Pseudomonas sp. CB-33이 생산하는 β-Xylosidase의 특성 = Characterizotion of β-xylosidase from Pseudomonas sp. CB-33

국문 초록 (Abstract)

Pseudomonas sp. CB-33 균주의 배양 상등액으로부터 ammonium sulfate 침전법, PEI 침전법, DEAE-Sephadex column chromatography, gel column chromatography 그리고 preparative disc gel electrophoresis를 이용하여 β-xylosidase를 정제하였다. 정제된 효소의 Km 값은 4.6 mM이었다. 효소활성의 최적 pH는 7.0이었으며 pH 6.5~9 범위에서 안정하였다. 최적 활성온도는 45℃였으며, 각 온도에서 30분 동안 정치하였을 때 35℃까지 안정하였다. SDS-PAGE에 의해 효소의 분자량을 측정해 본 결과 약 44,000dalton이었다. HG^2+, Cu^2+ 그리고 Zn^2+는 효소활성을 저해하였고, 여러 화학변형제로 효소에 처리해 본 결과 tyrosine과 tryptophan이 효소활성에 관여하고 있을 것으로 추정된다. 여러가지 기질에 대한 특이성을 조사해 본 결과 pNPX 뿐만 아니라 pNPA도 분해하였다. Xylan에 endoxylanase만을 작용시켰을 때보다 β-xylosidase와 endoxylanase를 혼합작용 시켰을 때 xylan의 가수분해도는 약 2배 정도 증가하였다. 그리고 Pseudomonas sp. CB-33 균주가 생산하는 xylan 분해효소는 oat spelt xylan보다 birchwood xylan에 보다 효과적으로 작용하였다.

다국어 초록 (Multilingual Abstract)

The β-xylosidase was purified 99-fold from the culture supernatant of Pseudomonas sp. CB-33 by ammonium sulfate precipitation, PEI precipitation, DEAE-Sephadex column chromatography, Sephadex G-75 gel filtration chromatography and preparative disc gel electrophoresis. Molecular weight of the enzyme was estimated to be 44,000 by SDS polyacrylamide gel electrophoresis. The enzyme has a pH optimum for activity at 7.0 and is stable over pH 6.5~9.0. The optimal temperature of the enzyme was 45℃, and it enzymatic activity was completely inactivated at 55℃ for 30 min. Km value of the enzyme for p-nitrophenyl- β-D-xylopyranoside was calculated to be 4.6 mM. The effect of various reagents on the β-xylosidase activity was investigated. The enzyme activity was completely inhibited by Hg^2+, Cu^2+, and Zn^2+. The β-xylosidase was inactivated by tryptophan-specific reagent, N-bromosuccinimide and the enzyme was competitively inhibited by xylose. The β-xylosidase and endoxylanase from Pseudomonas sp. CB-33 hydrolized xylan synergically. The purified enzyme also showed α-L-arabinofuranosidase activity.