WO2014142529A1 - Novel cellulase derived from metagenome, and preparation method therefor - Google Patents

Novel cellulase derived from metagenome, and preparation method therefor Download PDF

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WO2014142529A1
WO2014142529A1 PCT/KR2014/002024 KR2014002024W WO2014142529A1 WO 2014142529 A1 WO2014142529 A1 WO 2014142529A1 KR 2014002024 W KR2014002024 W KR 2014002024W WO 2014142529 A1 WO2014142529 A1 WO 2014142529A1
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cellulase
recombinant
celex
activity
gene
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송재준
최종현
고경철
한윤전
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한국생명공학연구원
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • the present invention relates to a novel cellulase derived from metagenome and a method for preparing the same. More specifically, cellulase (CelEx) selected from a metagenome library extracted from bovine lumen suspension using a robot-based ultrafast search system -BR12), the gene encoding the same and a method for producing a recombinant cellulase.
  • Cellulase selected from a metagenome library extracted from bovine lumen suspension using a robot-based ultrafast search system -BR12
  • Fibrous biomass is converted to sugar through pretreatment and enzymatic treatment, and then useful chemicals such as ethanol are produced by microbial fermentation. Since the main component of biomass is cellulose, the effective decomposition of cellulose into monosaccharides such as fermentable glucose is a very important process in the production of chemical products derived from biomass.
  • 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 (EC3.2.1.21).
  • 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 Hydrolysis of the reducing and non-reducing ends of the cellulose polymer produced by the kinase is in turn hydrolyzed to produce cellobiose with two molecules of glucose bound thereto. The cellobiose is hydrolyzed by beta-glucosidase to finally produce glucose.
  • metagenome As one of these approaches, research on metagenome has been attempted, which is defined as the generic name of all microorganisms in nature.
  • metagenomic studies consist of separating metagenomes from natural samples without culturing microorganisms, and then preparing them into libraries and introducing them into cultivable Escherichia coli. This is a method for securing useful products from microorganisms that have not been cultured, but it is difficult to obtain information on the microorganisms from which the genes are derived, but there is an advantage of simultaneously obtaining the products and genes of the microorganisms.
  • the present inventors have made intensive efforts to select enzymes having high cellulose resolution from metagenomes.
  • a celEx-BR12 gene having cellulase activity using a robot-based ultrafast search system from a metagenome library extracted from bovine lumen suspension. Selected, and confirmed the exolulase and endocellulase activity of the selected cellulase, to complete the present invention.
  • An object of the present invention is to provide a cellulase derived from metagenome (cellulase), a gene encoding the same, a recombinant vector containing the gene and a recombinant microorganism transformed with the gene or the recombinant vector.
  • Another object of the present invention is to provide a method for producing a recombinant cellulase by culturing the recombinant microorganism.
  • the present invention also provides a recombinant microorganism having a recombinant cellulase production ability, characterized in that the gene encoding the cellulase (celEx-BR12), the recombinant vector containing the gene, and the gene or the recombinant vector are introduced into a host microorganism. to provide.
  • the present invention also comprises the steps of (a) culturing a recombinant microorganism having the recombinant cellulase production capacity to produce a recombinant cellulase; And (b) provides a method for producing a recombinant cellulase comprising the step of recovering the resulting recombinant cellulase.
  • FIG. 1 is a diagram illustrating a robot-based ultrafast navigation system.
  • Figure 2 is a schematic diagram showing a metagenome-derived cellulase screening process using an ultrafast search system.
  • FIG. 3 is a graph showing the pFOS-CBR12 clone showing cellulase activity as a result of analyzing the metagenome library clone using the ultrafast search system.
  • FIG. 7 is a graph showing the stability according to (A) optimal pH, (B) pH, (C) optimal temperature and (D) temperature of recombinant CelEx-BR12 cellulase.
  • FIG. 8 shows data for measuring recombinant CelEx-BR12 cellulase degradation activity through thin-layer chromatography (TLC) analysis.
  • a metagenomic DNA library (metagenomic library) was produced by a molecular biological method ( Sambrook et al., Molecular cloning , 1989), and the production of a metagenomic library in bovine lumen suspension
  • the plasmid was linked to the pCC1FOS vector and packaged in a ⁇ DNA packaging kit and transduced into E. coli EPI300-T1.
  • the "metagenome” of the present invention is defined as "genome collection of all microorganisms in a specific natural environment,” and collectively called clones containing genomes or genes extracted from environmental samples are called metagenomes (Handelsman, J. et al., Chem Biol., 5: R245, 1998).
  • the metagenome library extracts genomes directly from the billions of microorganisms mixed in various environments such as soil, seawater, tidal flats, rivers, and animal intestines, and inserts them into a vector to clone.
  • plasmids that have been commonly used are also used, but BAC, YAC, Fosmid, and Cosmid, which can clone a larger gene or gene cluster, are used.
  • BAC, YAC, Fosmid, and Cosmid which can clone a larger gene or gene cluster, are used.
  • 'Fosmid' used in the present invention is capable of inserting a gene or genome of approximately 37-52kb in size and is known to have an advantage of exhibiting high transformation efficiency (Alduina, R. et al., FEMS Microbiol Lett. , 218: 181, 2003).
  • clones with cellulase activity using a robot-based ultrafast search system are screened (FIG. 3). 20,000 clones were analyzed, of which pFOS-CBR12 clones showing cellulase activity were selected (FIG. 4).
  • the cellulase selected from the metagenome derived from bovine lumen suspension by the method of the present invention is represented by the amino acid sequence of SEQ ID NO: 2, and is named CelEx-BR12 cellulase in the present invention.
  • the present invention relates to a cellulase represented by the amino acid of SEQ ID NO: 2 and a gene encoding the cellulase ( celEx-BR12 ).
  • the gene may be represented by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 5.
  • vector refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host.
  • Vectors can be plasmids, phage particles or simply potential genomic inserts. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since plasmids are the most commonly used form of current vectors, “plasmid” and “vector” are sometimes used interchangeably in the context of the present invention. For the purposes of the present invention, it is preferred to use plasmid vectors.
  • Typical plasmid vectors that can be used for this purpose include (a) a replication initiation point that allows for efficient replication to include hundreds of plasmid vectors per host cell, and (b) host cells transformed with the plasmid vector. It has a structure comprising an antibiotic resistance gene and (c) a restriction enzyme cleavage site into which foreign DNA fragments can be inserted. Although no appropriate restriction enzyme cleavage site is present, the use of synthetic oligonucleotide adapters or linkers according to conventional methods facilitates ligation of the vector and foreign DNA.
  • the recombinant microorganism according to the present invention may be prepared by inserting the gene on the chromosome of the microorganism or introducing the recombinant vector on the plasmid of the microorganism according to a conventional method.
  • the vector should be transformed into the appropriate host cell.
  • preferred host cells are prokaryotic cells.
  • Suitable prokaryotic host cells include E. coli XL-1Blue (Stratagene), E. coli DH5 ⁇ , E. coli JM101, E. coli K12, E. coli W3110, E. coli X1776, E. coli BL21 , and the like.
  • E. coli strains such as FMB101, NM522, NM538 and NM539 and other prokaryotic species and genera may also be used.
  • E. coli strains such as FMB101, NM522, NM538 and NM539 and other prokaryotic species and genera may also be used.
  • E. coli XL-1Blue (Stratagene)
  • E. coli DH5 ⁇ E. coli JM101
  • E. coli K12 E. coli W3110
  • E. coli X1776 E. coli BL21
  • Prokaryotic transformation can be readily accomplished using the calcium chloride method described in section 1.82 of Sambrook et al., Supra .
  • electroporation (Neumann et al., EMBO J. , 1: 841, 1982) can also be used for transformation of these cells.
  • a commonly known gene manipulation method may be used as a method of inserting the cellulase gene on the chromosome of the host cell.
  • physical methods include microinjection (direct DNA injection), liposomes, directed DNA uptake, receptor-mediated DNA transfer, or DNA transport using Ca ++ .
  • Gene transfer method using a lot has been used. Examples include the use of retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, herpes simplex virus vectors, poxvirus vectors, or lentivirus vectors.
  • retroviruses have high gene transfer efficiency and It can be used in a wide range of cells without binding by host DNA rearrangement (changes in host DNA-like regions resulting in changes in host DNA function).
  • DNA for a pre-sequence or secretion leader is operably linked to DNA for a polypeptide when expressed as a shear protein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the sequence;
  • the ribosomal binding site is operably linked to a coding sequence when it affects the transcription of the sequence;
  • the ribosomal binding site is operably linked to a coding sequence when positioned to facilitate translation.
  • "operably linked” means that the linked DNA sequence is in contact, and in the case of a secretory leader, is in contact and present within the reading frame.
  • enhancers do not need to touch. Linking of these sequences is performed by ligation (linking) at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers according to conventional methods are used.
  • celEx-BR12 cellulase to celEx-BR12 gene (SEQ ID NO: 1) celEx-BR12 gene having the base sequence of SEQ ID NO: 5 via a base sequence codon optimized (codon optimization) of the order It was synthesized and inserted into the pET-22b (+) vector (Novagen, USA) to prepare a recombinant vector pET-CBR12 containing a gene encoding cellulase, and then transformed into Escherichia coli BL21 (DE3).
  • SEQ ID NO: 1 celEx-BR12 gene having the base sequence of SEQ ID NO: 5 via a base sequence codon optimized (codon optimization) of the order It was synthesized and inserted into the pET-22b (+) vector (Novagen, USA) to prepare a recombinant vector pET-CBR12 containing a gene encoding cellulase, and then transformed into Escherichia coli BL21 (DE3).
  • a fosmid library was constructed by extracting DNA for producing a metagenomic library from a bovine lumen suspension.
  • extraction buffer 2% (w / v) CTAB, 20 mM EDTA, 1.4 M NaCl, 100 mM Tris-HCl, pH8.0
  • 100 ⁇ l proteinase K 10 mg / ml
  • Metanomic DNA was isolated using Q Sepharose to remove phenolic and humic acid, and DNA end-repair enzymes were isolated from the DNA.
  • the mixed solution (Epicentre, USA) was treated to repair the 5 'end with a blunt end.
  • the DNA repaired to the blunt end was loaded onto a 1% (w / v) low melting point agarose gel and then clamped homogeneous electric field (CHEF) -DR II system (Bio- Rad, USA) was used to perform pulsed field gel electrophoresis (PFGE) at 6 V / cm, and DNA having a size of 30 to 40 kb was separated using GELase (Epicentre Technologies, USA).
  • CHEF homogeneous electric field
  • PFGE pulsed field gel electrophoresis
  • Isolated 30-40kb sized DNA was linked to pCC1FOS phosphide vector (Epicentre Technologies, USA) using a phosmid library production kit (Copy Control Fosmid Library Production Kit, Epicentre Technologies, USA) and packaged in ⁇ DNA packaging kit E. coli EPI300-T1 (Epicentre Technologies, USA) was transduced and stored at 4 °C.
  • the metagenomic library was plated on LB plates containing 12.5 ⁇ g / ⁇ l of chloramphenicol and then cultured to form colonies.
  • PHSGC12 vector containing celEdx12 gene with cellulase activity was used as a positive control using E. coli XL1-Blue (Stratagene) using the shotgun method (Ko et al., Appl. Microbiol. Biotech ., 89: 1453, 2011). Inc., USA) and E. coli EPI300-T1 transformed with pCC1FOS vector without metagenome DNA inserted as a negative control.
  • Example 2 of the present invention pFOS-CBR12 hit clones were selected, and fosmids were isolated from the selected clones by alkaline lysis (HC Birnboim et al., Nuc. Acid Res). , 7: 1513, 1979), isolated DNA (chromosomal DNA) was digested with restriction enzyme Bfu CI (New England Biolabs, USA) (incubated at 65 ° C. for 20 minutes), followed by electrophoresis to size 4-5kb. DNA fragments were purified with a Gel Extraction kit (QIAGEN Inc, USA).
  • the purified DNA was treated with restriction enzyme BamHI (New England Biolabs, USA), then subcloned into a pHSG298 (Takara, USA) vector (pHSG-CBR12) to produce a shotgun library of celEx-BR12 gene (Ko et al. , Appl. Microbiol.Biotech. , 89: 1453, 2011).
  • DNA fragments containing the position of the start codon (ATG) in the nucleotide sequence of the celEx-BR12 gene (SEQ ID NO: 1) for expressing the CelEx-BR12 cellulase of the present invention, Nde I (New England Biolabs, USA) and Xho I ( New England Biolabs (USA) was amplified using PCR (TProfessional thermalcycle (Biometra, Germany) to include a restriction site, and the primer used in the PCR process was designed to amplify the celEx-BR12 gene.
  • the DNA amplified by PCR is about 1.2 kb, and the cloned DNA fragment contains NdeI and Xho I restriction enzymes, which are then inserted into the pET-22b (+) vector (Novagen, USA) to include a gene encoding cellulase.
  • PET-22b (+) vector includes a T7 promoter for expression of the inserted cellulase gene, and a hexa-histidine tag (His-) at the C-terminus for easy purification of the expressed cellulase. tag) sequence.
  • the cloned pET-22b (+) vector (pET-CBR12) was transformed into Escherichia coli BL21 (DE3) (Novagen, USA), and then confirmed the expression level of cellulase, but the expression state was confirmed to be insufficient. Then, celEx-BR12 gene was synthesized through codon optimization (SEQ ID NO: 5) to prepare a recombinant vector in the same manner as above, and then transformed into Escherichia coli ( Esherichia coli ) BL21 (DE3).
