Evaluation of Some Bioprocess Strategies for Control of Proteolyic Degradation of Raw Starch Digesting Amylase (RSD) of Aspergillus Carbonarius

Abstract

This study evaluates the presence of protease(s) in Aspergillus carbonarius submerged fermentation and increased specific raw starch digesting amylase (RSDA) productivity through control of proteolytic inhibition. The effect of cultural conditions on the production of protease and raw starch digesting amylase (RSDA) by Aspergillus carbonarius was also evaluated. Data obtained indicate that the type and concentration of medium carbon,nitrogen and divalent metal ions significantly (p < 0.001) influenced protease production. Potassium nitrate (KN03) and local rice starch used as nitrogen and carbon sources, respectively, reduced protease production very significantly with concomitant increase in RSDA activity. Maximum protease production was achieved with medium containing 5% glucose and 5% soybean meal in combination with 0.1% peptone as carbon and nitrogen sources, respectively and initial pH of 6.0. The time course of the protease production by the fungus showed that the enzyme synthesis followed logarithmic growth phase with maximum yield obtained at the 9'h day. The secreted protease from the fermentation broth was purified to apparent homogeneity by a four-step purification strategy comprising 4M sucrose concentration, ion-exchange chromatography on Q-Sepharose (Fast Flow), hydrophobic interaction chromatography on Phenyl Sepharose Fast Flow (High Sub) and gel filtration chromatography on Sephadex G-100. Approximately, 10-fold purification was achieved with specific activity of 485.47umg-' protein. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed a single migrating protein band corresponding to relative molecular weight of 72 kDa. The protease showed activity over a broad temperature range, 30 - 80°C, with temperature optimum at 40°C. The enzyme was stable at 50°C for 30 min. Only about 38% and 39% of the optimal activity was lost at 90°C and 100°C, respectively. The optimum pH of the protease was pH 3.0. The enzyme appeared to be 4 4' 4 r C 'relatively uninfluenced between pH 3.0 - 12.0. The enzyme was stable over a relatively broad range of pH (4.0 - 10.0) after 30 min. The protease activity was slightly stimulated by K+, ~ a ~ca2 ' and zn2' while ~ e ~ cu,2 ' and Mn2' significantly (p < 0.001) enhanced the enzyme activity. The protease activity was significantly (p < 0.001) repressed by H but slightly repressed by N a'. The activity of .the protease was not affected b y co2+, sr2+ and Mg2+. The protease activity was also significantly (p < 0.001) inhibited by both iodoacetate and dimethylsulfoxide, while EDTA and 2-mercaptoethanol appreciably enhanced the enzyme activity. The protease hydrolysed gelatin (K = 1.28mgmL; V = 0.14pmolmLI min-I), egg albumin (K = 1.61mgmL-; Vmax = 0.13pmolrn'minc')casein (K, =1 .92rngmL-'; Vmax = 0.11pmolrn'miann~d BSA (K, = 1.92mgmL-I; V = 0.1OpmolmLI min-I). The enzyme showed very strong affinity and hydrolytic potential for gelatin. Data obtained from this study indicate that manipulation of culture conditions and use of cysteine protease inhibitors could be applied as operational approach to reduce proteolytic enzyme and enhance RSDA production and stabilization in Aspergillus carbonarius. However, the unique physicochemical properties of the protease show that the enzyme could be equally assessed