 100 ml of LB medium containing 25 占 퐂 / ml of chloramphenicol at 37 占 폚 for 20 hours. Cells were harvested by centrifugation at 6000 xg for 15 min at 4 < 0 > C. DNA was extracted using Qiagen Plasmid Midi Kit (Qiagen, Valencia, Calif., USA) and shotgun sequencing was performed with two approaches (conventional Sanger sequencing and pyrosequencing). For shotgun sequencing by the Sanger method, 15 μg of fibrin phosmid clone DNA was used to obtain an arbitrary fragment of 2-3 kb. Fragmentation was performed using a HydroShear DNA Shearing Device (Genomic Solution, Waltham, Mass., USA) with the following parameters: sample volume 200 μl, speed code 11, and 20 shearing cycles. Small DNA fragments were removed using a CHROMA SPIN + TE1000 column (BD Biosciences Clontech, Heidelberg, Germany) followed by DNA polymerase and polynucleotide kinase. The prepared DNA fragment was dephosphorylated and ligated to the Smal site of pUC19. The ligation product was introduced into E. coli DH10B using electricity. Approximately 192 recombinant plasmids were randomly selected for sequencing in the shotgun DNA library. Each plasmid DNA was sequenced bi-directionally using the ABI 3730 automatic sequencer BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster, CA, USA). Sequence data were analyzed using Phred and phrap software. The other pyrosequencing was performed on a GS Junior sequencing system (Roche Diagnostics, Oakland, CA, USA). Briefly, 500 ng of the phosphide DNA sample was fragmented by spraying and the small fragment was removed by Agencourt AMPure XP bid (Beckman-Coulter, Beverly, MA, USA). RL adapters were ligated with fragmented DNA and quantified by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, Calif., USA). This library was amplified by cloning on capture beads of a water-in-oil emulsion microreactor. Five hundred thousand concentrated beads were deposited on a PicoTiterPlate device and pyrosequenced with a GS Junior device. Pyrosequencing data was analyzed using GS De Novo Assembler, version 2.7.

The nucleotide sequence of the enzyme-positive phosmid clone was determined using shotgun sequencing and pyrosequencing using the Sanger method. It was performed in the range of 2.7 times of the conventional shotgun sequencing using the Senter method, and the ~ 300kb pyrosequencing was produced. Thus, one contig of 42.453 kb could be obtained without the pCC1FOS vector sequence. Thirty-four ORFs were predicted in the flanking sequence of the phosphide clone Ad125D08, and the ORF containing the cellulase domain (KG35 gene) was identified and isolated within the clone.

(3) Enzyme positive Fosmid  Molecular analysis of clones

All contiguous sequences from traditional shotgun sequencing and pyrosequencing using the Sanger method were combined to construct all the family genomic-insert DNA sequences using SeqMan software in Lasergene version 7. Each adjacent gene prediction was performed using the basic parameters of MetaGeneMark. Function annotations were performed by searching the Pfam domain on the Pfam website (http://pfam.sanger.ac.uk). The amino acid sequence deduced from the nucleotide sequence of the enzyme-positive phosphite clone Ad125D08 was cloned using the BlastP on the NCBI website (http: // blast.cn) to find the protein most similar to the amino acid sequence from the identified open reading frames (ORFs). ncbi.nlm.nih.gov/Blast/). Multiple alignments of amino acid sequences deduced from sequences of ORFs and other proteins were performed using ClustalW software (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The PROSITE database (http://prosite.expasy.org/) was used to predict the module structure and the characterization sequence of the enzyme. The N-terminal region includes Signal IP software (http://www.cbs.dtu.dk/services/SignalP/) for signal peptide sequences and HMMTOP 2.0 software for the transmembrane domain (http://www.enzim.hu /hmmtop/html/submit.html).

Sequence analysis of the KG35 gene revealed that a single ORF predicted at molecular weights of 35.1 kDa and 5 pI is 963 bp long and encodes a single polypeptide of 320 amino acid residues. Signaling IP (Signal IP) and HMMTOP 2.0 were not able to detect a transient region in a known signal peptide sequence or KG35 protein. The PROSITE database module structure search predicted that the KG35 protein contains the conserved (amino acid) signature sequence VLYEICNEPN and the GH5 cellulase catalytic domain and that the second Glu residue is assumed to act as a catalyst (FIG. 1). The GH5 family cellulases have been reported to contain conserved amino acid sequence patterns [LIV] - [LIVMFYWGA] (2) - [DNEQG] - [LIVMGST] - {SENR} -NE- [PV] - [RHDNSTLIVFY].

