Abstract

A new amylase gene APGA1 was cloned from Aureobasidium pullulans NRRL 12974 and expressed in Pichia pastoris. This is the first report on cloning and expression of amylolytic gene from the industrially important microorganism A. pullulans. The purified recombinant protein with MW of 66 kDa and specific activity of 298.02 Umg^-1 protein was verified as a glucoamylase by its hydrolytic mode. This recombinant glucoamylase with optimal pH of 4.5, and temperature of 60°C, showed good hydrolytic activity against raw potato starch. At 60°C, 83.1% of raw potato starch slurry (150 gl^-1) was hydrolysed into glucose by 0.1 U mg^-1 starch purified recombinant glucoamylase in less than 2.5 h. This is the highest raw starch hydrolysis efficiency report about recombinant fungal glucoamylase. This useful property indicated that this glucoamylase may find important applications in the starch saccharification industry and in bioethanol production.

Key words: Glucoamylase, Aureobasidium pullulans, expression, raw starch degradation.

Abbreviation

MW, Molecular weight; LB, luria-bertani; NaCl, sodium chloride;PCR, polymerase chain reaction; BMGY, buffered glycerol-complex; BMMY, buffered methanol-complex; SDS-PAGE, sodium dodecyl sulfate polyacrylamidegel electrophoresis; DNS, 3,5-dinitrosalicylic acid; MALDITOF/TOF, matrix-assisted laser desorption/ionization time-of-flight/time-of-flight; HPLC, high-performance liquid chromatography.