Biotechnologically Relevant Enzymes and Proteins

Applied Microbiology and Biotechnology

, Volume 79, Issue 2, pp 225-233

First online:

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

  • Je-Ruei LiuAffiliated withDepartment of Animal Science and Technology, Institute of Biotechnology, National Taiwan University Email author 
  • , Chung-Hang DuanAffiliated withInstitute of BioAgricultural Sciences, Academia Sinica Email author 
  • , Xin ZhaoAffiliated withDepartment of Animal Science, McGill University
  • , Jason T. C. TzenAffiliated withGraduate Institute of Biotechnology, National Chung-Hsing University
  • , Kuo-Joan ChengAffiliated withInstitute of BioAgricultural Sciences, Academia Sinica
  • , Cheng-Kang PaiAffiliated withDepartment of Life Science, National Taiwan Normal University

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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