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Heterologous expression, purification and biochemical characterization of a new endo-1,4-β-xylanase from Rhodothermaceae bacterium RA.

Protein expression and purification (2019-08-04)
Kok Jun Liew, Chen Yi Ngooi, Mohd Shahir Shamsir, Rajesh Kumar Sani, Chun Shiong Chong, Kian Mau Goh
ABSTRACT

Xylanases (EC 3.2.1.8) are essential enzymes due to their applications in various industries such as textile, animal feed, paper and pulp, and biofuel industries. Halo-thermophilic Rhodothermaceae bacterium RA was previously isolated from a hot spring in Malaysia. Genomic analysis revealed that this bacterium is likely to be a new genus of the family Rhodothermaceae. In this study, a xylanase gene (1140 bp) that encoded 379 amino acids from the bacterium was cloned and expressed in Escherichia coli BL21(DE3). Based on InterProScan, this enzyme XynRA1 contained a GH10 domain and a signal peptide sequence. XynRA1 shared low similarity with the currently known xylanases (the closest is 57.2-65.4% to Gemmatimonadetes spp.). The purified XynRA1 achieved maximum activity at pH 8 and 60 °C. The protein molecular weight was 43.1 kDa XynRA1 exhibited an activity half-life (t1/2) of 1 h at 60 °C and remained stable at 50 °C throughout the experiment. However, it was NaCl intolerant, and various types of salt reduced the activity. This enzyme effectively hydrolyzed xylan (beechwood, oat spelt, and Palmaria palmata) and xylodextrin (xylotriose, xylotetraose, xylopentaose, and xylohexaose) to produce predominantly xylobiose. This xylanase is the first functionally characterized enzyme from the bacterium, and this work broadens the knowledge of GH10 xylanases.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Maltose solution, BioReagent, for molecular biology, ~20% in H2O
Sigma-Aldrich
Xylobiose, ≥90% (HPLC)