The conserved signature sequence of the KG35 protein will be located in the signature sequence pattern of the GH5 group described above. This result clearly shows that KG35 protein is strongly related to GH5 cellulase. The BlastP search, compared to the amino acid sequence of the KG35 protein with other proteins registered in the GenBank database, showed that this protein has a similarity of up to 58% with proteins derived from Eubacterium cellulosolvens (WP_051527028.1) Species shares less sequence identity with other GH5 family protein homologs and other GH5 family protein homologs from various microbial species (Table 1). The amino acid sequence of this homologous protein was also found to have characteristic functions that were conserved among GH5 family enzymes (Fig. 1). Overall, the KG35 endo-beta-l, 4-glucanase protein was up to 58% homologous to the homologous enzyme, indicating that the endo-beta-l, 4-glucanase found has a novel amino acid sequence it means.

(4) Recombination KG35  Endo-beta-1,4- Glucanase  Heterologous expression and purification

The KG35 gene (the complete ORF of the putative cellulase gene) was amplified from the enzyme-positive phosphide clone Ad125D08 using PCR. The amplicon was ligated into the expression vector pET24 (Novagen, Darmstadt, Germany) and the resulting recombinant vector was introduced into E. coli Rosetta-gamiTMNovagen. For further screening for the fibrinolytic activity of the enzyme produced by the transformed E. coli cells, the same CMC agar plate method was used to illuminate the transparent region around the colony after Congo red staining. Fibroblast E. coli cells were grown in 10 mL of Super Optimal Broth (SOB) at 37 DEG C with ampicillin (100 mu g / mL) and transgene expression was measured with 0.2 mM isopropyl-beta-D- D-1-thiogalactopyranoside (IPTG) until the absorbance of the cells at 600 nm reached 0.5. Cells were harvested, resuspended in 50 mM Tris-HCl buffer (pH-7.0), dispersed 5 times for 30 seconds each with ultrasound of 39% amplitude (750 Watt ultrasonic processor ; SONICS, Newtown, CT, USA). Overexpressed proteins were purified from cell-free extracts generated using the Chelating Excellose Spin Kit (Bioprogen, Daejeon, South Korea). 2 [mu] l of the purified protein was dispensed into CMC agar plates to confirm enzyme activity. Proteins were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) using the method of Lemmli (1970) and the protein concentration was measured using the Bradford Protein Assay Kit (Bio-Rad).

Zymogram analysis of recombinant KG35 endo-beta-1,4-gluconase was performed using 10% polyacrylamide gel containing 0.1% CMC under denaturing conditions. To remove the SDS of the enzyme, the gel was immersed in denaturing buffer (50 mM sodium phosphate, pH 7.0) overnight. After the washing step, the gel was incubated with 50 mM sodium phosphate buffer (pH 7.0) at 37 ° C for 1 hour and stained with 1% Congo red solution for 30 minutes. Decolorization was carried out using 1 M NaCl solution until a clear area was observed.

The putative endo-beta-1,4-glucanase gene (KG35 gene) was amplified from the fosmid clone Ad125D08 and cloned into the expression vector pET24a (+). The resulting recombinant vector was introduced into E. coli Rosetta-gamiTM for enzyme expression. The enzyme was overexpressed as a soluble protein with a C-terminal 6xHis tag and purified under natural conditions using an affinity column kit for the His tag: Chelating Excellose cSpin kit (Bioprogen). SDS-PAGE analysis of the purified recombinant KG35 endo-beta-1,4-glucanase protein revealed a single band with an apparent molecular weight of 35 kDa on the gel (FIG. 2). Zymogram analysis confirmed that a single band corresponding to the same molecular weight was a band of active endo-beta-1,4-glucanase (FIG. 2).