  • Escherichia coli having recombinant CelEx-BR12 sulfulase producing ability prepared by codon optimization in Example 4 was cultivated in LB medium to which 50 ⁇ g / ml Ampicillin was added at 30 ° C. to induce cellulase expression. 0.5 mM IPTG (isopropyl-d-1-thiogalactopyranoside) was added to incubate at 30 ° C. for 4 hours to produce recombinant CelEx-BR12 sulfulase.
  • IPTG isopropyl-d-1-thiogalactopyranoside
  • PBS buffer containing 500 mM sodium chloride (NaCl) was given a gradient of 0 to 500 mM imidazole, and the first purification was performed using a HiTrap chelating HP column (GE Healthcare, USA). Cellulase was subjected to secondary purification using a HiPrep 26/10 desalting column (GE Healthcare) to remove sodium chloride. All purification steps used an FPLC system (AKTA Explorer; GE Healthcare, USA).
  • the cellulase activity was measured according to the change of pH and temperature.
  • the buffer used for the optimal pH measurement was as follows: 100 mM sodium acetate (pH 3.0-6.0), 100 mM sodium phosphate (pH 6.0-8.0), 100 mM Tris-HCl (pH 8.0-9.0), 100 mM sodium bicarbonate (pH 9.0-11.0), 100 mM disodium phosphate (pH 11.0-12.0), 100 mM potassium chloride (pH 12.0-13.0).
  • the recombinant CelEx-BR12 sulfulase of the present invention is birch xylan (132.3 dl / mg), carboxymethylcellulose (105.9 dl / mg), mecanic-sfeldt xylan (67.9 dl / mg) and 2-hydroxy It was confirmed that high activity was shown in ethyl cellulose (26.3 dl / mg). However, it showed low activity on Avicel and no activity on laminarin, starch, curdlan, ⁇ -cellulose, D-gluconic acid and salicylic acid (Table 2).
  • TLC Thin-layer chromatography
  • TLC is a measure of the degree of degradation of cellooligosaccharides (cellobiose to cellopentaose) and carboxymethyl cellulose (CMC) by the cellulase contained in the cell extract.
  • CelEx-BR12 sulfulase In order to determine the effect on the activity of recombinant CelEx-BR12 sulfulase according to the type of metal ion, it was added and reacted with CelEx-BR12 sulfulase such that the final concentration of various metal ions was 1 mM.
  • CelEx-BR12 sulfulase reacted with metal ions was treated with EDTA to reach a final concentration of 1 mM and reacted at 4 ° C. for 3 hours, and then dialyzed using PBS buffer to remove EDTA, and then enzyme activity was measured. It was.
  • cellulase CelEx-BR12 selected in the present invention has exocellulase activity and endocellulase activity, it can be used for the production of bioethanol using fibrin-based biomass, and various fiber, detergent, feed, food, pulp and paper production, etc. Applicable to industrial fields.

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Abstract

The present invention relates to a novel cellulase derived from a metagenome, and a preparation method therefor, and more specifically, to: a cellulase (CelEx-CBR12) selected using a robot-based high-throughput screening system, from a metagenomic library extracted from suspended solids of bovine rumen; a gene encoding the same; and a method for preparing a recombinant cellulase. The cellulase CelEx-CBR12 selected in the present invention has exocellulase activity and endocellulase activity, and thus can be used for producing bioethanol using cellulosic biomass, and can be applied to various industries such as fibers, detergents, feed, food, pulp, paper production and the like.

Description

메타게놈 유래 신규 셀룰라아제 및 이의 제조방법Metagenome-derived novel cellulase and preparation method thereof
본 발명은 메타게놈(metagenomic) 유래 신규 셀룰라아제(cellulase) 및 이의 제조방법에 관한 것으로, 더욱 자세하게는 소의 루멘(rumen) 부유물에서 추출한 메타게놈 라이브러리로부터 로봇기반 초고속 탐색 시스템을 이용하여 선별된 셀룰라아제(CelEx-BR12), 이를 코딩하는 유전자 및 재조합 셀룰라아제의 제조방법에 관한 것이다.The present invention relates to a novel cellulase derived from metagenome and a method for preparing the same. More specifically, cellulase (CelEx) selected from a metagenome library extracted from bovine lumen suspension using a robot-based ultrafast search system -BR12), the gene encoding the same and a method for producing a recombinant cellulase.
화석 연료 고갈에 대비하기 위해서 원유 의존도가 가장 높은 수송용 연료 분야에 원유를 대체할 수 있는 에너지 기술 개발이 중요한 이슈로 떠오르고 있다. 바이오에탄올은 현존의 화석 연료대비 가격 경쟁력이 낮음에도 미국을 비롯한 브라질, EU 다수의 국가에서는 사용 중에 있으며 최근에는 동남 및 중국과 같은 서남아시아 일부의 국가에서 사회, 공익적 편익을 감안하여 정책적으로 개발하여 사용하기 위한 연구가 수행중이다. In order to prepare for the depletion of fossil fuels, the development of energy technologies that can replace crude oil in the transportation fuel sector, which is most dependent on crude oil, is emerging as an important issue. Bioethanol is used in many countries including the United States, Brazil and the EU even though its price competitiveness is lower than that of existing fossil fuels. Recently, it is developed in consideration of social and public benefits in parts of Southwest Asia such as Southeast and China. Research is underway for use.
국내 휘발유 중 바이오에탄올 5% 전국적 보급 시 CO2 약 105만톤/년 감축 예상이 예상되며, 국가 신 재생에너지 사용 비중이 약 0.11% 증가하고 2030년 45,323백만원 정도의 사회적 편익 발생할 것으로 예상된다. 옥수수 및 사탕수수와 같은 식량 자원을 이용한 바이오에탄올 생산은 식량 자원과의 충돌 및 원자재 가격 상승이라는 본질적인 취약점이 있기 때문에 곡물 자원과 경합이 없는 섬유소계 바이오매스 자원에서 바이오에탄올 생산 기술 개발이 주목받고 있다.Domestic gasoline of the bio-ethanol 5% CO 2 nationally advertised about 105 tons / year reduction is expected to expected increase in national renewable energy share of about 0.11%, and is expected to be approximately 45,323 social benefits of one million won in 2030. Bioethanol production using food resources such as corn and sugar cane has inherent weaknesses such as collisions with food resources and rising raw material prices. Therefore, the development of bioethanol production technology has been attracting attention from fiber-based biomass resources without competition with grain resources. .
섬유소계 바이오매스는 전처리 및 효소처리 과정 등을 거쳐서 당분으로 전환되고 이후 미생물 발효에 의해서 에탄올 등 유용한 화학제품들이 만들어지게 된다. 바이오매스의 주성분이 셀룰로오스이기 때문에 셀룰로오스를 발효 가능한 포도당과 같은 단당류로 효과적으로 분해하는 것은 바이오매스 유래의 화학제품 제조과정에 있어서 매우 중요한 과정이라고 할 수 있다.Fibrous biomass is converted to sugar through pretreatment and enzymatic treatment, and then useful chemicals such as ethanol are produced by microbial fermentation. Since the main component of biomass is cellulose, the effective decomposition of cellulose into monosaccharides such as fermentable glucose is a very important process in the production of chemical products derived from biomass.
셀룰로오스(cellulose)는 포도당이 베타-1,4-글리코시드 결합(beta-1,4-glycosidic bond)으로 연결된 고분자의 선형 다당류로서 자연계에 존재하는 가장 풍부한 유기물이며 재활용될 수 있는 에너지 자원으로 가치가 매우 높다. 셀룰로오스는 셀룰라아제(cellulase)라는 가수분해 효소에 의해서 구성성분인 포도당으로 분해된다. 현재까지 알려진 셀룰라아제는 크게 엔도-베타-1,4-글루카나아제(endo-beta-1,4-glucanase, EC 3.2.1.4), 엑소-베타-1,4-글루카나아제(exo-beta-1,4-glucanase, EC 3.2.1.91), 그리고 베타-글루코시다아제(beta-glucosidase, EC3.2.1.21)의 세 가지 형태로 분류될 수 있다. 엔도-베타-1,4-글루카나아제는 셀룰로오스 내부 사슬의 베타-1,4 결합을 무작위로 가수분해하고, 엑소-베타-1,4-글루카나아제는 엔도-베타-1,4-글루카나아제에 의해 생성된 셀룰로오즈 중합체의 환원말단 및 비환원말단을 차례로 가수분해하여 2 분자의 포도당이 결합된 셀로비오스(cellobiose)를 생산한다. 상기의 셀로비오스는 베타-글루코시다아제에 의하여 가수분해되어 최종적으로 포도당이 생성된다.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 (EC3.2.1.21). 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 Hydrolysis of the reducing and non-reducing ends of the cellulose polymer produced by the kinase is in turn hydrolyzed to produce cellobiose with two molecules of glucose bound thereto. The cellobiose is hydrolyzed by beta-glucosidase to finally produce glucose.
섬유소 분해균류로 알려진 트리코데르마 라이드(Trichoderma ride) 이외에도 다양한 다른 균류도 셀룰로오스 분해효소를 생산하고 셀룰로오스 화합물이나 카복실메틸셀롤로오스와 같은 용해되지 시운 셀룰로오스 유도체를 분해하는 것으로 알려져 있으나, 이들의 균류로부터 생산되는 셀룰라아제는 결정의 셀룰로오스 화합물에는 효과적으로 작용하지는 않는다. 아스파질러스(Aspergillus)로부터 생산되는 셀룰레이즈는 보통 높은 베타-글루코시데이즈 활성을 가지지만, 엔도-글루카네이즈 활성은 낮은 것으로 알려져 있으며, 트리코데르마(Trichoderma) 속 균주는 높은 엔도- 및 엑소-글루카네이즈 활성을 가지며, 베타-글루코시데이즈 활성은 낮은 것으로 알려져 있다. 기존의 셀룰라아제들은 셀룰로오스를 분해하는 과정에서 속한 유형에 따라 부분적인 역할만 할 수 있을 뿐 하나의 효소가 셀룰로오스를 포도당으로 직접 분해할 수는 없기 때문에 다수의 효소제를 혼합해서 사용해야 한다는 불편함을 가지고 있다.In addition to Trichoderma ride , also known as fibrinolytic fungi, a variety of other fungi are known to produce cellulose degrading enzymes and break down undissolved cellulose derivatives such as cellulose compounds or carboxymethylcellulose. The cellulase produced does not act effectively on the cellulose compound of the crystal. Cellulase produced from Aspergillus usually has high beta-glucosidase activity, but endo-glucanize activity is known to be low, and Trichoderma genus strains have high endo- and exo It is known to have glucanase activity and low beta-glucosidase activity. Existing cellulase has only the partial role of decomposing cellulose, but it is inconvenient to use a mixture of enzymes because one enzyme cannot directly break down cellulose into glucose. .
높은 셀룰로오스 분해활성을 가지는 셀룰라아제의 선별과정에서, 일반적인 셀룰라아제 기질로 사용되는 CMC(carboxymetyl cellulase)를 이용할 경우에는 엑소-셀룰라아제에 의한 활성으로 클리어 존(clear zone) 형성이 어렵기 때문에, 플레이트 어세이(plate assay)에서 클리어 존을 형성할 수 있는 기질의 선택이 어려워 엑소-셀룰라아제의 스크리닝은 그 방법이 매우 노동집약적이고, 시간 및 비용이 많이 소모되는 단점이 있어, 현재까지 연구된 셀룰라아제는 엔도-셀룰라아제에 비해 엑소-셀룰라아제의 수가 낮다. In the screening process of cellulase having high cellulolytic activity, CMC (carboxymetyl cellulase), which is used as a general cellulase substrate, is difficult to form a clear zone due to exo-cellulase activity. Screening of exo-cellulase is difficult due to the difficulty in selecting a substrate capable of forming a clear zone in a plate assay, which is disadvantageous in that the method is very labor intensive and time-consuming. The number of exo-cellulases is lower than.
그러나, 최근 분자미생물 생태학의 연구를 통해서 자연계에 존재하는 미생물의 99％ 이상이 전형적인 배지에서 배양되지 않는다는 사실이 증명되었다 (Amann et al., Microbiol. Rev., 59:143, 1995; Hugenholtz and Pace, Trends Biotechnol., 14:190, 1996; Ward et al., Nature, 345:63, 1990). 따라서, 실제로 배양되어 동정된 적이 없는 대다수 미생물의 효소를 찾으려는 노력이 필요하며, 이는 분자생물학적인 유전자 재조합 발현을 통하여 가능하리라 여겨지고 있다.However, recent studies of molecular microbial ecology have demonstrated that more than 99% of the microorganisms in nature are not cultured in typical media (Amann et al., Microbiol. Rev. , 59: 143, 1995; Hugenholtz and Pace , Trends Biotechnol., 14: 190, 1996; Ward et al., Nature, 345: 63, 1990). Therefore, efforts are needed to find enzymes of most microorganisms that have not been actually cultured and identified, which is thought to be possible through molecular biological recombinant expression.
