Purification and properties of an acetylxylan esterase from Thermobifida fusca

Abstract

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 p I 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 μM 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.