(5) Recombination KG35  Endo-beta-1,4- Glucanase  Enzyme property

The specific activities of the purified recombinant KG35 endo-beta-1,4-glucanase using various substrates were evaluated under the following standard conditions. The substrates used in this study were beta-mannan, p-nitrophenyl-D-glucopyranoside, and p-nitrophenyl-D-cellrobioside (p (xylan) derived from CMC barley glucan, laminarin, avicel and birchwood, which are originated from -nitrophenyl-D-cellobioside. In all assays, 10 μl of the appropriately diluted enzyme solution was added to 200 μl of sodium phosphate buffer (pH 7.0) containing 1% (w / v) of each substrate. Enzyme reactions proceeded at 50 ° C (different reaction times): 15 minutes CMC, glucan, ramirainin, avicel; Xylen and beta-mannan from 10 minutes Birchwood; 20 min P-nitro-D-glucopyranoside and p-nitrophenyl-D-cellrobioside. The specific activity was defined as the number of active units per milligram of protein. One unit of enzyme activity was defined as the amount of enzyme reduced by reducing sugar per minute and 1 μmol release. In addition, one unit of the enzyme activity toward the substrate (p-nitrophenyl-D-glucopyranoside and p-nitrophenyl-D-cellobioside) was defined as the amount of enzyme required to release 1 μmol of P nitrophenol per minute. The amounts of release reducing sugar and p-nitrophenol were determined by measuring the absorbance at 540 or 400 nm, respectively. All analyzes were performed in triplicate.

The optimum pH was determined by adding 100 mM sodium acetate buffer solution (pH 4.0 to 6.0), 100 mM sodium phosphate buffer solution (pH 6.0 to 8.0), 100 mM Tris-HCl buffer solution (pH 8.0 to 9.0), or 100 mM glycine- ) For 15 min at < RTI ID = 0.0 > 50 C < / RTI > by assaying the enzymatic activity of the recombinant KG35 endo-beta-l, 4-glucanase purified. To determine the optimal temperature, the enzyme activity was analyzed with 100 mM sodium phosphate buffer (pH 7.0) for 15 minutes at temperatures up to 20-70 ° C. All analyzes were performed in triplicate. The enzyme was preincubated at 4 ° C for 24 hours at pH 4.0 to 10.0, and the stability of pH was evaluated. The enzyme was preincubated for 1 hour in 100 mM sodium phosphate buffer (pH 7.0) at various temperatures as described above, and the thermal stability was evaluated. All analyzes were performed in triplicate.

The various biochemical functions of the purified recombinant KG35 endo-beta-1,4-glucanase were characterized. First, substrate specificity for various substrates was determined (Table 2).

Endo-beta-1,4-glucanase showed strong specific activity against CMC (8.40 U / mg) and barley glucan (5.40 U / mg). In contrast, the enzyme was found to contain laminarin (0.48 U / mg), p-nitrophenyl-D-cellobioside (0.51 U / mg) and avicel (0.12 U / mg). < / RTI > In addition, endo-beta-l, 4-glucanase was unable to hydrolyze p-nitro-D-glucopyranoside, xylan derived from birchwood or mannan.

CMC and barleyglucan are mainly based on β-1,4-glycosidic bonds and have an amorphous structure, which is most favorable as a substrate for recombinant KG35 endo-beta-1,4-glucanase. (Xylan) or beta-mannan from amorphous laminarin containing beta-1,3 / 1,6-glycosidic bonds and birchwood Containing hemicelluloses was not a specific substrate of recombinant KG35 endo-beta-1,4-glucanase. Although Avicel mainly contains a? -1,4-glycoside bond, it was not efficiently hydrolyzed by this enzyme. Although Avicel contains β-1,4-glycosidic bonds as other water-soluble cellulose substrates do, it has a crystal structure that can be efficiently hydrolyzed when the cellulase binding domain of the cellulase participates in the hydrolysis reaction (Bolam et al., 1998; Linder et al., 1996). Nevertheless, sequencing was unable to detect any cellulosic binding domain of KG35 endo-beta-1,4-glucanase. The absence of this region could explain the weak activity against avicel. For the two synthetic substrates used in this study, p-nitrophenyl-D-cellrobioside and p-nitro-D-glucopyranoside are synthetic substrates of exo-glucanase and glucosidase, respectively (Mukund et al., 1983; Wesley and Donal 1982). Thus, the recombinant KG35 endo-beta-1,4-glucanase showed some exo-glucanase activity and did not exhibit the? -Glucosidase activity. All these results for substrate specificity indicate that KG35 cellulase is highly likely to hydrolyze the? -1,4-glycoside bond of the amorphous region of cellulose and consequently functions as endo-beta-1,4-glucanase .

