Arginine mediated purification of trehalose-6-phosphate synthase (TPS) from Candida utilis: Its characterization and regulation

BackgroundTrehalose 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).MethodsIn the present study TPS is purified using a new methodology from Candida utilis cells by inclusion of 100 mM l-arginine during cell lysis and in the mobile phase of high performance gel filtration liquid chromatography (HPGFLC).ResultsAn 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 Vmax and lowest Km 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.General significanceSubstrate specificity, Vmax and Km 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.

► A 60 kDa protein having TPS activity was purified to electrophoretic homogeneity
► The specific activity of purified TPS was 2.03 U/mg protein.
► A new purification strategy was followed using l-arginine.
► N-terminal analysis revealed the similarity of purified TPS with TPS from S. cerevisiae.
► Purified TPS showed more affinity towards ADPG than its normal substrate UDPG.