Molecular cloning and characterization of a bifunctional xylanolytic enzyme from Neocallimastix patriciarum.
A cDNA encoding a bifunctional acetylxylan esterase/xylanase, XynS20E, was cloned from the ruminal fungus Neocallimastix patriciarum. A putative conserved domain of carbohydrate esterase family 1 was observed at the N-terminus and a putative conserved domain of glycosyl hydrolase family 11 was detected at the C-terminus of XynS20E. To examine the enzyme activities, XynS20E was expressed in Escherichia coli as a recombinant His(6) fusion protein and purified by immobilized metal ion-affinity chromatography. Response surface modeling combined with central composite design and regression analysis was then applied to determine the optimal temperature and pH conditions of the recombinant XynS20E. The optimal conditions for the highest xylanase activity of the recombinant XynS20E were observed at a temperature of 49 degrees C and a pH of 5.8, while those for the highest carbohydrate esterase activity were observed at a temperature of 58 degrees C and a pH of 8.2. Under the optimal conditions for the enzyme activity, the xylanase and acetylxylan esterase specific activities of the recombinant XynS20E toward birchwood xylan were 128.7 and 873.1 U mg(-1), respectively. To our knowledge, this is the first report of a bifunctional xylanolytic enzyme with acetylxylan esterase and xylanase activities from rumen fungus.
Pai
Cheng-Kang
CK
Department of Life Science, National Taiwan Normal University, Taipei, Taiwan.
Wu
Zong-Yuan
ZY
Chen
Ming-Ju
MJ
Zeng
Yi-Fang
YF
Chen
Jr-Wei
JW
Duan
Chung-Hang
CH
Li
Ming-Liang
ML
Liu
Je-Ruei
JR
D000115:Acetylesterase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002020:Buffaloes / Q000382:microbiology; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004271:DNA, Fungal / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D020088:Neocallimastix / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012417:Rumen / Q000382:microbiology; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature; D014990:Xylans / Q000378:metabolism*; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D018076:DNA, Complementary; D004271:DNA, Fungal; D011993:Recombinant Fusion Proteins; D014990:Xylans; D000115:Acetylesterase; C081851:acetylxylan esterase; D014995:Xylosidases