이러한 접근방법의 하나로 메타게놈(metagenome)에 관한 연구가 시도되고 있는데, 메타게놈은 자연계에 존재하는 모든 미생물의 유전체를 통칭하는 것으로 정의된다. 일반적으로, 메타게놈 연구는 자연계 시료로부터 미생물을 배양하지 않고 메타게놈을 분리한 후, 이들을 라이브러리로 작성하여 배양가능한 대장균에 도입하는 단계로 구성된다. 이는 배양이 불가능했던 미생물로부터 유용 산물을 확보하기 위한 방법으로, 유전자의 유래가 되는 미생물에 대한 정보는 얻기가 어려우나 미생물의 산물과 유전자를 동시에 확보할 수 있는 장점이 있다.As one of these approaches, research on metagenome has been attempted, which is defined as the generic name of all microorganisms in nature. In general, metagenomic studies consist of separating metagenomes from natural samples without culturing microorganisms, and then preparing them into libraries and introducing them into cultivable Escherichia coli. This is a method for securing useful products from microorganisms that have not been cultured, but it is difficult to obtain information on the microorganisms from which the genes are derived, but there is an advantage of simultaneously obtaining the products and genes of the microorganisms.
이에, 본 발명자들은 메타게놈으로부터 셀룰로오즈 분해능이 우수한 효소를 선별하기 위해 예의 노력한 결과, 소의 루멘(rumen) 부유물에서 추출한 메타게놈 라이브러리로부터 로봇기반 초고속 탐색 시스템을 이용하여 셀룰라아제 활성을 가지는celEx-BR12 유전자를 선별하였으며, 선별된 셀룰라아제의 엑소룰라아제 및 엔도셀룰라아제 활성을 확인하고, 본 발명을 완성하게 되었다. Accordingly, the present inventors have made intensive efforts to select enzymes having high cellulose resolution from metagenomes. As a result, we have identified a celEx-BR12 gene having cellulase activity using a robot-based ultrafast search system from a metagenome library extracted from bovine lumen suspension. Selected, and confirmed the exolulase and endocellulase activity of the selected cellulase, to complete the present invention.
발명의 요약Summary of the Invention
본 발명의 목적은 메타게놈(metagenomic) 유래의 셀룰라아제(cellulase), 이를 코딩하는 유전자, 상기 유전자를 함유하는 재조합 벡터 및 상기 유전자 또는 상기 재조합 벡터로 형질전환된 재조합 미생물을 제공하는 데 있다. An object of the present invention is to provide a cellulase derived from metagenome (cellulase), a gene encoding the same, a recombinant vector containing the gene and a recombinant microorganism transformed with the gene or the recombinant vector.
본 발명의 또 다른 목적은 상기 재조합 미생물을 배양하여 재조합 셀룰라아제를 제조하는 방법을 제공하는 데 있다.Another object of the present invention is to provide a method for producing a recombinant cellulase by culturing the recombinant microorganism.
상기 목적을 달성하기 위해서, 본 발명은 서열번호 2의 아미노산 서열로 표시되는 셀룰라아제를 제공한다. In order to achieve the above object, the present invention provides a cellulase represented by the amino acid sequence of SEQ ID NO: 2.
본 발명은 또한, 상기 셀룰라아제를 코딩하는 유전자(celEx-BR12), 상기 유전자를 포함하는 재조합 벡터 및 상기 유전자 또는 재조합 벡터가 숙주미생물에 도입되어 있는 것을 특징으로 하는 재조합 셀룰라아제 생산능을 가지는 재조합 미생물을 제공한다. The present invention also provides a recombinant microorganism having a recombinant cellulase production ability, characterized in that the gene encoding the cellulase (celEx-BR12), the recombinant vector containing the gene, and the gene or the recombinant vector are introduced into a host microorganism. to provide.
본 발명은 또한, (a) 상기 재조합 셀룰라아제 생산능을 가지는 재조합 미생물을 배양하여 재조합 셀룰라아제를 생성하는 단계; 및 (b) 생성된 재조합 셀룰라아제를 회수하는 단계를 포함하는 재조합 셀룰라아제의 제조방법을 제공한다. The present invention also comprises the steps of (a) culturing a recombinant microorganism having the recombinant cellulase production capacity to produce a recombinant cellulase; And (b) provides a method for producing a recombinant cellulase comprising the step of recovering the resulting recombinant cellulase.
도 1은 로봇 기반의 초고속 탐색 시스템을 나타낸 그림이다. 1 is a diagram illustrating a robot-based ultrafast navigation system.
도 2는 초고속 탐색 시스템을 이용한 메타게놈 유래 셀룰라아제 선별과정을 나타낸 모식도이다. Figure 2 is a schematic diagram showing a metagenome-derived cellulase screening process using an ultrafast search system.
도 3은 초고속 탐색 시스템을 이용하여 메타게놈 라이브러리 클론을 분석한 결과, 셀룰라아제 활성을 보이는 pFOS-CBR12 클론을 나타낸 그래프이다. 3 is a graph showing the pFOS-CBR12 clone showing cellulase activity as a result of analyzing the metagenome library clone using the ultrafast search system.
도 4는 본 발명에서 샷건 클로닝 방법으로 제조된 pHSG-CBR12의 셀룰라아제 유전자 구조 및 전사해독프레임을 도식화하여 나타낸 것이다. Figure 4 shows the schematic diagram of the cellulase gene structure and transcriptional detoxification frame of pHSG-CBR12 prepared by the shotgun cloning method in the present invention.
도 5는 CelEx-BR12 셀룰라아제의 상동성을 비교한 것이다.5 compares the homology of CelEx-BR12 cellulase.
도 6은 정제된 재조합 CelEx-BR12 셀룰라아제를 나타낸 것이다. 6 shows purified recombinant CelEx-BR12 cellulase.
도 7은 재조합 CelEx-BR12 셀룰라아제의 (A) 최적 pH, (B) pH에 따른 안정성, (C) 최적 온도 및 (D) 온도에 따른 안정성을 나타낸 그래프이다. 7 is a graph showing the stability according to (A) optimal pH, (B) pH, (C) optimal temperature and (D) temperature of recombinant CelEx-BR12 cellulase.
도 8은 TLC(thin-layer chromatography) 분석을 통해 재조합 CelEx-BR12 셀룰라아제 분해활성을 측정한 데이타이다. FIG. 8 shows data for measuring recombinant CelEx-BR12 cellulase degradation activity through thin-layer chromatography (TLC) analysis. FIG.
발명의 상세한 설명 및 바람직한 구현예Detailed Description of the Invention and Preferred Embodiments
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술 분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로 본 명세서에서 사용된 명명법은 본 기술 분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless defined otherwise, 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.
본 발명에서는 신규한 셀룰라아제(exocellulase)를 선별하기 위하여, 분자생물학적 방법(Sambrook et al., Molecular cloning, 1989)으로 메타게놈 DNA 라이브러리(metagenomic library)를 제작하였으며, 소의 루멘 부유물에서 메타게놈 라이브러리 제작을 위한 DNA를 추출하여 포스미드 라이브러리(fosmid library)를 구축하기 위해, 포스미드 pCC1FOS 벡터에 연결한 후 λDNA 패키징 키트에 포장하여 대장균(E. coli) EPI300-T1에 형질도입하였다.In the present invention, in order to select a novel cellulase (exocellulase), a metagenomic DNA library (metagenomic library) was produced by a molecular biological method ( Sambrook et al., Molecular cloning , 1989), and the production of a metagenomic library in bovine lumen suspension In order to construct a fosmid library by extracting the DNA, the plasmid was linked to the pCC1FOS vector and packaged in a λDNA packaging kit and transduced into E. coli EPI300-T1.
본 발명의 '메타게놈'은 "특정 자연환경에 존재하는 모든 미생물의 유전체 집합"으로 정의되고 있으며, 환경시료로부터 추출한 유전체 또는 유전자를 포함하는 클론을 총칭하여 메타게놈이라고 부르고 있다 (Handelsman, J. et al., Chem Biol., 5:R245, 1998). 메타게놈 라이브러리는 일반적으로, 토양, 해수, 갯벌, 하천, 동물 장내 등 다양한 환경에 혼재되어 있는 수백억 개의 미생물로부터 유전체를 직접 추출하여 벡터에 삽입하여 클로닝을 하게 된다. 벡터(vector)로는 일반적으로 많이 사용되어온 플라스미드도 사용되고 있지만 보다 큰 크기의 유전자 또는 유전자 클러스터(gene cluster)를 클로닝할 수 있는 BAC, YAC, 포스미드(Fosmid), 코스미드(Cosmid) 등이 사용되고 있으며, 본 발명에서 사용한 '포스미드(Fosmid)'는 대략 37~52kb 크기의 유전자 또는 유전체를 삽입할 수 있으며 높은 형질전환 효율을 나타내는 장점이 있는 것으로 알려져 있다 (Alduina, R. et al., FEMS Microbiol Lett., 218:181, 2003). The "metagenome" of the present invention is defined as "genome collection of all microorganisms in a specific natural environment," and collectively called clones containing genomes or genes extracted from environmental samples are called metagenomes (Handelsman, J. et al., Chem Biol., 5: R245, 1998). In general, the metagenome library extracts genomes directly from the billions of microorganisms mixed in various environments such as soil, seawater, tidal flats, rivers, and animal intestines, and inserts them into a vector to clone. As a vector, plasmids that have been commonly used are also used, but BAC, YAC, Fosmid, and Cosmid, which can clone a larger gene or gene cluster, are used. , 'Fosmid' used in the present invention is capable of inserting a gene or genome of approximately 37-52kb in size and is known to have an advantage of exhibiting high transformation efficiency (Alduina, R. et al., FEMS Microbiol Lett. , 218: 181, 2003).
본 발명의 일 실시예에서, 상기 메타게놈 라이브러리에서 셀룰라아제 활성을 지닌 클론을 선별하기 위해, 로봇 기반의 초고속 탐색 시스템(도 1)을 이용한 셀룰라아제 활성을 지닌 클론을 스크리닝 방법(도 3)으로, 총 20,000개의 클론을 분석하였으며, 이 중 셀룰라아제 활성을 보이는 pFOS-CBR12 클론을 선별하였다 (도 4).In one embodiment of the present invention, in order to screen clones with cellulase activity in the metagenome library, clones with cellulase activity using a robot-based ultrafast search system (FIG. 1) are screened (FIG. 3). 20,000 clones were analyzed, of which pFOS-CBR12 clones showing cellulase activity were selected (FIG. 4).
본 발명의 일 실시예에서는, 높은 셀룰라아제 활성을 보인 pFOS-CBR12 클론을 선별하였으며, 선별된 클론에서 포스미드(fosmid)를 분리한 다음, 정제된 celEx-BR12 유전자를 pHSG298 벡터에 서브클로닝하여 샷건 라이브러리(pHSG-CBR12)를 제작하였으며, 샷건 클로닝된 pHSG-CBR12는 대장균(E. coli) XL1-Blue에 형질전환시킨 후에, 셀룰라아제 활성을 가진 유전자가 삽입된 재조합 플라스미드를 분리하여, 염기서열 및 아미노산 서열을 분석하였다. In one embodiment of the present invention, pFOS-CBR12 clones showing high cellulase activity were selected, phosphmids were isolated from the selected clones, and then the purified celEx-BR12 gene was subcloned into the pHSG298 vector for shotgun libraries. (pHSG-CBR12) was prepared, and shotgun-cloned pHSG-CBR12 was transformed into E. coli XL1-Blue, and then the recombinant plasmid containing the gene having cellulase activity was isolated, and the nucleotide sequence and amino acid sequence. Was analyzed.
본 발명에서 샷건 클로닝된 celEx-BR12 유전자의 전사해독프레임(open reading frame; ORF)을 분석한 결과(도 4 및 표 1), 샷건 클로닝된 celEx-BR12의 유전자는 4kb로 이루어져 있으며, 2개의 ORF로 구성되어져 있다. ORF1은 약 1.8 kb로 이루어져 있으며 프리보텔라 루미니콜라(Prevotella ruminicola) 23의 페니실린 결합 단백질(penicillin-binding protein)과 약 84% 상동성을 보이는 것을 확인하였다. 또한, ORF2는 380개의 아미노산으로 이루어져 있으며, 난배양성 미생물(uncultured microorganism)의 셀로덱스트리나아제(cellodextrinase)와 약 92%, 프리보텔라 루미니콜라(Prevotella ruminicola) 23의 글라이코실 가수분해효소(glycosyl hydrolase) family 5와 약 83%의 상동성을 보이는 것을 확인하였다 (도 5).Analysis of the open reading frame (ORF) of the shotgun cloned celEx-BR12 gene in the present invention (Fig. 4 and Table 1), the gene of the shotgun cloned celEx-BR12 is composed of 4kb, two ORF It consists of. ORF1 consists of about 1.8 kb and shows about 84% homology with the penicillin-binding protein of Prevotella ruminicola 23. ORF2 also consists of 380 amino acids, about 92% of cell cultures of uncultured microorganisms, and glycosyl hydrolysing enzyme of Prevotella ruminicola 23. hydrolase) family 5 and about 83% homology was confirmed (Fig. 5).
따라서, 본 발명의 방법으로 소의 루멘 부유물 유래의 메타게놈에서 선별한 셀룰라아제는 서열번호 2의 아미노산 서열로 표시되며, 본 발명에서는 CelEx-BR12 셀룰라아제로 명명하였다. Therefore, the cellulase selected from the metagenome derived from bovine lumen suspension by the method of the present invention is represented by the amino acid sequence of SEQ ID NO: 2, and is named CelEx-BR12 cellulase in the present invention.
본 발명은 일 관점에서, 서열번호 2의 아미노산으로 표시되는 셀룰라아제 및 상기 셀룰라아제를 코딩하는 유전자(celEx-BR12)에 관한 것이다. In one aspect, the present invention relates to a cellulase represented by the amino acid of SEQ ID NO: 2 and a gene encoding the cellulase ( celEx-BR12 ).