Optimal pH and temperature of the recombinant enzyme were determined at various pH and temperature conditions (Fig. 3).

The optimal pH and temperature for CMC of recombinant KG35 endo-beta-1,4-glucanase were found to be about 6.0-7.0 and 30-50 ° C, respectively. In addition, enzyme stability was tested under various pH and temperature conditions (Figure 4).

Endo-beta-1,4-glucanase maintained activity over 70% after incubation at pH 5.0-7.0 for 24 hours. The same enzymes showed thermal stability after 1-H incubation at 30-50 ° C, but lost more than 60% activity after 1 hour incubation at 50 ° C or higher. Our empirical data have shown that recombinant enzymes collectively maintain pH and temperature stability within the optimal pH and temperature ranges, respectively. The optimum and stability characteristics of recombinant enzyme were similar to those of other cellulases from other ruminant ruminal microorganisms. Other cellulases of rumen microbes have been reported to have an optimum pH range of 5 to 7 and an optimal temperature close to 50 ° C (Nguyen et al., 2010; Zhao et al. Cellulase of the ruminal strain was also reported to maintain 70% activity in the pH range of 5.0 to 7.0 and the temperature range of 30 to 50 ° C (Xia et al. 2012). The ruminal pH of ruminants is below 5 to 7 under dietary conditions and the temperature of the rumen is 38 to 41 ° C (Mackie et al., 1999). The cellulose reaction of ruminal-bacterial cellulase was reported to be relatively strong at pH 6.5-7.0 in culture, but the reaction weakened with decreasing culture pH (Russell and Dombrowski 1980).

KG35 endo-beta-1,4-glucanase was found to have a relatively high activity and stability at pH and temperature conditions of rumen rats including recombinant KG35 endo-beta-1,4-glucanase This match seems to fit well into the rumen environment of black goats. The above observation can explain why recombinant KG35 endo-beta-l, 4-glucanase functions at a mild acid / neutral pH and moderate temperature than acid / alkaline pH or strong non-physiological temperature.

The preferred embodiments of the present invention have been described in detail above. It will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the appended claims.

Therefore, the scope of the present invention is defined by the appended claims rather than the foregoing detailed description, and all changes or modifications derived from the meaning, range, and equivalence of the claims are included in the scope of the present invention. .

