Cloning of a rumen fungal xylanase gene and purification of the recombinant enzyme via artificial oil bodies.
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 1,008 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 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 1,982.8 U mg(-1). The recombinant XynS20 was stable in the mild acid pH range from 5.0 to 6.0, and the optimum pH was 6.0. The optimal reaction temperature of XynS20 was 45 degrees C; at temperatures below 30 and above 55 degrees C, enzyme activity was less than 50% of that at the optimal temperature.
Liu
Je-Ruei
JR
Department of Animal Science and Technology, Institute of Biotechnology, National Taiwan University, 4F, No. 81 Chang-Xing Street, Taipei, Taiwan. jrliu@ntu.edu.tw
Duan
Chung-Hang
CH
Zhao
Xin
X
Tzen
Jason T C
JT
Cheng
Kuo-Joan
KJ
Pai
Cheng-Kang
CK
D000818:Animals; D003001:Cloning, Molecular; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D005800:Genes, Fungal; D020088:Neocallimastix / Q000201:enzymology* / Q000235:genetics; D009821:Oils / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012417:Rumen / Q000201:enzymology / Q000378:metabolism / Q000382:microbiology; D013696:Temperature; D014995:Xylosidases / Q000737:chemistry / Q000378:metabolism*
D009821:Oils; D011993:Recombinant Fusion Proteins; D014995:Xylosidases; D043364:Endo-1,4-beta Xylanases