한우의 반추위에서 게놈 DNA를 분리하여 메타게놈 은행을 구축한 다음 섬유소분해효소를 암호화하는 유전자를 클로닝 및 유전자를 선별하였다. 선별된 유전자의 DNA 염기서열 및 아미노산 서열을 분석하고 유전산물의 생화학적인 특성을 조사하였다. $cel$5C 유전자는 1,125 bp로 374개의 아미노산 잔기를 가진 단백질을 암호화하였으며 이 단백질 분자량은 42 kDa이었다. 이 효소의 최적 pH는 4 근방이었으며 최적 온도는 $50^{\circ}C$ 부근이었다. $cel$5C 유전자의 internal primer를 사용하여 인공적으로 배양할 수 있는 49종의 반추세균에서 분리한 게놈 DNA을 주형으로 PCR 분석한 결과 해당하는 밴드를 확인할 수 없었다. Cel5C는 현재로서는 배양할 수 없는 반추 미생물로 추정된다.
한우의 반추위에서 게놈 DNA를 분리하여 메타게놈 은행을 구축한 다음 섬유소분해효소를 암호화하는 유전자를 클로닝 및 유전자를 선별하였다. 선별된 유전자의 DNA 염기서열 및 아미노산 서열을 분석하고 유전산물의 생화학적인 특성을 조사하였다. $cel$5C 유전자는 1,125 bp로 374개의 아미노산 잔기를 가진 단백질을 암호화하였으며 이 단백질 분자량은 42 kDa이었다. 이 효소의 최적 pH는 4 근방이었으며 최적 온도는 $50^{\circ}C$ 부근이었다. $cel$5C 유전자의 internal primer를 사용하여 인공적으로 배양할 수 있는 49종의 반추세균에서 분리한 게놈 DNA을 주형으로 PCR 분석한 결과 해당하는 밴드를 확인할 수 없었다. Cel5C는 현재로서는 배양할 수 없는 반추 미생물로 추정된다.
A metagenomic library of cow rumen in the pCC1FOS phage vector was screened in $E.$$coli$ EPI300 for cellulase activity on carboxymethyl cellulose agar plates. One clone was partially digested with $Sau$3AI, ligated into the $Bam$HI site of the pBluescrip...
A metagenomic library of cow rumen in the pCC1FOS phage vector was screened in $E.$$coli$ EPI300 for cellulase activity on carboxymethyl cellulose agar plates. One clone was partially digested with $Sau$3AI, ligated into the $Bam$HI site of the pBluescript II SK+ vector, and transformed into $E.$$coli$$DH5{\alpha}$. We obtained a 1.5 kb insert DNA, designated $cel$5C, which hydrolyzes carboxymethyl cellulose. The cel5C gene has an open reading frame (ORF) of 1,125 bp encoding 374 amino acids. It belongs to the glycosyl hydrolase family 5 with the conserved domain LIMEGFNEIN. The molecular mass of the Cel5C protein induced from $E.$$coli$$DH5{\alpha}$, as analyzed by CMC SDS-PAGE, appeared to be approximately 42 kDa. The enzyme showed optimum cellulase activity at pH 4.0, and $50^{\circ}C$. We examined whether the $cel$5C gene comes from the 49 identified cow rumen bacteria using PCR. No PCR bands were identified, suggesting that the $cel$5C gene came from the unidentified cow rumen bacteria.
A metagenomic library of cow rumen in the pCC1FOS phage vector was screened in $E.$$coli$ EPI300 for cellulase activity on carboxymethyl cellulose agar plates. One clone was partially digested with $Sau$3AI, ligated into the $Bam$HI site of the pBluescript II SK+ vector, and transformed into $E.$$coli$$DH5{\alpha}$. We obtained a 1.5 kb insert DNA, designated $cel$5C, which hydrolyzes carboxymethyl cellulose. The cel5C gene has an open reading frame (ORF) of 1,125 bp encoding 374 amino acids. It belongs to the glycosyl hydrolase family 5 with the conserved domain LIMEGFNEIN. The molecular mass of the Cel5C protein induced from $E.$$coli$$DH5{\alpha}$, as analyzed by CMC SDS-PAGE, appeared to be approximately 42 kDa. The enzyme showed optimum cellulase activity at pH 4.0, and $50^{\circ}C$. We examined whether the $cel$5C gene comes from the 49 identified cow rumen bacteria using PCR. No PCR bands were identified, suggesting that the $cel$5C gene came from the unidentified cow rumen bacteria.
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대상 데이터
Cow’s rumen contents were obtained from a closed herd at the Gyeongnam National University of Science and Technology (Jinju, Korea).
The animals were rumen-fistulated Korean cows (HANWOO) with the body weight 400±10 kg, and fed twice by a mixed ration (rice hull and concentrate in a 4:1 ratio) for a day.
이론/모형
In this study, the cellulase of a new type was isolated and characterized from bovine rumen metagenome by using cosmid library and subcloning by using shotgun method.
Nucleotide sequencing was done by the dideoxy-chain termination method using the PRISM Ready Reaction Dye terminator/primer cycle sequencing kit (Perkin-Elmer, USA). The samples were analyzed with an automated DNA sequencer (Applied Biosystems, USA).
The 1.5 kb inserted fragment was sequenced using the dideoxy chain-termination method. It contains one complete open reading frame.
The optimal pH and temperature for the cellulase activity were determined in citric/sodium phosphate buffer containing 1% (w/v) carboxymethyl cellulose. The cellulase activity in the recombinant DNA clones was measured using the dinitrosalicylic acid method. Cellulase activity was measured at temperature 10-80℃ and at pH 3-11 over 30 min in absorbance at 510 nm.
성능/효과
The cloning and characterization of more cel genes from cow rumen metagenome should bring us closer to an answer for this. The experimental results shown in this paper demonstrate that the possibility of the breeding of rumen bacteria and novel useful genes from rumen. The ability to the breeding of rumen bacteria and novel useful genes from rumen will expand the use of enzymes in industrial application.
후속연구
Further study is anticipated in order to find the reason for multiple cell wall degrading enzymes. The cloning and characterization of more cel genes from cow rumen metagenome should bring us closer to an answer for this.
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