Heterologous expression and biochemical characterization of a thermostable xylulose kinase from Bacillus coagulans IPE22

Abstract

Xylulose kinase is an important enzyme involved in xylose metabolism, which is considered as essential biocatalyst for sustainable lignocellulosic-derived pentose utilization. Bacillus coagulans IPE22 is an ideal bacterium for refinery due to its strong ability to ferment xylose at high temperature. However, the B. coagulans xylose utilization mechanism remains unclear and the related promising enzymes need to be developed. In the present study, the gene coding for xylulose kinase from B. coagulans IPE22 (Bc-XK) was expressed in Escherichia coli BL21 (DE3). Bc-XK has a 1536 bp open reading frame, encoding a protein of 511 amino acids (56.15 kDa). Multiple sequence alignments were performed and a phylogenetic tree was built to evaluate differences among Bc-XK and other bacteria homologs. Bc-XK showed a broad adaptability to high temperature and the enzyme displayed its best performance at pH 8.0 and 60 °C. Bc-XK was activated by Mg²⁺ , Mn²⁺ , and Co²⁺ . Meanwhile, the enzyme could keep activity at 60 °C for at least 180 min. K_M values of Bc-XK for xylulose and ATP were 1.29 mM and 0.76 mM, respectively. The high temperature stability of Bc-XK implied that it was an attractive candidate for industrial application.

Keywords: Bacillus coagulans; bioinformatic analysis; lactic acid; thermostability; xylulose kinase.