본 발명에 있어서, 상기 유전자는 서열번호 1 또는 서열번호 5의 염기서열로 표시되는 것을 특징으로 할 수 있다. In the present invention, the gene may be represented by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 5.
본 발명은 다른 관점에서, 상기 셀룰라아제를 코딩하는 유전자(celEx-BR12)를 함유하는 재조합 벡터, 상기 셀룰라아제를 코딩하는 유전자 또는 상기 재조합 벡터가 숙주미생물에 도입되어 있는 것을 특징으로 하는 재조합 셀룰라아제 생산능을 가지는 재조합 미생물 및 상기 재조합 미생물을 배양하는 것을 특징으로 하는 재조합 셀룰라아제의 제조방법에 관한 것이다. In another aspect, the present invention provides a recombinant cell comprising the cellulase-encoding gene ( celEx-BR12 ), the cellulase-encoding gene or the recombinant vector is introduced into a host microorganism. Eggplant relates to a recombinant microorganism and a method for producing a recombinant cellulase, characterized in that the culturing the recombinant microorganism.
본 발명에서, "벡터 (vector)"는 적합한 숙주 내에서 DNA를 발현시킬 수 있는 적합한 조절 서열에 작동가능하게 연결된 DNA 서열을 함유하는 DNA 제조물을 의미한다. 벡터는 플라스미드, 파지 입자 또는 간단하게 잠재적 게놈 삽입물일 수 있다. 적당한 숙주로 형질전환되면, 벡터는 숙주 게놈과 무관하게 복제하고 기능할 수 있거나, 또는 일부 경우에 게놈 그 자체에 통합될 수 있다. 플라스미드가 현재 벡터의 가장 통상적으로 사용되는 형태이므로, 본 발명의 명세서에서 "플라스미드 (plasmid)" 및 "벡터 (vector)"는 때로 상호 교환적으로 사용된다. 본 발명의 목적상, 플라스미드 벡터를 이용하는 게 바람직하다. 이러한 목적에 사용될 수 있는 전형적인 플라스미드 벡터는 (a) 숙주세포당 수백 개의 플라스미드 벡터를 포함하도록 복제가 효율적으로 이루어지도록 하는 복제 개시점, (b) 플라스미드 벡터로 형질전환된 숙주세포가 선발될 수 있도록 하는 항생제 내성 유전자 및 (c) 외래 DNA 절편이 삽입될 수 있는 제한효소 절단부위를 포함하는 구조를 지니고 있다. 적절한 제한효소 절단부위가 존재하지 않을지라도, 통상의 방법에 따른 합성 올리고뉴클레오타이드 어댑터 (oligonucleotide adaptor) 또는 링커 (linker)를 사용하면 벡터와 외래 DNA를 용이하게 라이게이션 (ligation)할 수 있다. In the present invention, "vector" refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host. Vectors can be plasmids, phage particles or simply potential genomic inserts. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since plasmids are the most commonly used form of current vectors, "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purposes of the present invention, it is preferred to use plasmid vectors. Typical plasmid vectors that can be used for this purpose include (a) a replication initiation point that allows for efficient replication to include hundreds of plasmid vectors per host cell, and (b) host cells transformed with the plasmid vector. It has a structure comprising an antibiotic resistance gene and (c) a restriction enzyme cleavage site into which foreign DNA fragments can be inserted. Although no appropriate restriction enzyme cleavage site is present, the use of synthetic oligonucleotide adapters or linkers according to conventional methods facilitates ligation of the vector and foreign DNA.
본 발명에 따른 상기 재조합 미생물은 통상의 방법에 따라 상기 유전자를 미생물의 염색체(chromosome) 상에 삽입시키거나, 상기 재조합 벡터를 미생물의 플라스미드(plasmid) 상에 도입시킴으로써 제조할 수 있다. The recombinant microorganism according to the present invention may be prepared by inserting the gene on the chromosome of the microorganism or introducing the recombinant vector on the plasmid of the microorganism according to a conventional method.
라이게이션 후에, 벡터는 적절한 숙주세포로 형질전환되어야 한다. 본 발명에 있어서, 선호되는 숙주세포는 원핵세포이다. 적합한 원핵 숙주세포는 E. coli XL-1Blue(Stratagene), E. coli DH5α, E. coli JM101, E. coli K12, E. coli W3110, E. coli X1776, E. coli BL21 등을 포함한다. 그러나 FMB101, NM522, NM538 및 NM539와 같은 E.coli 균주 및 다른 원핵생물의 종(speices) 및 속(genera) 등이 또한 사용될 수 있다. 상기 E. coli에 덧붙여, 아그로박테리움 A4와 같은 아그로박테리움 속 균주, 바실루스 섭틸리스(Bacillus subtilis)와 같은 바실리(bacilli), 살모넬라 타이피뮤리움(Salmonella typhimurium) 또는 세라티아 마르게센스(Serratia marcescens)와 같은 또 다른 장내세균 및 다양한 슈도모나스(Pseudomonas) 속 균주가 숙주세포로서 이용될 수 있다.After ligation, the vector should be transformed into the appropriate host cell. In the present invention, preferred host cells are prokaryotic cells. Suitable prokaryotic host cells include E. coli XL-1Blue (Stratagene), E. coli DH5α, E. coli JM101, E. coli K12, E. coli W3110, E. coli X1776, E. coli BL21 , and the like. However , E. coli strains such as FMB101, NM522, NM538 and NM539 and other prokaryotic species and genera may also be used. In addition to E. coli, strains of the genus Agrobacterium, such as Agrobacterium A4, bacilli , such as Bacillus subtilis , Salmonella typhimurium or Serratia marcescens ) is another variety of enteric bacteria and Pseudomonas (Pseudomonas) in the strain, such as may be used as host cells.
원핵세포의 형질전환은 Sambrook et al., supra의 1.82 섹션에 기술된 칼슘 클로라이드 방법을 사용해서 용이하게 달성될 수 있다. 선택적으로, 전기천공법(electroporation)(Neumann et al., EMBO J., 1:841, 1982) 또한 이러한 세포들의 형질전환에 사용될 수 있다.Prokaryotic transformation can be readily accomplished using the calcium chloride method described in section 1.82 of Sambrook et al., Supra . Alternatively, electroporation (Neumann et al., EMBO J. , 1: 841, 1982) can also be used for transformation of these cells.
본 발명에서 셀룰라아제 유전자를 숙주세포의 염색체상에 삽입하는 방법으로는 통상적으로 알려진 유전자조작방법을 사용할 수 있다. 예를 들어, 물리적인 방법으로서, microinjection(세포에 DNA를 직접 넣는 것), liposome, directed DNA uptake, receptor~mediated DNA transfer 또는 Ca++을 이용한 DNA 운반 방법 등이 있으며, 최근에는 바이러스(virus)를 이용한 유전자 운반 방법이 많이 사용되고 있다. 일례로는 레트로바이러스 벡터, 아데노바이러스 벡터, 아데노-연관 바이러스 벡터, 헤르페스 심플렉스 바이러스 벡터, 폭스바이러스 벡터 또는 렌티바이러스 벡터를 이용하는 방법 등이 있으며, 특히, 레트로바이러스는 유전자 전달 효율이 높고 gross deletion이나 숙주 DNA와 재정렬(rearrangement : 숙주 DNA 중 자기 DNA와 유사한 부위를 바꾸는 것으로 숙주 DNA 기능의 변화를 초래함)에 의한 결합 없이 넓은 범위의 세포들에서 사용할 수 있다. In the present invention, as a method of inserting the cellulase gene on the chromosome of the host cell, a commonly known gene manipulation method may be used. For example, physical methods include microinjection (direct DNA injection), liposomes, directed DNA uptake, receptor-mediated DNA transfer, or DNA transport using Ca ++ . Gene transfer method using a lot has been used. Examples include the use of retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, herpes simplex virus vectors, poxvirus vectors, or lentivirus vectors. In particular, retroviruses have high gene transfer efficiency and It can be used in a wide range of cells without binding by host DNA rearrangement (changes in host DNA-like regions resulting in changes in host DNA function).
핵산은 다른 핵산 서열과 기능적 관계로 배치될 때 "작동가능하게 연결(operably linked)"된다. 이것은 적절한 분자 (예를 들면, 전사 활성화 단백질)가 조절 서열(들)에 결합될 때 유전자 발현을 가능하게 하는 방식으로 연결된 유전자 및 조절 서열(들)일 수 있다. 예를 들면, 전서열 (pre-sequence) 또는 분비 리더 (leader)에 대한 DNA는 폴리펩타이드의 분비에 참여하는 전단백질로서 발현되는 경우 폴리펩타이드에 대한 DNA에 작동가능하게 연결되고; 프로모터 또는 인핸서는 서열의 전사에 영향을 끼치는 경우 코딩서열에 작동가능하게 연결되거나; 또는 리보좀 결합 부위는 서열의 전사에 영향을 끼치는 경우 코딩 서열에 작동가능하게 연결되거나; 또는 리보좀 결합 부위는 번역을 용이하게 하도록 배치되는 경우 코딩 서열에 작동가능하게 연결된다. 일반적으로, "작동가능하게 연결된"은 연결된 DNA 서열이 접촉하고, 또한 분비 리더의 경우 접촉하고 리딩 프레임 내에 존재하는 것을 의미한다. 그러나, 인핸서 (enhancer)는 접촉할 필요가 없다. 이들 서열의 연결은 편리한 제한 효소 부위에서 라이게이션 (연결)에 의해 수행된다. 그러한 부위가 존재하지 않는 경우, 통상의 방법에 따른 합성 올리고뉴클레오티드 어댑터 (oligonucleotide adaptor) 또는 링커 (linker)를 사용한다. Nucleic acids are "operably linked" when placed in a functional relationship with other nucleic acid sequences. This may be genes and regulatory sequence (s) linked in such a way as to enable gene expression when the appropriate molecule (eg, transcriptional activation protein) is bound to the regulatory sequence (s). For example, DNA for a pre-sequence or secretion leader is operably linked to DNA for a polypeptide when expressed as a shear protein that participates in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when positioned to facilitate translation. In general, "operably linked" means that the linked DNA sequence is in contact, and in the case of a secretory leader, is in contact and present within the reading frame. However, enhancers do not need to touch. Linking of these sequences is performed by ligation (linking) at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers according to conventional methods are used.
본 발명에 있어서, 상기 미생물은 Agrobacterium 속, Aspergillus 속, Acetobacter 속, Aminobacter 속, Agromonas 속, Acidphilium 속, Bulleromyces 속, Bullera 속, Brevundimonas 속, Cryptococcus 속, Chionosphaera 속, Candida 속, Cerinosterus 속, Escherichia 속, Exisophiala 속, Exobasidium 속, Fellomyces 속, Filobasidium 속, Geotrichum 속, Graphiola 속, Gluconobacter 속, Kockovaella 속, Curtzmanomyces 속, Lalaria 속, Leucospoidium 속, Legionella 속, Psedozyma 속, Paracoccus 속, Petromyc 속, Rhodotorula 속, Rhodosporidium 속, Rhizomonas 속, Rhodobium 속, Rhodoplanes 속, Rhodopseudomonas 속, Rhodobacter 속, Sporobolomyces 속, Spridobolus 속, Saitoella 속, Schizosaccharomyces 속, Sphingomonas 속, Sporotrichum 속, Sympodiomycopsis 속, Sterigmatosporidium 속, Tapharina 속, Tremella 속, Trichosporon 속, Tilletiaria 속, Tilletia 속, Tolyposporium 속, Tilletiposis 속, Ustilago 속, Udenlomyce 속, Xanthophilomyces 속, Xanthobacter 속, Paecilomyces 속, Acremonium 속, Hyhomonus 속, Rhizobium 속 등을 예시할 수 있으며, 대장균(Escherichia coli)인 것이 바람직하다.In the present invention, the microorganism of the genus Agrobacterium , Aspergillus , Acetobacter , Aminobacter , Agromonas , Acidphilium , Bulleromyces , Bullera , Brevundimonas , Cryptococcus , Chionosphaera , Candida , Cerinosterus , Escherichia , Exisophiala genus, Exobasidium genus, Fellomyce s genus, Filobasidium genus, Geotrichum genus, Graphiola genus, Gluconobacter genus, Kockovaella genus, Curtzmanomyces genus, Lalaria genus, Leucospoidium genus, Legionell a genus, Psedozyma genus, Paracoccus genus, Petromyc genus, Rhodotorula Rhodosporidium in, Rhizomonas in, Rhodobium in, Rhodoplanes in, Rhodopseudomonas in, Rhodobacter genus, Sporobolomyces in, Spridobolus in, Saitoella in, Schizosaccharomyces genus, Sphingomonas genus, Sporotrichum in, Sympodiomycopsis in, Sterigmatosporidium in, Tapharina in, Tremella genus, Trichosporon in , Tilletiaria in, Tilletia genus, Tolyposporium in, Tilletiposis in, Ustilago genus, Udenlomyce in, Xanthophilomyces in, Xanthobacter in, Paecilomyces genus, Acremonium genus, Hyhomonus genus, Rhizobium genus and the like can be exemplified, Escherichia coli is preferred.
