Molecular Cloning and Characterization of Rumen Fungal Xylanolytic Enzymes
A gene encoding a xylanase,named xynS20,was cloned from the ruminal fungus Neocallimastix patriciarum.The DNA sequence of xynS20 revealed that the gene was 1008 bp in size and encoded amino acid sequences with a predicted molecular weight of 36 kDa.The amino acid sequence alignment showed that the highest sequence identity (28.4％) is with insect gut xylanase XYL6805.According to the sequence-based classification,a putative conserved domain of glycosyl hydrolase (GH) family 11 was detected at the N-terminus of XynS20,and a putative conserved domain of family 1 carbohydrate-binding module (CBM) was observed at the C-terminus of XynS20.An Asn-rich linker sequence was found between the N-terminal catalytic domain and the C-terminal CBM of XynS20.To examine the activity of the gene product,xynS20 gene was cloned as an oleosin-fused protein,expressed in Escherichia coli,affinity-purified by formation of artificial oil bodies,released from oleosin by intein-mediated peptide cleavage,and finally harvested by concentration of the supernatant.The specific activity of purified XynS20 toward oat spelt xylan was 1982.8 U/mg.The recombinant XynS20 was stable in the mild acid pH range from 5.0 ～ 6.0,and the optimum pH was 6.0.The optimal reaction temperature of XynS20 was 45℃ ; at temperatures below 30 and above 55℃,enzyme activity was less than 50％ of that at the optimal temperature.
Rumen Neocallimastix patriciarum Xylanase Artificial oil body
Je-Ruei Liu
Department of Animal Science and Technology, Institute of Biotechnology, National Taiwan University, Taipei 222
国内会议
广州
英文
183-196
2009-11-01（万方平台首次上网日期，不代表论文的发表时间）