Heterologous expression of β-γ-type cyclodextrin glycosyltransferase of newly isolated alkaliphilicBacillussp. SD5 inPichia pastoris

Abstract

Cyclodextrin glycosyltransferase (CGTase) enzyme is used industrially to obtain cyclodextrins. In this study, a newly isolated alkaliphilic CGTase producing Bacillus sp. SD5 was used as the source organism for the gene encoding for CGTase. The gene was amplified and sequenced in preparation for cloning into Pichia pastoris for methanol-induced expression and extracellular secretion. Three strategies were employed in designing the amplification primers for obtaining the fragments to be used in transformation. Hence, three P. pastoris X33 clones were obtained: N2 (with natural gene sequence), S3 (also encoding the c-myc epitope and polyhistidine tail), and M5 (encoding the polyhistidine tail). Extracellular CGTase activity was monitored throughout fermentation for each clone. The molecular weights of the enzymes were in the range of 85–90 kDa and all three clones showed maximum CGTase activity upon 24 h of induced fermentation. The CGTase enzyme obtained from the M5 clone had Km and Vmax values 13.59 mg/mL and 1153 µmol/(mg min), respectively. The enzyme was purified and displayed optimum activity at 50°C and pH 6 and pH 9. HPLC analysis of reaction products following 10 min of incubation in a reaction solution containing 4% starch showed 852.5 mg/L β-cyclodextrin and 89.6 mg/L γ-cyclodextrin being produced.