title: Purification and characterization of a trehalase-invertase enzyme with dual activity from Candida utilis.
abstract: Trehalose and sucrose, two important anti-stress non-reducing natural disaccharides, are catabolized by two enzymes, namely trehalase and invertase respectively. In this study, a 175 kDa enzyme protein active against both substrates was purified from wild type Candida utilis and characterized in detail. Substrate specificity assay and activity staining revealed the enzyme to be specific for both sucrose and trehalose. The ratio between trehalase and invertase activity was found to be constant at 1:3.5 throughout the entire study. Almost 40-fold purification and 30% yield for both activities were achieved at the final step of purification. The presence of common enzyme inhibitors, thermal and pH stress had analogous effects on its trehalase and invertase activity. Km values for two activities were similar while Vmax and Kcat also differed by a factor of 3.5. Competition plot for both substrates revealed the two activities to be occurring at the single active site. N-terminal sequencing and MALDI-TOF data analysis revealed higher similarity of the purified protein to previously known neutral trehalases. While earlier workers mentioned independent purification of neutral trehalase or invertase from different sources, the present study reports the purification of a single protein showing dual activity.
authors.affiliation: Drug Development Diagnostics & Biotechnology Division, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700032, India.
authors.forename: Sagar
authors.initials: S
authors.lastname: Lahiri
authors.forename: Arghya
authors.initials: A
authors.lastname: Basu
authors.forename: Shinjinee
authors.initials: S
authors.lastname: Sengupta
authors.forename: Shakri
authors.initials: S
authors.lastname: Banerjee
authors.forename: Trina
authors.initials: T
authors.lastname: Dutta
authors.forename: Dhananjay
authors.initials: D
authors.lastname: Soren
authors.forename: Krishnananda
authors.initials: K
authors.lastname: Chattopadhyay
authors.forename: Anil K
authors.initials: AK
authors.lastname: Ghosh
mesh_terms: D000595:Amino Acid Sequence; D002175:Candida / Q000201:enzymology*; D020134:Catalytic Domain; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016133:Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014198:Trehalase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D043324:beta-Fructofuranosidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D043324:beta-Fructofuranosidase; D014198:Trehalase