Cellulosimicrobium sp. YB-43의 mannanase B 유전자 클로닝과 특성 분석

Abstract

두 종류의 mannanases를 생산하는 Cellulosimicrobium sp. YB-43로부터 mannanase 유전자를 클로닝하고 그 염기서열을 결정하였다. Mannanase 유전자는 manB로 명명되었으며, 427 아미노 잔기로 구성된 단백질을 코드하는 1,284개 염기로 구성되었다. ManB는 추론된 아미노산 배열에 근거해서 glycosyl hydrolase family 5에 속하는 mannanase와 상동성이 높은 활성영역과 함께 2개의 탄수화물 결합영역을 포함하고 있는 다영역 효소로 확인되었다. Cellulosimicrobium sp. YB-43의 manB 유전자를 함유한 재조합 대장균의 균체 파쇄상등액으로부터 정제된 ManB의 아미노 말단 배열이 QGASAASDG로 결정되었으며 이는 SignalP4.1 server로 그람 음성균을 기준으로 예측된 signal peptide의 결과와 정확하기 일치하였다. 정제된 ManB의 최적 반응조건은 <TEX>$55^{\circ}C$</TEX>와 pH 6.5-7.0이며 locust bean gum (LBG), konjac과 guar gum을 가수분해 하였으며, 셀룰로스, 자일란, 전분과 para-nitrophenyl-<TEX>${\beta}$</TEX>-mannopyranoside에 대해서는 분해활성이 없었다. ManB의 활성은 <TEX>$Mg^{2+}$</TEX>, <TEX>$K^+$</TEX>와 <TEX>$Na^+$</TEX>에 의해 약간 저해되었으며 <TEX>$Cu^{2+}$</TEX>, <TEX>$Zn^{2+}$</TEX>, <TEX>$Mn^{2+}$</TEX>과 SDS에 의해서는 크게 저해되었다. 또한 이 효소는 mannobiose 보다 큰 중합도를 갖는 만노올리고당을 가수분해하였으며, LBG와 만노올리고당을 가수분해하였을 때 mannobiose가 가장 많은 양으로 생성되었다. A mannanase gene was cloned into Escherichia coli from Cellulosimicrobium sp. YB-43, which had been found to produce two kinds of mannanase, and sequenced completely. This mannanase gene, designated manB, consisted of 1,284 nucleotides encoding a polypeptide of 427 amino acid residues. Based on the deduced amino acid sequence, the ManB was identified to be a modular enzyme including two carbohydrate binding domains besides the catalytic domain, which was highly homologous to mannanases belonging to the glycosyl hydrolase family 5. The N-terminal amino acid sequence of ManB, purified from a cell-free extract of the recombinant E. coli carrying a Cellulosimicrobium sp. YB-43 manB gene, has been determined as QGASAASDG, which was correctly corresponding to signal peptide predicted by SignalP4.1 server for Gram-negative bacteria. The purified ManB had a pH optimum for its activity at pH 6.5~7.0 and a temperature optimum at <TEX>$55^{\circ}C$</TEX>. The enzyme was active on locust bean gum (LBG), konjac and guar gum, while it did not exhibit activity towards carboxymethylcellulose, xylan, starch, and para-nitrophenyl-<TEX>${\beta}$</TEX>-mannopyranoside. The activity of enzyme was inhibited very slightly by <TEX>$Mg^{2+}$</TEX>, <TEX>$K^+$</TEX>, and <TEX>$Na^+$</TEX>, and significantly inhibited by <TEX>$Cu^{2+}$</TEX>, <TEX>$Zn^{2+}$</TEX>, <TEX>$Mn^{2+}$</TEX>, and SDS. The enzyme could hydrolyze mannooligosaccharides larger than mannobiose, which was the most predominant product resulting from the ManB hydrolysis for mannooligosaccharides and LBG.