Paenibacillus woosongensis로부터 대장균에 Xylanase 10A의 유전자 클로닝과 정제 및 특성분석
Gene Cloning, Purification and Characterization of Xylanase 10A from Paenibacillus woosongensis in Escherichia coli
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Paenibacillus woosongensis의 xylanase 유전자를 클로닝하고 그 염기서열을 결정하였다. Xylanase 유전자는 xyn10A 로 명명되었으며, 481 아미노잔기로 구성된 단백질을 코드하는 1,446개 뉴클레오티드로 구성되었다. 추론된 아미노산배열에 따르면 Xyn10A는 glycosyl hydrolase family 10 xylanase와 상동성이 높은 활성영역과 카르복실 말단에 탄수화물을 결합하는 것으로 추정되는 영역이 포함된 다영역효소로 확인되었다. DEAE-Sepharose와 Phenyl-Separose 컬럼 크로마토그래피 과정을 통해 P. woosongensis xyn10A 유전자를 함유한 재조합 대장균의 균체 파쇄상등액으로부터Xyn10A를 정제하였다. 정제된 Xyn10A의 아미노 말단 배열이 GIANGSKF로 결정되었으며 이는 SignalP5.0 server로예측된 signal peptide의 다음 아미노산 배열과 정확하게 일치하였다. 정제된 Xyn10A는 33 kDa 크기의 절단된 단백질이며 균체내 분해에 의해 카르복시 말단에서 CBM이 제거된 것으로 판단된다. 정제된 효소는 최적 pH와 온도가 6.0 과 55−60℃이며 oat spelt xylan에 대한 반응 동력학적 계수Vmax와 Km이 298.8 U/mg과 2.47 mg/ml로 각각 나타났다. 효소는 birchwood xylan이나 oat spelt xylan보다 arabinoxylan 에 대한 활성이 높았으며 para-nitrophenyl-β-xylopyranoside에대해 낮은 활성을 보였다. Xyn10A의 활성은 Cu2+, Mn2+과SDS에 의해서 크게 저해되었으며 K+, Ni2+과 Ca2+에 의해는 상당하게 증진되었다. 또한 이 효소는 xylobiose 보다 중합도가 큰 자일로올리고당을 분해하였으며, 자일로올리고당의 최종 가수분해 산물은 xylose와 xylobiose로 확인되었다.
Abstract
A gene coding for the xylanase was cloned from Paenibacillus woosongensis, followed by determination of its complete nucleotide sequence. This xylanase gene, designated as xyn10A, consists of 1,446 nucleotides encoding a polypeptide of 481 amino acid residues. Based on the deduced amino acid sequence, Xyn10A was identified to be a modular enzyme composed of a catalytic domain highly homologous to the glycosyl hydrolase family 10 xylanase and a putative carbohydrate-binding module (CBM) in the C-terminus. By using DEAE-sepharose and phenyl-sepharose column chromatography, Xyn10A was purified from the cellfree extract of recombinant Escherichia coli carrying a P. woosongensis xyn10A gene. The N-terminal amino acid sequence of the purified Xyn10A was identified to exactly match the sequence immediately following the signal peptide predicted by the Signal5.0 server. The purified Xyn10A was a truncated protein of 33 kDa, suggesting the deletion of CBM in the C-terminus by intracellular hydrolysis. The purified enzyme had an optimum pH and temperature of 6.0 and 55−60℃, respectively, with the kinetic parameters Vmax and Km of 298.8 U/mg and 2.47 mg/ml, respectively, for oat spelt xylan. The enzyme was more active on arabinoxylan than on oat spelt xylan and birchood xylan with low activity for p-nitrophenyl-β-xylopyranoside. Xylanase activity was significantly inhibited by 5 mM Cu2+, Mn2+, and SDS, and was noticeably enhanced by K+, Ni2+, and Ca2+. The enzyme could hydrolyze xylooligosaccharides larger than xylobiose. The predominant products resulting from xylooligosaccharide hydrolysis were xylobiose and xylose.
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