본 발명의 일 실시예에서는, CelEx-BR12 셀룰라아제를 발현시키기 위하여 celEx-BR12 유전자(서열번호 1)의 염기서열을 코돈 최적화(codon optimization)를 통해 서열번호 5의 염기서열을 가지는 celEx-BR12 유전자를 합성하여, pET-22b(+) 벡터 (Novagen, 미국) 에 삽입하여, 셀룰라아제를 코딩하는 유전자를 포함하는 재조합 벡터 pET-CBR12을 제조한 다음, 대장균(Escherichia coli) BL21(DE3)에 형질전환시켰다. In one embodiment of the invention, expressing the CelEx-BR12 cellulase to celEx-BR12 gene (SEQ ID NO: 1) celEx-BR12 gene having the base sequence of SEQ ID NO: 5 via a base sequence codon optimized (codon optimization) of the order It was synthesized and inserted into the pET-22b (+) vector (Novagen, USA) to prepare a recombinant vector pET-CBR12 containing a gene encoding cellulase, and then transformed into Escherichia coli BL21 (DE3). .
본 발명의 pET-22b(+) 벡터는 삽입된 셀룰라아제 유전자가 발현될 수 있도록 T7 프로모터를 포함하며, 발현된 셀룰라아제를 쉽게 정제할 수 있도록 C-말단에 hexa-histidine tag(His-tag) 서열을 포함하는 것을 특징으로 한다.  The pET-22b (+) vector of the present invention includes a T7 promoter for expressing the inserted cellulase gene, and has a hexa-histidine tag (His-tag) sequence at the C-terminus so that the expressed cellulase can be easily purified. It is characterized by including.
본 발명에서는 상기에서 제조된 재조합 CelEx-BR12 설룰라아제 생산능을 가지는 대장균을 배양하여, 재조합 CelEx-BR12 설룰라아제를 Ni-NTA 흡착 크로마토그래피방법을 사용하여 정제하였으며, 상기 정제된 CelEx-BR12 설룰라아제는 SDS-PAGE(sodium dodecyl sulfate polyacrylamide gel electrophoresis)로 약 42kDa의 크기를 가지고 있는 것을 확인하였다 (도 6).In the present invention was cultured E. coli having a recombinant CelEx-BR12 sulfulase production capacity prepared above, the recombinant CelEx-BR12 sulfulase was purified using Ni-NTA adsorption chromatography method, the purified CelEx-BR12 The sulfulase was confirmed to have a size of about 42 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (FIG. 6).
본 발명의 방법으로 생산된 재조합 CelEx-BR12 설룰라아제의 효소활성을 측정하기 위하여, pH 및 온도변화에 따른 셀룰라아제 활성을 특정한 결과, 약 35℃, pH5.0에서 최대활성을 보이는 것을 확인하였으며, pH7 내지 9의 중성부분에서 안정성이 높게 유지되는 것을 확인하였다. 또한, CelEx-BR12 셀룰라아제는 20 내지 30℃에서 2시간 이상 활성이 유지되는 것을 확인하였으며, 40℃이상에서는 불안정한 것을 확인하였다 (도 7).In order to measure the enzymatic activity of the recombinant CelEx-BR12 sulfulase produced by the method of the present invention, it was confirmed that the cellulase activity according to the pH and temperature changes showed a maximum activity at about 35 ° C. and pH 5.0. It was confirmed that the stability is maintained high in the neutral portion of pH 7-9. In addition, CelEx-BR12 cellulase was confirmed that the activity is maintained for 2 hours or more at 20 to 30 ℃, it was confirmed that unstable above 40 ℃ (Fig. 7).
본 발명의 재조합 CelEx-BR12 설룰라아제의 기질특이성을 확인한 결과, 자작나무 자일란(132.3Ｕ/㎎), 카복실메틸셀룰로오즈(105.9Ｕ/㎎), 오트-스펠트 자일란(67.9Ｕ/㎎) 및 2-하이드록시에틸-셀룰로오즈(26.3Ｕ/㎎)에 높은 활성을 보이는 것을 확인하였지만, 아비셀에는 낮은 활성을 보였으며, 라미나린, 녹말, 커들란, α-셀룰로오즈, D-글루콘산 및 살리신에는 활성을 보이지 않았다 (표 2).As a result of confirming the substrate specificity of the recombinant CelEx-BR12 sulfulase of the present invention, birch xylan (132.3 dl / mg), carboxymethyl cellulose (105.9 dl / mg), oat-sfeldt xylan (67.9 dl / mg) and 2 It showed high activity in hydroxyethyl-cellulose (26.3 dl / mg), but showed low activity in Avicel, and activity in laminarin, starch, curdlan, α-cellulose, D-gluconic acid and salicycin. Not visible (Table 2).
CelEx-BR12 설룰라아제의 미카엘리스-멘텐 상수(Michaelis-Menten constants; K m) 및 최대 반응 속도(maximal reaction velocities; V max)를 라인웨버-버크식(Lineweaver-Burk) 방법에 따라 측정한 결과, 표 3에 나타난 바와 같이, 본원발명의 방법으로 제조된 재조합 CelEx-BR12 설룰라아제의 Ｋ m 값은 12.92μM 및 Ｖ max 값은 1.55 ×10-4μmol/min을 나타내는 것을 확인하였다. The Michaelis-Menten constants ( K m ) and maximum reaction velocities ( V max ) of CelEx-BR12 sulfulase were measured according to the Lineweaver-Burk method. , as shown in Table 3, K m values for the recombinant CelEx BR12-sulfonic Lula kinase prepared by the method of the present invention 12.92μM and V max values were confirmed to exhibit a 1.55 × 10 -4 μmol / min.
또한, 본 발명의 재조합 CelEx-BR12 설룰라아제의 효소활성을 측정하기 위해 TLC(thin-layer chromatography) 분석을 수행한 결과, 셀로트리오스(cellotriose; G3), 셀로테트로스(cellotetrose; G4), 셀로펜토오스(cellopentaose; G5)를 각각 분해하는 것으로 보아 엑소셀룰라아제(exocellulase)의 활성 및 엔도셀룰라아제(endocellulase)의 활성을 동시에 보유함을 확인하였으며(도 8), 금속이온의 종류에 따라 재조합 CelEx-BR12 설룰라아제의 활성에 미치는 영향을 알아보기 위하여, 다양한 금속이온의 최종농도가 1mM이 되도록 CelEx-BR12 설룰라아제에 첨가하여 반응시킨 결과, 대부분의 금속이온에서 상대적인 셀룰라아제의 활성이 130 내지 150% 정도 증가한 것을 확인하였고, 아연이온(Zn2+)을 처리한 경우에는 최대 177% 셀룰라아제 활성이 증가한 것을 확인하였다. 하지만, 철이온(Fe2+) 및 수은이온(Hg2+)에서는 오히려 셀룰라아제 활성이 감소하였다 (표 4). In addition, as a result of performing thin-layer chromatography (TLC) analysis to measure the enzymatic activity of the recombinant CelEx-BR12 sulfulase of the present invention, cellotriose (G3), cellotetrose (G4), Decomposing cellopentaose (G5), respectively, it was confirmed that the activity of exocellulase (endocellulase) and endocellulase activity (endocellulase) at the same time (Fig. 8), depending on the type of metal ion recombinant CelEx- In order to investigate the effect on the activity of BR12 sulfulase, the reaction was performed by adding CelEx-BR12 sulfulase to the final concentration of various metal ions to 1 mM, and the relative cellulase activity of most metal ions was 130 to 150. It was confirmed that the percent increase, when treated with zinc ions (Zn 2+ ) it was confirmed that up to 177% cellulase activity increased. However, cellulase activity was decreased in iron ions (Fe 2+ ) and mercury ions (Hg 2+ ).
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.
실시예Example
실시예 1 : 메타게놈 라이브러리 제작 Example 1 Preparation of Metagenome Library
본 발명에서는 신규한 셀룰라아제를 선별하기 위하여, 메타게놈 라이브러리를 제작하였으며, 이전에 알려진 분자생물학적 방법을 사용하여 메타게놈 DNA로부터 라이브러리를 제작하였다 (Sambrook et al., Molecular cloning, 1989; a laboratory manual, 3rd ed. Cold Spring Habor Laboratory., Cold Spring Habor, NY.).In the present invention, in order to select novel cellulase, a metagenomic library was constructed, and a library was prepared from metagenomic DNA using previously known molecular biological methods (Sambrook et al., Molecular cloning , 1989; a laboratory manual, 3rd ed.Cold Spring Habor Laboratory., Cold Spring Habor, NY.).
먼저, 소(한우)의 루멘 부유물에서 메타게놈 라이브러리 제작을 위한 DNA를 추출하여 포스미드 라이브러리(fosmid library)를 구축하였다. 샘플 10ｇ에 27㎖의 추출버퍼(extraction buffer; 2%(w/v)CTAB, 20mM EDTA, 1.4 M NaCl, 100 mM Tris-HCl, pH8.0) 및 100㎕의 프로테나아제 K(proteinase K; 10㎎/㎖)을 첨가한 다음, 200rpm에서 30분 동안 교반하였다.First, a fosmid library was constructed by extracting DNA for producing a metagenomic library from a bovine lumen suspension. In 10 g of sample, 27 ml of extraction buffer (2% (w / v) CTAB, 20 mM EDTA, 1.4 M NaCl, 100 mM Tris-HCl, pH8.0) and 100 μl proteinase K; 10 mg / ml) was added and then stirred at 200 rpm for 30 minutes.
각 샘플에 6㎖의 10%(w/v) SDS(sodium dodecyl sulfate)를 첨가하여 65℃에서 2시간 동안 부드럽게 흔들어준 다음, 4℃에서 16,000×ｇ 조건으로 20분 동안 원심분리하였으며, 각 상등액을 새로운 튜브에 옮기고, 동량의 페놀/클로로포름/이소아밀알콜(phenol/chloroform/isoamyl alcohol; 25:24:1(v/v/v))을 첨가한 다음, -20℃에서 1 시간 동안 방치하였다. 그 후, 4℃에서 20,000×ｇ 조건으로 20분 동안 원심분리하여 남아있는 펠렛을 70% 에탄올(v/v)로 세척한 다음 200㎕의 TE 버퍼로 재현탁하였다. Each sample was added with 6 ml of 10% (w / v) sodium dodecyl sulfate (SDS) and gently shaken at 65 ° C. for 2 hours, followed by centrifugation at 4 ° C. under 16,000 × g for 20 minutes. Was transferred to a new tube, and the same amount of phenol / chloroform / isoamyl alcohol (25: 24: 1 (v / v / v)) was added and then left at -20 ° C for 1 hour. . Thereafter, the remaining pellets were washed with 70% ethanol (v / v) by centrifugation at 20,000 × g for 20 minutes at 4 ° C. and then resuspended in 200 μl of TE buffer.
메타지노믹 DNA(metagenomic DNA)는 페놀산(phenolic acid) 및 부식산(humic acid)을 제거하기 위해 Q 세파로오즈(Q-Sepharose)를 이용하여 분리하였으며, 분리된 DNA에 DNA end-repair 효소 혼합액(Epicentre, USA)을 처리하여 5' 말단을 블런트 엔드(blunt end)로 수선하였다.Metanomic DNA was isolated using Q Sepharose to remove phenolic and humic acid, and DNA end-repair enzymes were isolated from the DNA. The mixed solution (Epicentre, USA) was treated to repair the 5 'end with a blunt end.
블런트 엔드(blunt end)로 수선된 DNA를 1%(w/v) 저융점 아가로오즈겔(low melting point agarose gel)에 로딩한 후, CHEF(clamped homogeneous electric field)-DR II 시스템(Bio-Rad, 미국)을 사용하여 6V/cm 조건에서 펄스장겔전기영동법(pulsed field gel electrophoresis ; PFGE)을 수행하였으며, GELase(Epicentre Technologies, 미국)를 이용하여 30 내지 40kb 크기를 가지는 DNA를 분리하였다. The DNA repaired to the blunt end was loaded onto a 1% (w / v) low melting point agarose gel and then clamped homogeneous electric field (CHEF) -DR II system (Bio- Rad, USA) was used to perform pulsed field gel electrophoresis (PFGE) at 6 V / cm, and DNA having a size of 30 to 40 kb was separated using GELase (Epicentre Technologies, USA).
분리된 30 내지 40kb 크기를 가지는 DNA는 포스미드 라이브러리 제조 키트(Copy Control Fosmid Library Production Kit, Epicentre Technologies, 미국)를 이용하여 pCC1FOS 포스미드 벡터(Epicentre Technologies, 미국)에 연결한 후 λDNA 패키징 키트에 포장하여 대장균(E. coli) EPI300-T1(Epicentre Technologies, 미국)에 형질도입하여 4℃에서 보관하였다. Isolated 30-40kb sized DNA was linked to pCC1FOS phosphide vector (Epicentre Technologies, USA) using a phosmid library production kit (Copy Control Fosmid Library Production Kit, Epicentre Technologies, USA) and packaged in λDNA packaging kit E. coli EPI300-T1 (Epicentre Technologies, USA) was transduced and stored at 4 ℃.
