Bacillus licheniformis WL-12의 carboxymethyl cellulase (cellulase) 유전자를 함유한 대장균 균체 파쇄상등액으로부터 DEAE-Sepharose와 Q-Sepharose 컬럼 크로마토그래피를 통해 cellulase를 정제하였다. 정제된 효소의 비활성은 163 U/mg이었으며, SDS-PAGE에 의해 측정된 분자량은 약 49.5 kDa으로 나타났다. pH 5.5와 $55^{circ}C$에서 최대 반응활성을 보였으며, SDS (5mM)에 의해서는 cellulase의 활성이 완전히 저해되었고 $Cu^{2+}$5mM)에 의해서는 약간 증진되었다. 정제된 cellulase는 CMC, konjac, barley $eta$-glucan과 lichenan을 가수분해하였으나 xylan, locust bean gum 및 p-nitrophenyl-$eta$-glucopyranoside를 분해하지 못하였다. Cellooligosaccharides를 정제된 WL-12 cellulase로 분해하였을 때 cellobiose와 cellotriose가 주된 최종 반응산물로 관찰되었으며 cellobiose보다는 중합도가 큰 cellotriose, cellotetrasoe와 cellopentaose는 분해하였으나 cellobiose는 분해하지 못하는 것으로 확인되었다.
Carboxymethyl celluase (cellulase) was purified from cell-free extract of the recombinant Escherichia coli carrying a Bacillus licheniformis WL-12 cellulase gene by DEAE-Sepharose and phenyl-Sepharose column chromatography with specific activity of 163 U/mg protein. The molecular mass of the purified enzyme was estimated to be approximately 49.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme had a pH optimum at 5.5 and a temperature optimum at $55^{circ}C$. The activity of the enzyme was completely inhibited by SDS (5 mM), and slightly enhanced by $Cu^{2+}$ (5 mM). The cellulase was active on CMC, konjac, barely glucan and lichenan, while it did not exhibit activity towards xylan, locust bean gum, and p-nitrophenyl-$eta$-glucopyranoside. The predominant products resulting from the cellulase hydrolysis were cellobiose and cellotriose for cellooligosaccharides including cellotriose, cellotetraose and cellopentaose. The enzyme could hydrolyze cellooligosaccharides larger than cellobiose.
