Production Of Protease By Aspergillus Flavus In Solid State Fermentation
The study for the production of protease from Aspergillus flavus using wheat bran as substrate under Solid State Fermentation was conducted in University of Abuja, Department of Microbiology. Aspergillus flavus was isolated from spoilt bread and was identified on the basis of the morphological assessment such as macroscopic and microscopy. Among the characteristics used includes: colonial characteristics such surface appearance, texture and colour of the colonies. The protease activity increased with increase in the fermentation periods.
The quantities of the protease enzyme produced by the Aspergillus flavus in the basal medium were measured using UV-Spectrophotometer and the result is shown in Table 5. The protease activity was found to be higher at day 7 than day 5 and 3 with 4.51±10.06 proteaase Unit/mL, 8.63±0.12 U/mL and 18.93±1.20 AU/mL respectively. the extracellular protease produced by Aspergillus flavus isolated from spoilt bread in Gwagwalada were not significantly different at P= 0.05 level of significance. The study demonstrated thatAspergillus flavus wasable to produce extracellular protease enzymes important in the decomposition of protein materials.
1.1 Background of the Study
Protease constitutes a large and complex group of enzymes that plays an important nutritional and regulatory role in nature. Proteases are (physiologically) necessary for living organisms; they are ubiquitous and found in a wide diversity of sources. Protease is the most important industrial enzyme of interest accounting for about 60% of the total enzyme market in the world and account for approximately 40% of the total worldwide enzyme sale (Godfrey and West, 1996; Chouyyok et al., 2005). They are generally used in detergents (Barindra et al., 2006), food industries, leather, meat processing, cheese making, silver recovery from photographic film, production of digestive and certain medical treatments of inflammation and virulent wounds (Rao et al., 1998; Paranthaman et al., 2009). They also have medical and pharmaceutical applications.
Microbial proteases are degradative enzymes which catalyze the total hydrolysis of proteins (Raju et al., 1994; Haq et al., 2006). The molecular weight of proteases ranges from 18 – 90 kDa (Sidney and Lester, 1972). These enzymes are found in a wide diversity of sources such as plants, animals and microorganisms but they are mainly produced by bacteria and fungi. Microbial proteases are predominantly extracellular and can be secreted in the fermentation medium.
In the production of protease, it has been shown to be inducible and was affected by the nature of the substrate used in fermentation. Therefore, the choice of an appropriate inducing substrate is of great importance. Different carbon sources such as wheat bran, rice straw, rice bran, cotton and bagasse have been studied for the induction and biosynthesis of protease. However, wheat bran is a superior carbon source for the production of protease by Aspergillus flavus. So the further studies were carried out by using wheat bran as carbon source.
The use of agro-industrial residues as the basis for cultivation media is a matter of great interest, aiming to decrease the costs of enzyme production and meeting the increase in awareness on energy conservation and recycling (Singh et al., 2009). Major impediments to the exploitation of commercial enzymes are their yield, stability, specificity and the cost of production. New enzymes for use in commercial applications with desirable biochemical and physiochemical characteristics and low production cost have been focus of much research (Kabli, 2007). Solid state fermentation (SSF) was chosen for the present research because it has been reported to be of more grated productivity than that of submerged fermentation (Ghildyal et al., 1985; Hesseltine, 1972). Economically, SSF offers many advantages including superior volumetric productivity, use of simpler machinery, use of inexpensive substrates, simpler downstream processing, and lower energy requirements when compared with submerged fermentation (Paranthaman et al., 2009).
1.2 Aim of the Study
The aim of this study was to produce protease from Aspergillus flavus using wheat bran as a substrate under Solid State Fermentation.
1.3 Objectives of the Study
The objectives of the study include:
- To isolate Aspergillus flavus from spoilt bread in Gwagwalada.
- To determine the frequencies of occurrence of the isolated Aspergillus flavus from spoilt bread using simple percentages.
- To determine the proteolytic potential of the isolated fungi using basal medium.
- To determine the quantity of the protease enzyme produced by the isolated fungi using spectrophotometer.
A summary of purification steps for protease from A. flavus is given in Table 9. The purification of protease resulted in 2 fold purification with 66% of recovery by ammonium sulphate precipitation. The purification of crude enzyme through DEAE cellulose column chromatography gave 5.8 folds increase in purity with 3.2% recovery of protease from A. flavus. The similar observation was reported by Ogundero and Osunlaja (1986) for A. clavatus.
Fractions from the DEAE-Cellulose column which showed the highest activity were pooled and subjected to SDS- PAGE for determination of molecular weight of the protein. Purified enzyme preparation showed only one band corresponding to molecular weight of approximately 46 kDa (Fig. 10). Our results are more or less similar to that of Akel et al. (2009) who reported that the purified protease enzyme revealed a molecular mass of 49 kDa.
The maximum enzyme activity was found to be pH 7.0. Similar results were obtained for the optimum pH for enzymatic activity of other Bacillus species: pH 7.5 for Bacillus subtilis ITBCCB 148 (Yandri et al., 2008), Bacillus sp. HS08 (Huang et al., 2006) and Bacillus sp. S17110 (Jung et al., 2007); pH 8.0 for Bacillus cereus KCTC 3674 (Kim et al., 2001), Thermophilic Bacillus SMIA2 (Nascimento and Martins, 2004) and B. cereus BG1 (Ghorbel-Frikha et al., 2005).
The maximum enzyme activity was found to be 50°C. This was supported by Li et al. (1997) who reported that alkaline protease isolated from Thermomyces lanuginosus P134 had a broad temperature optimum of 50°C. Samal et al. (1991) also reported an alkaline protease from Tritirachium album lumber to be quite thermostable even up to 50°C. The protease activity was accelerated by Zn2+ and it was inhibited by Mg2+ and Ca2+. In contrast, Nehra et al. (2004) reported that Mg2+ was found to be an activator of the alkaline protease enzyme produced by Aspergillus sp. suggesting these metal ions had a capability to protect enzymes against denaturation.
Vmax and Km values for protease enzyme from Aspergillus flavus were determined from Line Weaver and Eadie-Hofstee plots. The results revealed that alkaline protease from A. flavus had a Vmax of 60.0 U/mg of protein and Km value of 0.6mg/ml. Matta et al. (1994) has reported proteases with lower Km values with casein substrate from Bacillus alcalophilus and Pseudomonas species, which showed Km values of 0.4 and 2.5 mg/ml, respectively. A slightly higher Km value of 3.7 mg/ml has been reported for the enzyme from B. polymyxa strain indicating higher affinity of the enzyme towards casein (Kaur et al., 1998).
We have characterized protease from a locally isolated fungus Aspergillus flavus. Its desirable characteristics such as broad substrate specificity, stability at high pH, stability at high temperature are significant characteristics of any enzyme for industrial application. Overall, the study provides that the wheat bran has a good potential to be used as solid state fermentation for protease production using A. flavus. The lab-scale study on protease production from wheat bran as a major substrate might give the basic information of further development for large scale production.
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