การหาลักษณะจำเพาะของยีนเซลลูเลสและไซลาเนสจากแบคทีเรียในกระเพาะหมักของวัว

Abstract

In this research, cellulase and xylanase producing bacteria were isolated from cow’s rumen fluid. Among twenty isolates, strain B4 showed the highest cellulolytic and hemicellulolytic activity. Analysis of a 16S rDNA sequence showed the highest homology with Bacillus subtilis (>99%). The isolate, strain B4 was designated as B. subtilis B4. Then both of the cellulase (BglC) and xylanase (XynA) genes of B. subtilis B4 were cloned into expression vector (pETDuet-1) and transformed into E. coli strain BL21 (DE3). The transformants named pETbglC and pETxynA showed cellulase and xylanase specific activity of 19.46 and 110.38 U/mg protein, respectively and about 3-fold of cellulase and 2-fold of xylanase grater than enzymes of the wild type. The cellulase and xylanse which produced by pETbglC and pETxynA were purified by ammonium sulfate precipitation, DEAD Toyopearl 650M and Butyl-Toyopearl 650M column. The molecular mass of purified cellulase and xylanase were estimated to be approximately 50 and 23 kDa on SDS-PAGE, respectively. The cellulase exhibited its optimum activity at 60 °C and pH 8.0. It was stable over a broad range at 20-80 °C and pH 5-10. Optimal temperature and pH of xylanase activity were 50 °C and pH 7.0. The enzyme showed stability at 20-50 °C and pH 4-9. Moreover, enzyme activity of both enzymes was stimulated by Fe3+ and was strongly inhibited by Mn2+ and Cu2+. Furthermore, cellulase and xylanase in this study have potential to be used for degradation of non-treated and pretreated lignocellulosic substates (corn stover, rice straw, paragrass and napier grass). Therefore, these enzymes could be useful in hydrolysis of lignocellulosic biomass into reducing sugar, which is finally able to ferment to ethanol.