Molecular characterization of a novel trehalose-6-phosphate hydrolase, TreA, from Bacillus licheniformis

作者：Chuang Tzu Ting; Ong Ping Lin; Wang Tzu Fan; Huang Hsien Bin; Chi Meng Chun; Lin Long Liu

来源：International Journal of Biological Macromolecules, 2012, 50(3): 459-470.

DOI：10.1016/j.ijbiomac.2012.01.011

• Trehalose-6-P-hydrolase
• Bacillus licheniformis
• Organic solvent-tolerant enzyme
• Thermal unfolding
• Chemical-induced denaturation

An unidentified Bacillus licheniformis trehalose-6-phosphate hydrolase (BlTreA) gene was cloned and heterologously expressed in Escherichia coli M15 cells. The over-expressed BlTreA was purified to apparent homogeneity by metal-affinity chromatography and its molecular mass was determined to be approximately 65.9 kDa. The temperature and pH optima for BlTreA were 30 degrees C and 8.0, respectively. The enzyme hydrolyzed p-nitrophenyl-alpha-D-glucopyranoside (pNPG) and trehalose-6-phosphate efficiently, but it was inactive toward five other p-nitrophenyl derivatives. Steady-state kinetics with pNPG showed that BlTreA had a K-M value of 5.2 mM and a k(cat) value of 30.2 s(-1). Circular dichroism analysis revealed that the secondary structures of BlTreA did not altered by 5-10% acetone and 10-20% ethanol, whereas 5-10% SDS had a detrimental effect on the folding of the enzyme. Thermal unfolding of this enzyme was found to be highly irreversible. The native enzyme started to unfold beyond similar to 0.14 M guanidine hydrochloride (GdnHCl) and reached the unfolded intermediates, [GdnHCl](0.5,N-1), and [GdnHCl](0.5.I-U), at 1.02 and 2.24 M, respectively. BlTreA was unfolded completely by 8 M urea with [urea](0.5,N-U) of 4.98 M, corresponding to a free energy change of 4.29 kcal/mol for the N --> U process. Moreover, the enzyme was unfolded by GdnHCl through a reversible pathway and the refolding reaction exhibited an intermediate state. Taken together, the characterization data provide a foundation for the future structure-function studies of BlTreA, a typical member of glycoside hydrolase family 13.

• 出版日期2012-4-1