Production and Partial Purification of a Neutral Metalloprotease by Fungal Mixed Substrate Fermentation

Alagarsamy Sumantha1, Chandran Sandhya1, George Szakacs2,
Carlos R. Soccol3 and Ashok Pandey1*

Biotechnology Division, Regional Research Laboratory (CSIR), Trivandrum, India
2Department of Agricultural Chemical Technology, Technical University of Budapest, H-1111 Budapest, Hungary
3Laboratory of Process Biotechnology, Federal University
of Parana, Curitiba, Brazil

Article history:

Received May 5, 2005

Accepted June 17, 2005

Key words:

neutral protease, solid-state fermentation, Aspergillus, agro-industrial residues


Five strains of fungi belonging to Aspergillus sp. were evaluated by casein agar plate assay and a wheat bran-based solid-state fermentation for selecting a neutral protease-producing culture. Based on the results, A. oryzae NRRL 2217 was selected for further studies. Sixteen different agro-industrial residues were evaluated for their potential to serve as asubstrate for neutral protease production by this fungal strain. Results showed that a combination of coconut oil cake and wheat bran in the mass ratio of 1:3 was the best substratefor enzyme production. Various process parameters influencing protease production including fermentation time, initial moisture content, and fermentation temperature were optimised. The medium was supplemented with different nutrients in the form of organic and inorganic nitrogen and carbon sources. Supplementation of chitin increased the enzyme production significantly. Ammonium nitrate as inorganic nitrogen supplement slightly enhanced enzyme production. No organic nitrogen supplement was effective enhancer of enzyme production. Fermentation was performed under optimised conditions (initial moisture content V/m= 50 %, temperature 30 °C, 48 h). Partial purification of the enzyme resulted in a 3-fold increase in the specific activity of the enzyme. The partially purifiedenzyme was characterised by various features that govern the enzyme activity such as assay temperature, assay pH and substrate concentration. The effect of various metal ions and known protease inhibitors on the enzyme activity was also studied. The enzyme was found to be stable in pH range 7.0–7.5, and at temperature of 50 °C for 35 min. By the activating effect of divalent cations (Mg2+,Ca2+,Fe2+) and inhibiting effect of certain chelating agents (EGTA, EDTA), the enzyme was found to be a metalloprotease. 

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