실시예 2 : 초고속 탐색 시스템을 이용한 메타게놈 라이브러리 유래의 셀룰라아제 선별Example 2 Cellulase Selection from Metagenome Library Using an Ultrafast Search System
본 발명에서는 로봇기반의 초고속 탐색 시스템(High-throughput screening; HTS)(도 1)을 이용하여, 도 3의 초고속 탐색 시스템을 이용한 셀룰라아제 스크리닝 과정의 방법으로, 실시예 1에서 제작된 메타게놈 라이브러리에서 셀룰라아제활성을 가진 클론을 선별하였다In the present invention, using a robot-based high-throughput screening system (HTS) (FIG. 1), by the method of cellulase screening using the ultra-fast search system of FIG. 3, in the metagenome library prepared in Example 1 Clones with Cellulase Activity were Selected
먼저, 메타게놈 라이브러리에서 셀룰라아제 활성이 있는 재조합 플라스미드를 선별하기 위하여, 메타게놈 라이브러리를 12.5㎍/㎕의 클로람페니콜(chloramphenicol)이 포함된 LB 평판배지에 도말한 다음, 콜로니(colony)가 형성되도록 배양하였다. 양성 대조군으로 셀룰라아제 활성을 가진 celEdx12 유전자가 포함된 pHSGC12 벡터를 샷건 방법(Ko et al., Appl. Microbiol. Biotech., 89:1453, 2011)을 사용하여 대장균(E. coli) XL1-Blue(Stratagene Inc., 미국)에 형질전환시켜 사용하였으며, 음성대조군으로 메타게놈 DNA가 삽입되지 않은 pCC1FOS 벡터로 형질전환된 대장균(E.coli) EPI300-T1를 사용하였다. First, in order to select recombinant plasmids with cellulase activity from the metagenome library, the metagenomic library was plated on LB plates containing 12.5 μg / μl of chloramphenicol and then cultured to form colonies. . PHSGC12 vector containing celEdx12 gene with cellulase activity was used as a positive control using E. coli XL1-Blue (Stratagene) using the shotgun method (Ko et al., Appl. Microbiol. Biotech ., 89: 1453, 2011). Inc., USA) and E. coli EPI300-T1 transformed with pCC1FOS vector without metagenome DNA inserted as a negative control.
형광기질인 10μM 4-methylumbelliferyl-β-d-cellobioside (MeUmbG2: Sigma-Aldrich), 아라비노오스(arabinose) 및 12.5㎍/㎕의 클로람페니콜(chloramphenicol)이 포함된 LB 액체배지를 Janus Automated Workstation의 Janus Liquid Handler (Perkin Elmer, 미국)을 이용하여 96웰 플레이트(96well plate)에 200㎕씩 분주하였다. 그 후, 96웰 플레이트를 K3 colony picker(KBiosystems, 영국)로 옮겨 상기 LB 평판배지에 배양되어있는 각각의 콜로니를 96웰 플레이트에 접종한 다음, Liconic STX40 Automated Incubator(Woburn, 미국)를 이용하여 37℃에서 12 내지 16시간 배양하였다. 배양이 끝난 다음, 배양액(배지+균체) 100㎕를 0.5M 글라이신 버퍼(glycine buffer, pH10.4)가 100㎕씩 분주 되어 있는 블랙 96웰 플레이트(black 96well plate)에 넣어준 후, 셀룰라아제 활성에 따른 MeUmbG2의 형광 정도를 1420 VICTOR multilabel counter(Perkin Elmer, 미국)에서 λexcitation=365 nm, λemission≥460 nm로 측정하여 Workout software(Perkin Elmer, 미국)에서 분석하였다. LB liquid medium containing 10 μM 4-methylumbelliferyl-β-d-cellobioside (MeUmbG 2 : Sigma-Aldrich), arabinose, and 12.5 μg / μl chloramphenicol, the fluorescent substrate, Janus Automated Workstation 200 μl was dispensed into 96 well plates using a Liquid Handler (Perkin Elmer, USA). Subsequently, the 96 well plates were transferred to a K3 colony picker (KBiosystems, UK) and each colony cultured in the LB plate medium was inoculated into 96 well plates, followed by 37 using a Liconic STX40 Automated Incubator (Woburn, USA). Incubated at 16 ° C. for 12-16 hours. After incubation, 100 μl of the culture medium (medium + bacteria) was placed in a black 96 well plate in which 100 μl of 0.5 M glycine buffer (pH 10.4) was dispensed, followed by cellulase activity. The fluorescence intensity of MeUmbG 2 was measured by λexcitation = 365 nm and λemission≥460 nm on a 1420 VICTOR multilabel counter (Perkin Elmer, USA) and analyzed by Workout software (Perkin Elmer, USA).
본 발명에서는 총 20,000개의 클론을 분석하였으며, 이 중 셀룰라아제 활성을 보이는 pFOS-CBR12의 클론을 선별하였다 (도 4).In the present invention, a total of 20,000 clones were analyzed, and among them, clones of pFOS-CBR12 showing cellulase activity were selected (FIG. 4).
실시예 3 : CelEx-BR12의 샷건 라이브러리 제작 및 염기서열 분석Example 3 Shotgun Library Construction and Sequence Analysis of CelEx-BR12
본 발명의 실시예 2에서 pFOS-CBR12 클론(hit clone)을 선별하였으며, 선별된 클론에서 알칼리 용혈(alkali lysis)방법으로 포스미드(fosmid)를 분리하였으며(H.C. Birnboim et al., Nuc. Acid Res., 7:1513, 1979), 분리된 DNA(chromosomal DNA)는 제한효소인 BfuCI(New England Biolabs, 미국) (65℃에서 20분 동안 배양)으로 절단한 후, 전기영동하여 4 내지 5kb 크기를 가지는 DNA 단편을 겔 추출 키트(Gel Extraction kit ; QIAGEN Inc, 미국)로 정제하였다. 정제된 DNA는 제한효소 BamHI(New England Biolabs, 미국)을 처리한 다음, pHSG298 (Takara, 미국)벡터에 서브클로닝 하였으며(pHSG-CBR12) celEx-BR12 유전자의 샷건 라이브러리를 제작하였다 (Ko et al., Appl. Microbiol. Biotech., 89:1453, 2011). In Example 2 of the present invention, pFOS-CBR12 hit clones were selected, and fosmids were isolated from the selected clones by alkaline lysis (HC Birnboim et al., Nuc. Acid Res). , 7: 1513, 1979), isolated DNA (chromosomal DNA) was digested with restriction enzyme Bfu CI (New England Biolabs, USA) (incubated at 65 ° C. for 20 minutes), followed by electrophoresis to size 4-5kb. DNA fragments were purified with a Gel Extraction kit (QIAGEN Inc, USA). The purified DNA was treated with restriction enzyme BamHI (New England Biolabs, USA), then subcloned into a pHSG298 (Takara, USA) vector (pHSG-CBR12) to produce a shotgun library of celEx-BR12 gene (Ko et al. , Appl. Microbiol.Biotech. , 89: 1453, 2011).
상기에서 제조된 celEx-BR12 유전자의 샷건 라이브러리는 대장균(E. coli) XL1-Blue에 형질전환시켜 20㎍/㎖ 카나마이신(kanamycin) 및 10μM MeUmbG2이 포함된 LB 액체배지에서 배양하였다. 형질전환된 대장균(E. coli) XL1-Blue에서 플라스미트 미디 키트(Plasmid Midi kit ;QIAGEN, 미국)을 사용하여 셀룰라아제 활성을 가진 유전자가 삽입된 재조합 플라스미드(pHSG298)를 분리하여, 염기서열을 분석하였다 (솔젠트; SolGent Co, 한국), 분석된 염기서열은 NCBI(www.ncbi.nlm.nih.gov/blast/)에서 BLAST를 수행하였으며, BioEdit(version 7.0.9.0.)의 다중정렬 프로그램(multialignment program)으로 분석하였다. The shotgun library of the celEx-BR12 gene prepared above was transformed into E. coli XL1-Blue and cultured in LB liquid medium containing 20 μg / ml kanamycin and 10 μM MeUmbG 2 . In the transformed E. coli XL1-Blue, using a Plasmid Midi kit (QIAGEN, USA), a recombinant plasmid (pHSG298) containing a gene having cellulase activity was isolated and analyzed for sequencing. (Solgent; SolGent Co, Korea), the analyzed sequencing was performed by BLAST in NCBI (www.ncbi.nlm.nih.gov/blast/), and the multi-alignment program of BioEdit (version 7.0.9.0.) multialignment program).
표 1 메타게놈에서 선별된 셀룰라아제 상동성 비교
Vector Gene Homologous Similarity (%)
pHSG-CBR12 orf1 Penicillin-binding protein [Prevotellaruminicola23] 84
celEx-BR12 family 5 glycosyl hydrolase [Prevotellaruminicola23] 83
Table 1 Comparison of Cellulase Homology Selected from Metagenome
Vector Gene Homologous Similarity (%)
pHSG-CBR12 orf1 Penicillin-binding protein [ Prevotellaruminicola23 ] 84
celEx-BR12 family 5 glycosyl hydrolase [ Prevotellaruminicola23 ] 83
celEx-BR12의 유전자는 4 kb로 이루어져 있으며, 2개의 ORF로 구성되어져 있다. ORF1은 약 1.8 kb로 이루어져 있으며 프리보텔라 루미니콜라(Prevotella ruminicola) 23의 페니실린 결합 단백질(penicillin-binding protein)과 약 84% 상동성을 가진다. ORF2는 380개의 아미노산으로 이루어져 있으며, 난배양성 미생물(uncultured microorganism)의 셀로덱스트리나아제(cellodextrinase)와 약 92%, 프리보텔라 루미니콜라(Prevotella ruminicola) 23의 글라이코실 가수분해효소(glycosyl hydrolase) family 5와 약 83%의 상동성을 보이는 것을 확인하였으며(도 5), 본 발명에서는 이 효소를 CelEx-BR12 셀룰라아제로 명명(서열번호 1의 염기서열, 서열번호 2의 아미노산 서열)하였다. The gene of celEx-BR12 is 4 kb and consists of two ORFs. ORF1 consists of about 1.8 kb and has about 84% homology with the penicillin-binding protein of Prevotella ruminicola 23. ORF2 consists of 380 amino acids, about 92% of cell cultures of uncultured microorganisms, and glycosyl hydrolase of Prevotella ruminicola 23. About 83% homology with family 5 was confirmed (FIG. 5). In the present invention, the enzyme was named CelEx-BR12 cellulase (SEQ ID NO: 1, SEQ ID NO: 2).
실시예 4 : 셀룰라아제를 코딩하는 유전자를 포함하는 재조합 벡터 및 재조합 미생물 제작Example 4 Construction of Recombinant Vectors and Recombinant Microorganisms Comprising Genes Encoding Cellulase
본 발명의 CelEx-BR12 셀룰라아제를 발현시키기 위하여 celEx-BR12 유전자(서열번호 1)의 염기서열에서 개시코돈(ATG)의 위치를 포함하는 DNA 단편들을 NdeI(New England Biolabs, 미국) 와 XhoI(New England Biolabs, 미국) 제한효소 부위가 포함되도록 PCR(TProfessional thermalcycle(Biometra, 독일)을 사용하여 증폭시켰으며, PCR과정에 사용된 프라이머는 celEx-BR12 유전자를 증폭할 수 있도록 디자인하였다. DNA fragments containing the position of the start codon (ATG) in the nucleotide sequence of the celEx-BR12 gene (SEQ ID NO: 1) for expressing the CelEx-BR12 cellulase of the present invention, Nde I (New England Biolabs, USA) and Xho I ( New England Biolabs (USA) was amplified using PCR (TProfessional thermalcycle (Biometra, Germany) to include a restriction site, and the primer used in the PCR process was designed to amplify the celEx-BR12 gene.
celEx-BR12 프라이머 celEx-BR12 primer
[서열번호 3] CBR12-F : 5’- GGAATTCCAT ATG CGGAAGAATTCCTTTAAA-3’[SEQ ID NO 3] CBR12-F: 5'- GGAATTC CAT ATG CGGAAGAATTCCTTTAAA-3 '
[서열번호 4] CBR12-R : 5’-CCGCTCGAGTTTCTCTAGGGGCTTTCCT-3’[SEQ ID NO 4] CBR12-R: 5'-CCG CTCGAG TTTCTCTAGGGGCTTTCCT-3 '
(NdeI site : CATATG, XhoI site : CTCGAG, 굵은 글씨 ATG 개시코돈)(NdeI site: CATATG, Xho I site: CTCGAG, bold ATG start codon)
PCR로 증폭된 DNA는 약 1.2kb이며, 클로닝된 DNA 단편은 NdeI 와 XhoI 제한효소를 처리한 다음, pET-22b(+) 벡터 (Novagen, 미국) 에 삽입하여, 셀룰라아제를 코딩하는 유전자를 포함하는 재조합 벡터를 제작하였으며, pET-22b(+) 벡터는 삽입된 셀룰라아제 유전자가 발현될 수 있도록 T7 프로모터를 포함하며, 발현된 셀룰라아제를 쉽게 정제할 수 있도록 C-말단에 hexa-histidine tag(His-tag) 서열을 포함한다. The DNA amplified by PCR is about 1.2 kb, and the cloned DNA fragment contains NdeI and Xho I restriction enzymes, which are then inserted into the pET-22b (+) vector (Novagen, USA) to include a gene encoding cellulase. PET-22b (+) vector includes a T7 promoter for expression of the inserted cellulase gene, and a hexa-histidine tag (His-) at the C-terminus for easy purification of the expressed cellulase. tag) sequence.
상기 클로닝된 pET-22b(+) 벡터(pET-CBR12)는 대장균(Escherichia coli) BL21(DE3)(Novagen, 미국)에 형질전환시킨 후, 셀룰라아제의 발현정도를 확인하였으나, 발현상태가 미비한 것을 확인하고, 코돈 최적화(codon optimization)를 통해 celEx-BR12 유전자를 합성(서열번호 5)하여 상기와 같은 방법으로 재조합 벡터를 제조한 다음, 대장균(Escherichia coli) BL21(DE3)에 형질전환시켰다. The cloned pET-22b (+) vector (pET-CBR12) was transformed into Escherichia coli BL21 (DE3) (Novagen, USA), and then confirmed the expression level of cellulase, but the expression state was confirmed to be insufficient. Then, celEx-BR12 gene was synthesized through codon optimization (SEQ ID NO: 5) to prepare a recombinant vector in the same manner as above, and then transformed into Escherichia coli ( Esherichia coli ) BL21 (DE3).
