Overexpression and characterization of alkaliphilic Bacillus firmus strain K-1 xylanase

Authors

  • Karntichar Mongkorntanyatip Department of Microbiology, Faculty of Science, King Mongkut's University of Technology Thonburi, Bangmod, Bangkok, 10140, Thailand
  • Puangpen Limsakul School of Bioresources and Technology, King Mongkut's University of Technology Thonburi, Bangkuntien, Bangkok, 10150, Thailand
  • Khanok Ratanakhanokchai School of Bioresources and Technology, King Mongkut's University of Technology Thonburi, Bangkuntien, Bangkok, 10150, Thailand
  • Pongsak Khunrae Department of Microbiology, Faculty of Science, King Mongkut's University of Technology Thonburi, Bangmod, Bangkok, 10140, Thailand

Keywords:

Bacillus firmus K-1, Expression and purification, Recombinant enzyme, Xyn11A, Xylanase

Abstract

The alkaliphilic Bacillus firmus strain K-1 produces an alkaliphilic xylanase (Xyn11A) which can be stable across a wide pH range and active at high temperatures. However, the enzyme suffers from low activity when isolated directly from the culture broth using corn husk. A method was developed which employed recombinant DNA technology to produce recombinant Xyn11A (rXyn11A) in an Escherichia coli (E. coli) expression system. The thioredoxin-fused Xyn11A (Thio-rXyn11A) could be expressed as a soluble form in BL21 (DE3). The expressed protein was tobacco etch virus (TEV) cleavaged to remove the thioredoxin tag and subjected to further purification using Ni2+-NTA affinity chromatography followed by gelfiltration chromatography. Activity of rXyn11A was shown to be under the same conditions as the native enzyme isolated directly from the K-1, having a broad range of active pH (5.0-12.0), with the maximum activity obtained from pH 5.0 at 60oC. Interestingly, the obtained rXyn11A exhibited a very large increase in specific activity (3034 U/mg), which was 84-times higher than that reported in the native enzyme when observed under the same conditions. Also seen in the xylan hydrozation assay, the rXyn11A hydrolyzed insoluble xylans around 100-times more effectively than the native enzyme. The results from this study demonstrated a successful method for generating the enzyme rXyn11A with much improved activity, making it feasible for industrial applications.

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Published

2017-12-31

Issue

Section

Research Article