Original scientific paper
Production and Partial Purification of a Neutral Metalloprotease by Fungal Mixed Substrate Fermentation
Alagarsamy Sumantha
; Biotechnology Division, Regional Research Laboratory (CSIR), Trivandrum, India
Chandran Sandhya
; Biotechnology Division, Regional Research Laboratory (CSIR), Trivandrum, India
George Szakacs
; Department of Agricultural Chemical Technology, Technical University of Budapest, H-1111 Budapest, Hungary
Carlos R. Soccol
; Laboratory of Process Biotechnology, Federal University of Parana, Curitiba, Brazil
Ashok Pandey
; Biotechnology Division, Regional Research Laboratory (CSIR), Trivandrum, India
Abstract
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 a substrate 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 substrate for 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 purified enzyme 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.
Keywords
neutral protease; solid-state fermentation; Aspergillus; agro-industrial residues
Hrčak ID:
110561
URI
Publication date:
15.12.2005.
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