Abstract
A bacterial strain producing high level of a phytase was isolated from cattle feces and identified as Bacillus subtilis, and designated as Bacillus sp. CF 5-26. The production of the phytase from Bacillus sp. CF 5-26 reached the highest level after 72 hours at $37^{\circ}C$. The optimum condition of the media for the production of phytase was 10% rice bran extract, 0.1% whey protein powder, $0.01%\;CaCl_{2},\;0.01%\;KH_{2}PO_4$. The phytase was purified 20.3 folds with ethanol precipitation, Sephadex G-100, CM Sepharose CL-6B and Sephacryl S-100-HR column chromatography. The molecular weight of the purified enzyme was estimated to be 66 kDa on SDS-polyacrylamide gel electrophoresis. The purified phytase activity was stable up pH 5.0, 7.0, 11.0 and the remaining activity was 50% when it was treated at $100^{\circ}C$ for 1 hour. The substrate specificity of phytase was most active against sodium phytate and inositol polyphosphate compound. And the phytase hydrolysed tripolyphosphate and pyrophosphate a little. The Km value for the sodium phytate was 0.64 mM and the Vmax value was $4.41\;{\mu}mol/min$.
단위 동물의 사료 내 phytate 인의 효율적 이용으로 배출되는 인의 양을 줄여 환경오염을 감소시키기 위해 phytate의 분해활성이 뛰어난 phytase 효소 분비 미생물을 탐색하기 위하며 우분으로부터 phytate 분해활성이 뛰어난 phytase를 생산하는 균주를 분리하였다. 분리균주를 동정한 결과, API 50 CHB test에 의해 B. circulans로 분류되었고, 16S rRNA sequencing 결과, B. subtilis로 동정되어 Bacillus sp. CF 5-26으로 명명하였다. 효소생산을 위한 최적배지 조성은 10% rice bran extract, 0.1%, whey protein powder, $0.01%\;CaCl_{2},\;0.01%\;KH_{2}PO_4$ 이었다. Bacillus sp. CF 5-26이 생산하는 phytase는 ethanol 침전, Sephadex G-100, CM Sepharose CL-6B, Sephacryl S-100-HR column chromatography를 통하여 정제도 20.3배, 수율 5.6% 정제되었고, SDS-PAGE에서 분자량 66 kDa의 단일 band를 확인하였다. 정제된 phytase는 pH 5.0, 7.0, 11.0에서 안정하였으며, $100^{\circ}C$에서 1시간 처리하였을 때 50%의 잔존활성을 보였다. 기질 특이성은 inositol polyphosphate인 sodium phytate 분해활성이 뛰어났으며, tripolyphosphate와 pyrophosphate에도 약간의 활성을 보였다. Bacillus sp. CF 5-26이 생산하는 phytase의 sodium phytate에 대한 Km은 0.64 mM 이었고, Vmax는 $4.41{\mu}mol/min$ 이었다.
