title: Molecular characterization of a novel trehalose-6-phosphate hydrolase, TreA, from Bacillus licheniformis.
abstract: 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 °C and 8.0, respectively. The enzyme hydrolyzed p-nitrophenyl-α-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 5.2mM and a k(cat) value of 30.2s(-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 ~0.14 M guanidine hydrochloride (GdnHCl) and reached the unfolded intermediates, [GdnHCl](0.5,N-I) and [GdnHCl](0.5,I-U), at 1.02 and 2.24 M, respectively. BlTreA was unfolded completely by 8M 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.
authors.affiliation: Department of Biochemical Science and Technology, National Chiayi University, 300 Syuefu Road, Chiayi County 60004, Taiwan.
authors.forename: Tzu-Ting
authors.initials: TT
authors.lastname: Chuang
authors.forename: Ping-Lin
authors.initials: PL
authors.lastname: Ong
authors.forename: Tzu-Fan
authors.initials: TF
authors.lastname: Wang
authors.forename: Hsien-Bin
authors.initials: HB
authors.lastname: Huang
authors.forename: Meng-Chun
authors.initials: MC
authors.lastname: Chi
authors.forename: Long-Liu
authors.initials: LL
authors.lastname: Lin
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D003001:Cloning, Molecular; D019295:Computational Biology; D004186:Disaccharidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D019791:Guanidine / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011401:Promoter Regions, Genetic / Q000235:genetics; D011489:Protein Denaturation / Q000187:drug effects; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D012997:Solvents / Q000737:chemistry / Q000494:pharmacology; D013696:Temperature; D014508:Urea / Q000494:pharmacology; D014867:Water / Q000737:chemistry
chemical_list: D012997:Solvents; D014867:Water; D012967:Sodium Dodecyl Sulfate; D014508:Urea; D004186:Disaccharidases; C040631:alpha, alpha-phosphotrehalase; D019791:Guanidine