Molecular cloning and characterization of a bifunctional xylanolytic enzyme from Neocallimastix patriciarum

Appl Microbiol Biotechnol. 2010 Feb;85(5):1451-62. doi: 10.1007/s00253-009-2175-5. Epub 2009 Aug 19.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylesterase / chemistry
  • Acetylesterase / genetics
  • Acetylesterase / isolation & purification
  • Acetylesterase / metabolism*
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Binding Sites
  • Buffaloes / microbiology
  • Chromatography, Affinity
  • Cloning, Molecular*
  • DNA, Complementary
  • DNA, Fungal / genetics
  • Escherichia coli / genetics
  • Kinetics
  • Molecular Sequence Data
  • Neocallimastix / enzymology*
  • Neocallimastix / genetics*
  • Neocallimastix / metabolism
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Rumen / microbiology
  • Sequence Alignment
  • Substrate Specificity
  • Temperature
  • Xylans / metabolism*
  • Xylosidases / chemistry
  • Xylosidases / genetics
  • Xylosidases / isolation & purification
  • Xylosidases / metabolism*


  • DNA, Complementary
  • DNA, Fungal
  • Recombinant Fusion Proteins
  • Xylans
  • Acetylesterase
  • acetylxylan esterase
  • Xylosidases

Associated data

  • GENBANK/FJ529209