Characterization of xylanase gene from Clostridium acetobutylicum
Afifa Husna, Yo-Chia Chen
National Pingtung University of Science and Technology
Abstract
Background: Clostridium acetobutylicum is an anaerobic Gram negative bacterium known to have the ability to degrade and utilize hemicellulose as a substrate for its metabolism. In order to use carbon source other than sugar, this bacteria harbor some glycosyl hydrolase enzymes as secondary metabolites. One of the enzymes is xylanase (endo-β-1,4-xylanase) which can breakdown xylan. In industry, this enzyme could be used to produce many kind of xylan derivatives, such as xylooligosaccharides, which has high economical value. Aims: Therefore, the aim of this study is to clone xylanase gene and identify the ability of the expressed xylanase from Clostridium acetobutylicum to breakdown any kind of xylan sources. Material and Methods: Xylanase gene from Clostridium acetobutylicum ATCC 824 was amplified using Polymerase Chain Reaction (PCR) and cloned into pET 21a(+) expression vector, and then the constructs was transformed into Escherichia coli BL21 competent cells by heat shock. Protein expression was induced by the addition of 0.5 mM IPTG for 4 hours. The expressed recombinant protein was identified using SDS-PAGE and its activity towards various kind of substrates. Results: PCR amplification result showed that recombinant xylanase gene from C. acetobutylicum has 1371 bp and encodes 456 amino acids protein. BLAST analysis result showed that the recombinant gene has catalytic site and belongs to glycosyl hydrolase family 43. SDS-PAGE was then carried out to confirm the expression of the cloned gene and production of an approximately 42 kDa protein was confirmed from the cloned gene. However, the ability of the expressed protein to degrade any kind of xylan sources needs to be further evaluated. Conclusion: Xylanase gene from C. acetobutylicum was successfully cloned and expressed, resulted in approximately 42 kDa recombinant protein, but its activity to breakdown xylan substrates needs to be further investigated.
Keywords: Cloning, Clostridium acetobutylicum, overexpression, xylanase
Topic: Environmentally Sustainable Agriculture