Genetic and biochemical characterization of thermophilic β-cyclodextrin glucanotransferase from Gracilibacillus alcaliphilus SK51.001.

Abstract

Gracilibacillus alcaliphilus SK51.001, a strain that produces β-CGTase (β-cyclodextrin glucanotransferase) (EC 2.4.1.19), was screened and isolated from Sudanese soil. The objective of this study was to sequence and characterize the β-CGTase gene from G. alcaliphilus SK51.001. According to 16S rRNA analysis of the strain and its morphological shape, it was identified as G. alcaliphilus. The β-CGTase gene was successfully cloned, sequenced, and expressed in Escherichia coli BL21. This gene showed 706 amino acid residues including 33 amino acids as a signal peptide. The active site residues of G. alcaliphilus SK51.001CGTase were described using enzyme modeling and docking with the products. The estimated molecular mass of G. alcaliphilus SK51.001CGTase was approximately 74 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the evaluation of the gel filtration showed approximately 85 kDa, which means G. alcaliphilus SK51.001CGTase is a monomer. The optimum temperature and pH of G. alcaliphilus SK51.001CGTase were 60 °C and 7.0 respectively. Gracilibacillus alcaliphilus SK51.001CGTase was comparatively stable at a pH levels between 6.0 and 9.0 and temperatures of 30-50 °C. The activity of G. alcaliphilus SK51.001CGTase was increased by Ni2+ , and Co2+ but inhibited by Al3+ and Fe3+ . The kinetic parameters of Km and Vmax were 2068.52 μg mL-1 and 0.13 μmol mL-1 min-1 , respectively. Gracilibacillus alcaliphilus SK51.001CGTase could hydrolyze soluble starch into α-, β-, and γ-cyclodextrin in a ratio of 2: 83: 15% respectively. This high ratio production of β-CD could allow the enzyme to be used in β-CD production. © 2020 Society of Chemical Industry.