실험실 배양 조건에서는 발현되지 않는 대장균 K-12의 endochitinase 유전자(yheR)를 PCR로 증폭하여 pET28c와 pQE9벡터에 각각 클로닝 하였다. yheB유전자를 가진 pET28c와 pQE9 벡터를 함유한 대장균에서 생산된 endochitinase는 생장배지 내로 일부 분리되었다. 과량 생산된 endochitinase를 His-affinity 크로마토그래피와 DE-52 크로마토그래피로 부분 정제하였으며 SDS-PAGE에 의한 단백질의 분자량은 약 97,000 이었다. 정제된 효소의 최적 pH는 6이었으며 최적 온도는 $40^{circ}C$이었다.
The putative endochitinase gene, yheB of Escherichia coli K-12 is not expressed under lab culture conditions. The endochitinase gene was amplified by PCR and subcloned into pET28c vector and pQE9 vector, respectively. The endochitinase produced in E. coli harboring pET28c containing yheB or pQE9 vector containing yheE was partly released into the growth medium. The overproduced endochitinase was partially purified by His affinity column chromatography and DE-52 column chromatography. The apparent molecular weight of the endochitinase determined by SDS-polyacrylamide gel electrophoresis was about 97,000. The purified E. coli endochitinase showed maximal chitinolytic activity at pH 6 and $40^{circ}C$.
