Applied Microbiology and Biotechnology

, Volume 79, Issue 2, pp 225–233

Cloning of a rumen fungal xylanase gene and purification of the recombinant enzyme via artificial oil bodies

Biotechnologically Relevant Enzymes and Proteins

Abstract

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°C; at temperatures below 30 and above 55°C, enzyme activity was less than 50% of that at the optimal temperature.

Keywords

Rumen Neocallimastix patriciarum Xylanase Artificial oil body 

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Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  1. 1.Department of Animal Science and Technology, Institute of BiotechnologyNational Taiwan UniversityTaipeiTaiwan
  2. 2.Institute of BioAgricultural SciencesAcademia SinicaTaipeiTaiwan
  3. 3.Department of Animal ScienceMcGill UniversityMontrealCanada
  4. 4.Graduate Institute of BiotechnologyNational Chung-Hsing UniversityTaichungTaiwan
  5. 5.Department of Life ScienceNational Taiwan Normal UniversityTaipeiTaiwan

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