[feed] Atom [feed] RSS 1.0 [feed] RSS 2.0

Acid protease from Aspergillus oryzae: Structure-stability and enhancement of the activity by physical, chemical and molecular biological approaches

Vishwanatha, K. S. (2009) Acid protease from Aspergillus oryzae: Structure-stability and enhancement of the activity by physical, chemical and molecular biological approaches. PhD thesis, University of Mysore.


Download (4MB)


The focus of this investigation has been to screen fungal sources for aspartate proteases, which are used in food industries as food protein modifiers and as replacers of animal rennets in cheese making. The isolation, characterization and stability parameters of two high active aspartic proteases from Aspergillus oryzae MTCC 5341 are reported for the first time. Aspergillus oryzae MTCC 5341 produced maximum aspartic protease production (8.6 105 U/g bran of acid protease and 40, 000 U/g bran of milk clotting enzyme.) by solid state fermentation. Process parameters for solid state fermentation for production of these enzymes have been optimized and downstream parameters standardized. The acid protease from A. oryzae has a molecular weight of 47 kDa and is rich in  structure (~60%) like other aspartic proteases. Milk clotting enzyme from A. oryzae is a 34 kDa aspartic protease with 49⁰C as its midpoint of thermal denaturation. Pepstatin inhibited acid protease completely; other class specific protease inhibitors had no effect on proteolytic activity. Pepstatin is a competitive inhibitor with a Ki of 3.7x10-7M. Acid protease activated trypsinogen to active trypsin, Val-(Asp)4-Lys, a hexapeptide towards N-terminal of precursor trypsinogen is cleaved to release trypsin. v Acid protease was stable in a pH range of 2-6 and on either side, enzyme lost activity and structure due to collapse in hydrophobic interactions. Temperature induced aggregation of the molecule due to autolysis. Chaotropic salts induced unfolding of the molecule and thereby resulting in aggregation, molecular chaperons like SCN-1, β and α- casein gave thermal protection- involvement of ionic interactions in maintenance of structure Chemical denaturants induced unfolding curves were cooperative and biphasic- N→U. GuHCl obeyed “two fold” rule in unfolding of the molecule ([denaturant]1/2 were Urea- 1.74 M and GuHCl- 0.85 M). Loss of proteolytic activity upon unfolding coincides with loss in tertiary and secondary structure reflecting good correlation between activity and structural integrity of the molecule Rennet hydrolyzed all the three casein fractions (α-, β- and κ- casein) showing little specificity for hydrolysis. Higher rate of hydrolysis towards κ – Casein than α and β caseins. Hydrolysis of α-, β- caseins by the protease action adds bitter peptides essential for flavor developing during ripening. Cheese preparation compared with control (Mucor rennet). Clotting time, yield and surface characteristics were on par with control These studies on aspartic proteases from A. oryzae may lead to new sources of high activity enzymes for use in food industry. These enzymes with improved stability help in understanding the structure-function relation of aspartic proteases.

Item Type: Thesis (PhD)
Uncontrolled Keywords: aspartate proteases, fungal sources, structure-function, food industries
Subjects: 500 Natural Sciences and Mathematics > 07 Life Sciences > 04 Microbiology > 04 Fungi
600 Technology > 08 Food technology > 16 Nutritive value > 05 Enzymes
Divisions: Protein Chemistry and Technology
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 01 Mar 2011 07:28
Last Modified: 28 Dec 2011 10:21
URI: http://ir.cftri.com/id/eprint/9946

Actions (login required)

View Item View Item