실시예 5 : 재조합 미생물에서 생산된 재조합 셀룰라아제의 생산 및 정제Example 5 Production and Purification of Recombinant Cellulase Produced in Recombinant Microorganisms
실시예 4에서 코돈 최적화를 통해 제조한 재조합 CelEx-BR12 설룰라아제 생산능을 가지는 대장균을 30℃에서 50㎍/㎖ 앰피실린(Ampicillin)이 첨가된 LB 배지에서 배양하였으며, 셀룰라아제 발현을 유도하기 위해 0.5mM IPTG(isopropyl-d-1-thiogalactopyranoside)를 첨가하여 30℃에서 4시간 동안 배양하여 재조합 CelEx-BR12 설룰라아제를 생산하였다. 그 다음, 7,000×ｇ 조건으로 10분 동안 원심분리하여, 남아있는 세포 펠렛을 차가운(ice-cold) PBS 버퍼(200 mM NaCl, 3 mM KCl, 2 mM KH2PO4, and 1 mM Na2HPO4; pH 7.5)로 세척하였다. 세척된 세포는 VCX750 소니케이터(VCX750 sonicator; Sonics Materials. Inc., 미국)를 이용하여 파쇄하였으며, 파쇄된 세포 추출액에서 C-말단에 His-tag이 포함된 CelEx-BR12 설룰라아제를 분리하기 위해, Ni-NTA 흡착 크로마토그래피방법을 사용하여 정제하였다. Escherichia coli having recombinant CelEx-BR12 sulfulase producing ability prepared by codon optimization in Example 4 was cultivated in LB medium to which 50 µg / ml Ampicillin was added at 30 ° C. to induce cellulase expression. 0.5 mM IPTG (isopropyl-d-1-thiogalactopyranoside) was added to incubate at 30 ° C. for 4 hours to produce recombinant CelEx-BR12 sulfulase. The remaining cell pellet was then centrifuged at 7,000 × g for 10 minutes to ice-cold PBS buffer (200 mM NaCl, 3 mM KCl, 2 mM KH 2 PO 4 , and 1 mM Na 2 HPO). 4 ; pH 7.5). The washed cells were crushed using a VCX750 sonicator (Sonics Materials. Inc., USA), to separate CelEx-BR12 sulfulase containing His-tag at the C-terminus from the crushed cell extract. For the purification, Ni-NTA adsorption chromatography was used.
500mM 염화나트륨(NaCl)이 포함된 PBS 버퍼에 0 내지 500mM 이미다졸(imidazole)의 농도변화(gradient)를 주어 HiTrap chelating HP 컬럼(GE Healthcare, 미국)을 이용하여 1차 정제를 하였으며, 1차 정제된 셀룰라아제는 염화나트륨을 제거하기 위해 HiPrep 26/10 탈염(desalting) 컬럼(GE Healthcare)를 사용하여 2차 정제를 하였다. 모든 정제 단계는 FPLC 시스템(AKTA Explorer; GE Healthcare, 미국)을 사용하였다.PBS buffer containing 500 mM sodium chloride (NaCl) was given a gradient of 0 to 500 mM imidazole, and the first purification was performed using a HiTrap chelating HP column (GE Healthcare, USA). Cellulase was subjected to secondary purification using a HiPrep 26/10 desalting column (GE Healthcare) to remove sodium chloride. All purification steps used an FPLC system (AKTA Explorer; GE Healthcare, USA).
상기 정제된 CelEx-BR12 설룰라아제는 SDS-PAGE(sodium dodecyl sulfate polyacrylamide gel electrophoresis)로 약 42kDa의 크기를 가지고 있는 것을 확인하였으며(도 6), 정제된 CelEx-BR12 설룰라아제는 BSA를 스탠다드로 사용하여 Bio-Rad protein assay kit(Bio-Rad Laboratories, 미국)로 정량하였다.The purified CelEx-BR12 sulfulase was confirmed to have a size of about 42 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (FIG. 6), and the purified CelEx-BR12 sulfulase used BSA as a standard. Quantification was performed using a Bio-Rad protein assay kit (Bio-Rad Laboratories, USA).
실시예 6 : 재조합 CelEx-BR12 설룰라아제의 효소활성 및 기질특이성 측정Example 6 Determination of Enzyme Activity and Substrate Specificity of Recombinant CelEx-BR12 Sululase
6-1 : 재조합 CelEx-BR12 설룰라아제 특성 확인6-1: Characterization of recombinant CelEx-BR12 sulfulase
상기 실시예 5에서 정제한 CelEx-BR12 셀룰라아제의 반응 최적 조건을 확인하기 위해, pH 및 온도의 변화에 따른 셀룰라아제 활성을 측정하였다. In order to confirm the optimum reaction conditions of the CelEx-BR12 cellulase purified in Example 5, the cellulase activity was measured according to the change of pH and temperature.
CelEx-BR12 셀룰라아제의 최적 pH를 확인하기 위해, pH3.0 내지 pH13.0의 다양한 버퍼에 0.1mM MeUmbG2를 첨가하여 37℃에서 20분 동안 반응시켰으며, 효소 활성은 엑소셀룰라아제 기질로 사용되는 4-methylumbelliferyl-β-d-cellobioside (MeUmbG2 ;MeUmb glycosides)가 효소에 의해 분해되어 생성되는 MeUmb(4-methylumbelliferylliferone)의 양을 측정하였다. CelEx-BR12의 pH 안정성은 각각의 버퍼에서 4℃에서 24시간 반응시킨 후 잔존활성을 측정하였다. To determine the optimal pH of CelEx-BR12 cellulase, 0.1 mM MeUmbG2 was added to various buffers ranging from pH3.0 to pH13.0 and reacted at 37 ° C. for 20 minutes, and the enzyme activity was 4- used as an exocellulase substrate. The amount of MeUmb (4-methylumbelliferylliferone) produced by methylumbelliferyl-β-d-cellobioside (MeUmbG 2 ; MeUmb glycosides) was analyzed by enzymes. The pH stability of CelEx-BR12 was measured for 24 hours at 4 ° C. in each buffer and the residual activity was measured.
최적 pH 측정을 위하여 사용된 버퍼는 다음과 같다 : 100mM 소듐 아세테이트(sodium acetate; pH 3.0-6.0), 100mM 소듐 포스페이트(sodium phosphate; pH 6.0-8.0), 100mM Tris-HCl(pH 8.0-9.0), 100mM 소듐 바이카보네이트(sodium bicarbonate; pH 9.0-11.0), 100mM 디소듐 포스페이트(disodium phosphate; pH 11.0-12.0), 100mM 포타슘 클로라이드(potassium chloride; pH 12.0-13.0). The buffer used for the optimal pH measurement was as follows: 100 mM sodium acetate (pH 3.0-6.0), 100 mM sodium phosphate (pH 6.0-8.0), 100 mM Tris-HCl (pH 8.0-9.0), 100 mM sodium bicarbonate (pH 9.0-11.0), 100 mM disodium phosphate (pH 11.0-12.0), 100 mM potassium chloride (pH 12.0-13.0).
CelEx-BR12 셀룰라아제의 최적 반응온도를 측정하기 위해, 100mM 소듐 아세테이트(sodium acetate; pH5.0) 버퍼에 0.1mM MeUmbG2를 첨가하여 20 내지 60℃에서 0 내지 120분 동안 반응시킨 후 잔존활성을 측정하였다. In order to determine the optimum reaction temperature of CelEx-BR12 cellulase, 0.1 mM MeUmbG2 was added to 100 mM sodium acetate (pH 5.0) buffer and reacted at 20 to 60 ° C. for 0 to 120 minutes. .
그 결과, 본 발명의 CelEx-BR12 셀룰라아제는 약 35℃, pH5.0에서 최대활성을 보이는 것을 확인하였으며, pH 7 내지 9의 중성부분에서 안정성이 높게 유지되는 것을 확인하였다. 또한, CelEx-BR12 셀룰라아제는 20 내지 30℃에서 2시간 이상 활성이 유지되는 것을 확인하였으며, 40℃이상에서는 불안정한 것을 확인하였다 (도 7).As a result, it was confirmed that the CelEx-BR12 cellulase of the present invention exhibited maximum activity at about 35 ° C. and pH 5.0, and it was confirmed that stability was maintained at a neutral portion of pH 7 to 9. In addition, CelEx-BR12 cellulase was confirmed that the activity is maintained for 2 hours or more at 20 to 30 ℃, it was confirmed that unstable above 40 ℃ (Fig. 7).
6-2 : 재조합 CelEx-BR12 설룰라아제 기질특이성6-2: Recombinant CelEx-BR12 Sululase Substrate Specificity
상기 실시예 5에서 정제한 CelEx-BR12 설룰라아제의 기질특이성(substrate specificity)을 측정하기 위해, 0.1mM MeUmbG2 및 1%(w/v) 아비셀(avicel), 1%(w/v) 자작나무 자일란(birch wood xylan), 1%(w/v) α-셀룰로오즈(α-cellulose), 1%(w/v) 카복실메틸셀룰로오즈(carboxymethyl cellulose; CMC), 1%(w/v) 커들란(curdlan), 1%(w/v) 2-하이드록시에틸-셀룰로오즈(2-hydroxyethyl-cellulose), 1%(w/v) 라미나린(laminarin), 1%(w/v) 오트-스펠트 자일란(oat-spelt xylan), 0.5%(w/v) 살리신(salicin), 50μM D-글루콘산(D-gluconic acid) 및 1%(w/v) 녹말(starch)를 포함하는 다양한 탄수화물을 이용하여 기질 특이성을 측정하였다. In order to measure the substrate specificity of CelEx-BR12 sulfulase purified in Example 5, 0.1 mM MeUmbG 2 and 1% (w / v) Avicel, 1% (w / v) Birch wood xylan, 1% (w / v) α-cellulose, 1% (w / v) carboxymethyl cellulose (CMC), 1% (w / v) curdlan (curdlan), 1% (w / v) 2-hydroxyethyl-cellulose, 1% (w / v) laminarin, 1% (w / v) haute-spell Using a variety of carbohydrates including oat-spelt xylan, 0.5% (w / v) salicin, 50 μM D-gluconic acid and 1% (w / v) starch Substrate specificity was measured.
표 2 재조합 CelEx-BR12 설룰라아제의 기질 특이성 측정
Substrate Specific activity (Ｕ/㎎)
CMC 105.9
Avicel pH101 LA
Birch wood xylan 132.3
Oat spelt xylan 67.9
Laminarin NA
Starch NA
2-hydroxyethyl-cellulose 26.3
Curdlan NA
α-cellulose NA
Salicin NA
d-Gluconic acid NA
MeUmbG2 0.3
TABLE 2 Determination of Substrate Specificity of Recombinant CelEx-BR12 Sululase
Substrate Specific activity (Ｕ / mg)
CMC 105.9
Avicel pH101 LA
Birch wood xylan 132.3
Oat spelt xylan 67.9
Laminarin NA
Starch NA
2-hydroxyethyl-cellulose 26.3
Curdlan NA
α-cellulose NA
Salicin NA
d-Gluconic acid NA
MeUmbG 2 0.3
그 결과, 본 발명의 재조합 CelEx-BR12 설룰라아제는 자작나무 자일란(132.3Ｕ/㎎), 카복실메틸셀룰로오즈(105.9Ｕ/㎎), 오트-스펠트 자일란(67.9Ｕ/㎎) 및 2-하이드록시에틸-셀룰로오즈(26.3Ｕ/㎎)에 높은 활성을 보이는 것을 확인하였다. 하지만, 아비셀에는 낮은 활성을 보였으며, 라미나린, 녹말, 커들란, α-셀룰로오즈, D-글루콘산 및 살리신에는 활성을 보이지 않았다 (표 2).As a result, the recombinant CelEx-BR12 sulfulase of the present invention is birch xylan (132.3 dl / mg), carboxymethylcellulose (105.9 dl / mg), haute-sfeldt xylan (67.9 dl / mg) and 2-hydroxy It was confirmed that high activity was shown in ethyl cellulose (26.3 dl / mg). However, it showed low activity on Avicel and no activity on laminarin, starch, curdlan, α-cellulose, D-gluconic acid and salicylic acid (Table 2).
6-3 : 재조합 CelEx-BR12 설룰라아제 효소활성 측정6-3: Determination of Recombinant CelEx-BR12 Sululase Enzyme Activity
상기 실시예 5에서 정제한 CelEx-BR12 설룰라아제의 효소활성을 측정하기 위해, 셀룰라아제에 100mM 소듐 아세테이드(sodium acetate) 버퍼에 MeUmbG2의 최종농도가 0.1 mM이 되도록 처리한 후, pH5.0, 37℃에서 20분 동안 반응시켰으며, 4-methylumbelliferylliferone의 표준용액은 0.05 내지 1nM로 설정하여 측정하였다. In order to measure the enzymatic activity of the CelEx-BR12 sulfulase purified in Example 5, the final concentration of MeUmbG2 in 100 mM sodium acetate buffer in cellulase was treated to 0.1 mM, and then pH5.0 The reaction was carried out at 37 ° C. for 20 minutes, and the standard solution of 4-methylumbelliferylliferone was measured at 0.05 to 1 nM.
