title: Arginine mediated purification of trehalose-6-phosphate synthase (TPS) from Candida utilis: Its characterization and regulation.
abstract: BACKGROUND
  Trehalose is the most important multifunctional, non-reducing disaccharide found in nature. It is synthesized in yeast by an enzyme complex: trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP).
  
  
  METHODS
  In the present study TPS is purified using a new methodology from Candida utilis cells by inclusion of 100mM l-arginine during cell lysis and in the mobile phase of high performance gel filtration liquid chromatography (HPGFLC).
  
  
  RESULTS
  An electrophoretically homogenous TPS that was purified was a 60 kDa protein with 22.1 fold purification having a specific activity of 2.03 U/mg. Alignment of the N-terminal sequence with TPS from Saccharomyces cerevisiae confirmed the 60 kDa protein to be TPS. Optimum activity of TPS was observed at a protein concentration of 1 μg, at a temperature of 37°C and pH 8.5. Aggregation mediated enzyme regulation was indicated. Metal cofactors, especially MnCl₂, MgCl₂ and ZnSO₄, acted as stimulators. Metal chelators like CDTA and EGTA stimulated enzyme activity. Among the four glucosyl donors, the highest V(max) and lowest K(m) values were calculated as 2.96 U/mg and 1.36 mM when adenosine di phosphate synthase (ADPG) was used as substrate. Among the glucosyl acceptors, glucose-6-phosphate (G-6-P) showed maximum activity followed by fructose-6-phosphate (F-6-P). Polyanions heparin and chondroitin sulfate were seen to stimulate TPS activity with different glucosyl donors.
  
  
  CONCLUSIONS
  Substrate specificity, V(max) and K(m) values provided an insight into an altered trehalose metabolic pathway in the C. utilis strain where ADPG is the preferred substrate rather than the usual substrate uridine diphosphaphate glucose (UDPG). The present work employs a new purification strategy as well as highlights an altered pathway in C. utilis.
authors.affiliation: Biotechnology Division, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata 700 032, India.
authors.forename: Shinjinee
authors.initials: S
authors.lastname: Sengupta
authors.forename: Sagar
authors.initials: S
authors.lastname: Lahiri
authors.forename: Shakri
authors.initials: S
authors.lastname: Banerjee
authors.forename: Bipasha
authors.initials: B
authors.lastname: Bashistha
authors.forename: Anil K
authors.initials: AK
authors.lastname: Ghosh
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry*; D002175:Candida / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005964:Glucosyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity
chemical_list: D001120:Arginine; D005964:Glucosyltransferases; C023180:trehalose-6-phosphate synthase