Extraction and Purification of Glucoamylase and Protease Produced by Aspergillus awamori in a Single-Stage Fermentation

Sangeeta Negi¹*, Suneel Gupta¹ and Rintu Banerjee²

¹Biotechnology Section, Applied Mechanics Department, Motilal Nehru National Institute of Technology, Teliyarganj, Allahabad, 221004 Uttar Pradesh, India
²Agriculture and Food Engineering Department, Microbial Biotechnology and Downstream Processing Laboratory, Indian Institute of Technology, Kharagpur, 721302 West Bengal, India

Article history:
Received: November 20, 2010
Accepted: February 24, 2011

Key words:
extraction, purification, amylase, protease, Aspergillus awamori

Summary:
Simultaneous extraction and purification of glucoamylase and protease produced concomitantly by Aspergillus awamori Nakazawa MTCC 6652 in a single fermentor using solid-state fermentation (SSF) has been studied. Soaking for 2 h at room temperature (around 30 °C) in 10 % glycerol was found to be most suitable for optimum simultaneous extraction of glucoamylase and protease with the yield of 8645.8 U/g of glucoamylase and 798.6 U/g of protease in dry substrate. Crude extract to acetone ratio of 1:2 yielded optimum simultaneous precipitation of glucoamylase (35.3 %) and protease (61.9 %) with 4.06- and 7.17-fold purification, respectively. Ion exchange chromatography showed specific activities of purified fractions of 253.2 U/mg of glucoamylase and 59.7 U/mg of protease, with 22.1 and 40.8 % recovery, respectively. After gel filtration chromatography specific activity, recovery and purification of glucoamylase were found to be 306.8 U/mg, 4.6 % and 6.25-fold, respectively, whereas those of protease were 85.6 U/mg, 12.9 % and 17.0-fold, respectively. SDS-PAGE and zymogram studies of the purified enzymes indicated the presence of three starch-hydrolyzing isoforms of glucoamylase with molecular mass of approx. 109.6, 87.1, and 59.4 kDa and two types of acid protease with molecular mass of approx. 47.9 and 35.5 kDa. These findings can be very useful for enzyme industry, where glucoamylases and proteases are used concurrently.

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