Purification and properties of an acetylxylan esterase from Thermobifida fusca

摘要

An acetylxylan esterase from Thermobifida fusca NTU22 was purified 51-fold as measured by specific activity from crude culture filtrate by ultrafiltration concentration,Sepharose CL-6B and DEAE-Sepharose CL-6B column chromatography.The overall yield of the purified enzyme was 14.4%.The purified enzyme gave an apparent single protein band on an SDS-PAGE.The molecular mass of purified enzyme as estimated by SDS-PAGE and by gel filtration on Sepharose CL-6B was found to be 30 and 28 kDa,respectively,indicating that the acetylxylan esterase from T.fusca NTU22 is a monomer.The pI value of the purified enzyme was estimated to be 6.55 by isoelectric focusing gel electrophoresis.The N-terminal amino acid sequence of the purified esterase was ANPYERGP.The optimum pH and temperature for the purified enzyme were 8.0 and 80°C,respectively.The Zn~(2+),Hg~(2+),PMSF and DIPF inhibited the enzyme activity.The K_m value for p-nitrophenyl acetate and acetylxylan were 1.86 mu M and 0.15%,respectively.Co-operative enzymatic degradation of oat-spelt xylan by purified acetylxylan esterase and xylanase significantly increased the acetic acid liberation compared to the acetylxylan esterase action alone.