셀룰라아제에 의해 MeUmbG2가 분해되어 생성된 MeUmb의 형광 정도는 1420 VICTOR multilabel counter(Perkin Elmer, 미국)에서 λexcitation=365 nm, λemission≥460 nm로 측정하여 Workout software(Perkin Elmer, 미국)에서 분석하였다. The fluorescence of MeUmbG 2 generated by cellulase was analyzed in the Workout software (Perkin Elmer, USA) by measuring λexcitation = 365 nm and λemission≥460 nm on a 1420 VICTOR multilabel counter (Perkin Elmer, USA).
분석과정은 0.1mM MeUmb glycosides이 포함된 100mM 소듐 아세테이즈 버퍼(pH5.0) 100㎕를 반응용액으로 하여 수행하였으며, 500mM 글라이신(glycine) 버퍼(pH10.4)를 100㎕ 첨가하여 반응을 종결시켰다. 효소의 유닛(U)은 단위시간 당(1min) 당 공여체의 1μmol당 수용체에 전이하는데 필요한 효소의 양으로 정의된다.The assay was performed with 100 μl of 100 mM sodium acetate buffer (pH5.0) containing 0.1 mM MeUmb glycosides as the reaction solution. The reaction was terminated by adding 100 μl of 500 mM glycine buffer (pH10.4). I was. The unit of enzyme (U) is defined as the amount of enzyme required to transfer to the receptor per μmol of donor per unit time (1 min).
CelEx-BR12 설룰라아제의 미카엘리스-멘텐 상수(Michaelis-Menten constants; K m) 및 최대 반응 속도(maximal reaction velocities; V max)는 라인웨버-버크식(Lineweaver-Burk) 방법에 따라 측정되었다. The Michaelis-Menten constants ( K m ) and the maximum reaction velocities ( V max ) of CelEx-BR12 sulfulase were measured according to the Lineweaver-Burk method.
표 3 CelEx-BR12 설룰라아제의 효소 활성
Ｋm(μM) Ｖ max (μmol/min) Ｋ cat(/min) Ｋ cat/Ｋ m(/min/μM)
12.92 1.55×10-4 1.2×10-5 9.29×10-7
TABLE 3 Enzymatic Activity of CelEx-BR12 Sululase
Ｋm ( μM) Ｖ max (μmol / min) Ｋ cat (/ min) Ｋ cat / Ｋ m (/ min / μM)
12.92 1.55 × 10 -4 1.2 × 10 -5 9.29 × 10 -7
표 3에 나타난 바와 같이, 본원발명의 방법으로 제조된 재조합 CelEx-BR12 설룰라아제의 Ｋ m 값은 12.92μM 및 Ｖ max 값은 1.55 ×10-4μmol/min을 나타내는 것을 확인하였다. As shown in Table 3, K m values for the recombinant CelEx BR12-sulfonic Lula kinase prepared by the method of the present invention 12.92μM and V max values were confirmed to exhibit a 1.55 × 10 -4 μmol / min.
6-4 : TLC 분석을 통한 재조합 CelEx-BR12 설룰라아제 효소활성 측정6-4: Determination of Recombinant CelEx-BR12 Sululase Enzyme Activity by TLC Analysis
상기 실시예 5에서 정제한 CelEx-BR12 설룰라아제의 효소활성을 측정하기 위해 TLC(thin-layer chromatography) 분석을 수행하였다. Thin-layer chromatography (TLC) analysis was performed to measure the enzymatic activity of CelEx-BR12 sulfulase purified in Example 5.
TLC는 세포 추출물에 포함된 셀룰라아제에 의해 셀룰로올리고사카라이드(cellooligosaccharides; cellobiose to cellopentaose) 및 카복실메틸셀룰로오즈(carboxymethyl cellulose; CMC)의 분해정도를 측정한 것으로, 효소와 반응된 각각의 기질은 실리카 겔(silica gel) 60(Merck, 미국)에서 용매(1-부탄올:아세트산:물 = 2:1:1(v/v/v)) 조건에서 분리시킨 다음, 에탄올로 희석시킨 5% 황산(H2SO4(v/v))으로 도포 후 200℃에서 5분간 발색시켜 최종산물을 확인하였다. TLC is a measure of the degree of degradation of cellooligosaccharides (cellobiose to cellopentaose) and carboxymethyl cellulose (CMC) by the cellulase contained in the cell extract. (silica gel) 60 (Merck, USA), isolated under solvent (1-butanol: acetic acid: water = 2: 1: 1 (v / v / v)) conditions, and then diluted with ethanol 5% sulfuric acid (H 2 SO 4 (v / v)) and then the color development at 200 ℃ 5 minutes to confirm the final product.
그 결과, 도 8에 나타난 바와 같이, 본 발명의 재조합 CelEx-BR12 설룰라아제는 셀로트리오스(cellotriose; G3), 셀로테트로스(cellotetrose; G4), 셀로펜토오스(cellopentaose; G5)를 각각 분해하는 것으로 보아 엑소셀룰라아제(exocellulase)의 활성 및 엔도셀룰라아제(endocellulase)의 활성을 동시에 보유함을 확인하였다. As a result, as shown in FIG. 8, the recombinant CelEx-BR12 sulfulase of the present invention degrades cellotriose (G3), cellotetrose (G4), and cellopentaose (G5), respectively. It was confirmed that the activity of the exocellulase (endocellulase) and endocellulase (endocellulase) at the same time.
6-5 : 금속이온에 따른 재조합 CelEx-BR12 설룰라아제 효소활성6-5: Recombinant CelEx-BR12 Sululase Enzyme Activity According to Metal Ion
금속이온의 종류에 따라 재조합 CelEx-BR12 설룰라아제의 활성에 미치는 영향을 알아보기 위하여, 다양한 금속이온의 최종농도가 1mM이 되도록 CelEx-BR12 설룰라아제에 첨가하여 반응시켰다. In order to determine the effect on the activity of recombinant CelEx-BR12 sulfulase according to the type of metal ion, it was added and reacted with CelEx-BR12 sulfulase such that the final concentration of various metal ions was 1 mM.
금속이온과 반응시킨 CelEx-BR12 설룰라아제는 최종농도가 1mM이 되도록 EDTA를 처리하여 4℃에서 3시간 동안 반응시킨 후, EDTA를 제거하기 위해 PBS 버퍼를 이용하여 투석시킨 다음, 효소 활성을 측정하였다. CelEx-BR12 sulfulase reacted with metal ions was treated with EDTA to reach a final concentration of 1 mM and reacted at 4 ° C. for 3 hours, and then dialyzed using PBS buffer to remove EDTA, and then enzyme activity was measured. It was.
표 4 금속이온에 따른 재조합 CelEx-BR12 설룰라아제 효소활성
Metallic ion Relative activity (%)
None 100
Ca2+ 139
Co2+ 147
Cu2+ 137
Fe2+ 45
Hg2+ 0
Mg2+ 142
Mn2+ 143
Ni2+ 149
K+ 149
Rb+ 149
Zn2+ 177
Table 4 Recombinant CelEx-BR12 Sululase Enzyme Activity According to Metal Ion
Metallic ion Relative activity (%)
None 100
Ca 2+ 139
Co 2+ 147
Cu 2+ 137
Fe 2+ 45
Hg 2+ 0
Mg 2+ 142
Mn 2+ 143
Ni 2+ 149
K + 149
Rb + 149
Zn 2+ 177
그 결과, 대부분의 금속이온에서 상대적인 셀룰라아제의 활성이 130 내지 150% 정도 증가한 것을 확인하였으며, 아연이온(Zn2+)을 처리한 경우에는 최대 177% 셀룰라아제 활성이 증가한 것을 확인하였다. 하지만, 철이온(Fe2+) 및 수은이온(Hg2+)에서는 오히려 셀룰라아제 활성이 감소하였다 (표 4). As a result, it was confirmed that the relative cellulase activity was increased by about 130 to 150% in most metal ions. When zinc ions (Zn 2+ ) were treated, it was confirmed that up to 177% cellulase activity was increased. However, cellulase activity was decreased in iron ions (Fe 2+ ) and mercury ions (Hg 2+ ) (Table 4).
본 발명에서 선별한 셀룰라아제 CelEx-BR12는 엑소셀룰라아제 활성 및 엔도셀룰라아제 활성을 가지고 있으므로, 섬유소계 바이오매스를 이용한 바이오에탄올 생산에 이용될 수 있으며, 섬유, 세제, 사료, 식품, 펄프 및 종이 생산 등 다양한 산업 분야에 적용할 수 있다. Since the cellulase CelEx-BR12 selected in the present invention has exocellulase activity and endocellulase activity, it can be used for the production of bioethanol using fibrin-based biomass, and various fiber, detergent, feed, food, pulp and paper production, etc. Applicable to industrial fields.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
전자파일 첨부하였음.Electronic file attached.

Claims (8)

  1. 서열번호 2의 아미노산으로 표시되는 셀룰라아제.Cellulase represented by the amino acid of SEQ ID NO: 2.
  2. 제1항의 셀룰라아제를 코딩하는 유전자(celEx-BR12).The gene encoding the cellulase of claim 1 ( celEx-BR12 ).
  3. 제2항에 있어서, 서열번호 1 또는 서열번호 5의 염기서열로 표시되는 것을 특징으로 하는 유전자(celEx-BR12).The gene ( celEx-BR12 ) according to claim 2, wherein the gene is represented by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 5.
  4. 제2항 또는 제3항의 셀룰라아제를 코딩하는 유전자를 함유하는 재조합 벡터.A recombinant vector containing a gene encoding the cellulase of claim 2.
  5. 제4항에 있어서, 상기 재조합 벡터는 pET-CBR12인 것을 특징으로 하는 재조합 벡터.The recombinant vector of claim 4, wherein the recombinant vector is pET-CBR12.
  6. 제2항의 셀룰라아제를 코딩하는 유전자 또는 상기 유전자를 함유하는 재조합 벡터가 숙주미생물에 도입되어 있는 것을 특징으로 하는 재조합 셀룰라아제 생산능을 가지는 재조합 미생물.A recombinant microorganism having recombinant cellulase production ability, wherein the gene encoding the cellulase of claim 2 or a recombinant vector containing the gene is introduced into a host microorganism.
  7. 제6항에 있어서, 상기 숙주미생물은 대장균(Escherichia coli)인 것을 특징으로 하는 재조합 미생물.The recombinant microorganism of claim 6, wherein the host microorganism is Escherichia coli .
  8. 다음의 단계를 포함하는 재조합 셀룰라아제의 제조방법;A method for producing a recombinant cellulase comprising the following steps;
    (a) 제6항의 재조합 셀룰라아제 생산능을 가지는 재조합 미생물을 배양하여 재조합 셀룰라아제를 생성하는 단계; 및(a) culturing a recombinant microorganism having the recombinant cellulase producing ability of claim 6 to produce a recombinant cellulase; And
    (b) 생성된 재조합 셀룰라아제를 회수하는 단계.(b) recovering the resulting recombinant cellulase.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110643620A (en) * 2019-10-22 2020-01-03 怀化学院 High-activity poria cocos cellulose endonuclease gene and protein and recombinant vector thereof
CN114214306A (en) * 2022-01-26 2022-03-22 西北农林科技大学 Ruminant rumen protozoan specific cellulase OCCel1A and application thereof

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101698927B1 (en) * 2015-08-06 2017-01-23 한국생명공학연구원 Method for displaying multifunctional cellulolytic enzyme in bacterial cell surface and use thereof

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
DATABASE NCBI 6 May 2013 (2013-05-06), accession no. GK74985.1 *
DEL POZO, MERCEDES V. ET AL.: "Microbial beta-glucosidases from cow rumen metagenome enhance the saccharification of lignocellulose in combination with commercial cellulase cocktail", BIOTECHNOLOGY FOR BIOFUELS, vol. 5, no. 73, 2012, pages 1 - 13 *
DUAN, C. -J. ET AL.: "Isolation and partial characterization of novel genes encoding acidic cellulases from metagenomes of buffalo rumens", JOURNAL OF APPLIED MICROBIOLOGY, vol. 107, 2009, pages 245 - 256, XP055087932, DOI: doi:10.1111/j.1365-2672.2009.04202.x *
DUAN, CHENG-JIE ET AL.: "Mining metagenomes for novel cellulase genes", BIOTECHNOLOGY LETTERS, vol. 32, no. 12, 2010, pages 1765 - 1775, XP019859052, DOI: doi:10.1007/s10529-010-0356-z *
FENG, YI ET AL.: "Cloning and identification of novel cellulase genes from uncultured microorganisms in rabbit cecum and characterization of the expressed cellulases", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 75, no. 2, 2007, pages 319 - 328, XP019513648, DOI: doi:10.1007/s00253-006-0820-9 *
KO, KYONG-CHEOL ET AL.: "Strategy for screening metagenomic resources for excellulase activity using a robotic, high-throughput screening system", JOURNAL OF MICROBIOLOGICAL METHODS, vol. 94, no. 3, 24 July 2013 (2013-07-24), pages 311 - 316, XP028700699, DOI: doi:10.1016/j.mimet.2013.07.010 *
PANG, HAO ET AL.: "Identification of cellulase genes from the metagenomes of compost soils and functional characterization of one novel endoglucanase", CURRENT MICROBIOLOGY, vol. 58, no. 4, 2009, pages 404 - 408, XP019708349 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110643620A (en) * 2019-10-22 2020-01-03 怀化学院 High-activity poria cocos cellulose endonuclease gene and protein and recombinant vector thereof
CN114214306A (en) * 2022-01-26 2022-03-22 西北农林科技大学 Ruminant rumen protozoan specific cellulase OCCel1A and application thereof

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