<110> Kyonggi University Industry & Academia Cooperation Foundation <120> A novel endo-beta-1,4-glucanases from metagenomic resources and          method for preparing the same <130> NP16-08127 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 320 <212> PRT <213> Artificial Sequence <220> <223> Endo-beta-1,4-glucanase <400> 1 Met Asn Lys Asn Asp Glu Arg Lys Gly Glu Ala Thr Met Asn Thr Cys   1 5 10 15 Phe Asp Arg His Gly Ala Leu Arg Val Ser Gly Thr Gln Leu Leu Asp              20 25 30 Glu Ala Glu Arg Arg Val Gln Leu Arg Gly Val Ser Thr Leu Gly Leu          35 40 45 Ala Trp Tyr Pro Glu Tyr Val Asn Glu Ala Ala Phe Arg Thr Leu Arg      50 55 60 Asp Gly Trp Gly Ala Asn Thr Val Arg Leu Ala Met Tyr Thr Cys Glu  65 70 75 80 Ser Gly Gly Tyr Met Thr Gly Gly Asp Arg Ala Leu Glu Ala Leu                  85 90 95 Ile Asp Arg Gly Val Lys Leu Cys Ala Ala Leu Gly Leu Tyr Val Ile             100 105 110 Ile Asp Trp His Ile Leu Ser Asp Gly Asp Pro Asn Thr His Ala Asp         115 120 125 Ala Ala Glu Asp Phe Phe Gly Arg Ile Ser Ala Arg Tyr Ala Gly Gln     130 135 140 Pro His Val Leu Tyr Glu Ile Cys Asn Glu Pro Asn Gly Pro Ala Ala 145 150 155 160 Thr Trp Pro Ala Val Lys Arg Tyr Ala Arg Arg Ile Ile Pro Val Ile                 165 170 175 Arg Ala Asn Ala Pro Asp Ala Val Ile Leu Cys Gly Thr Pro Asn Trp             180 185 190 Ser Gln Asp Val Asp Leu Ala Ala Ala Asp Pro Leu Asp Asp Gly Asn         195 200 205 Leu Met Tyr Ala Leu His Phe Tyr Ala Ala Thr His Lys Glu Ala Leu     210 215 220 Arg Glu Lys Ala Glu Arg Ala Leu Ala Ala Gly Ala Pro Leu Phe Ile 225 230 235 240 Thr Glu Phe Ser Ile Cys Asp Ala Ser Gly Asp Gly Ala Ile Asp Tyr                 245 250 255 Asp Ser Ala Ala Ala Trp Arg Ala Leu Ile Ala Lys His Gly Leu Ser             260 265 270 Tyr Val Ala Trp Ser Leu Ser Asn Arg Asp Glu Ser Ala Ala Leu Ile         275 280 285 Arg Ala Asp Cys Pro Arg Thr Ser Asp Trp Thr Asp Asp Asp Leu Thr     290 295 300 Asp Thr Gly Arg Trp Leu Lys Ala Ala Leu Asn Glu Asp Arg Leu Gln 305 310 315 320 <210> 2 <211> 963 <212> DNA <213> Artificial Sequence <220> Nucleotide sequence encoding endo-beta-1,4-glucanase <400> 2 atgaacaaga acgacgagcg aaagggagaa gcgacgatga atacctgttt tgaccgacac 60 ggcgcgctgc gcgtgtccgg cacccagctg ttggacgagg cggagaggag ggtccagctg 120 ggccgccttt 180 cggacccttc gcgacggctg gggcgcgaac accgtgcgcc tggcgatgta cacctgcgag 240 tcgggcggct acatgaccgg cggcgacagg gcggcgctcg aggcgctgat cgaccggggc 300 gtgaagctct gcgcggcgct gggcctgtac gtcatcatcg actggcacat tttaagcgac 360 ggcgacccga acacccacgc cgatgccgcc gaggatttct ttggccggat cagcgcgcgc 420 tatgccggcc agccccacgt gctctacgag atctgcaacg agccgaacgg ccccgcggcg 480 acctggccgg cggtgaagcg ctacgccagg cggatcatcc ccgtgatccg cgccaacgcg 540 cccgacgcgg tgatcctgtg cgggacgccg aactggtccc aggacgtgga cctcgcggcg 600 gccgatcccc tggacgacgg gaacctcatg tacgcgctgc acttctacgc cgcgacccat 660 aaggaggcgc tgcgggagaa ggcggagcgc gcgctcgcgg ccggcgcgcc gctgtttatc 720 accgaattca gcatctgcga tgcctcgggc gacggcgcga tcgactatga ctccgccgcg 780 gcgtggcgcg ctctgatcgc gaagcacggg ctctcctatg tggcgtggag cctgtccaac 840 cgggatgaga gcgccgcgct gatccgcgcc gattgcccga ggacgtccga ctggaccgac 900 gacgacctca ccgacaccgg cagatggctc aaggcggcgc tgaacgagga cagattgcag 960 taa 963

Claims (8)

delete delete delete delete delete delete delete A DNA fragment containing the sequence of SEQ ID NO: 2 derived from the rumen of the black goat was cloned from the phosphide clone into the amplification and expression vector pET24a (+) to generate a recombinant vector, and the resulting recombinant vector was introduced into Escherichia coli Rosetta-gami And the enzyme was overexpressed with a soluble protein having a C-terminal 6xHis tag and purified under natural conditions using an affinity column kit for His tag: Chelating Excellose cSpin kit (Bioprogen) to produce beta-mannan (p-nitrophenyl-D-cellobioside), CMC (carboxymethyl) -methionine, p-nitrophenyl-D-glucopyranoside, only carboxymethyl cellulose (CMC) and barley glucan in xylan derived from cellulose, barley glucan, laminarin, avicel and birchwood, Enzyme activity Out, Endo for holding a 24-hour incubation of 70% or more activity after at pH 5.0-7.0 - process for producing a beta-1,4-glucanase of claim (endo-β-1,4-glucanases).

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