Production of Protease by Aspergillus Flavus in Solid State Fermentation

Production of Protease by Aspergillus Flavus in Solid State Fermentation

Chapter One

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.

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 flavusfrom 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.

CHAPTER TWO

Literature Review

Introduction

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.

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). Fungi elaborate a wide variety of proteolytic enzymes than bacteria. The filamentous fungi have a potential to grow under varying environmental conditions such as time course, pH and temperature, utilizing a wide variety of substrates as nutrients (Haq et al., 2006). Several species of strains including fungi (Aspergillus flavus, Aspergillus melleu, Aspergillus niger, Chrysosporium keratinophilum, Fusarium graminarum, Penicillium griseofulvin, Scedosporium apiosermum) and bacteria (Bacillus licheniformis, Bacillus firmus, Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus proteolyticus, Bacillus subtilis, Bacillus thuringiensis) are reported to produce proteases (Ellaiah et al., 2002).

The environmental conditions of the fermentation medium play a vital role in the growth and metabolic production of a microbial population. The most important among these are the medium, incubation temperature and pH. The pH of the fermentation medium is reported to have substantial effect on the production of proteases (Al- Shehri, 2004). It can affect growth of the microorganisms either indirectly by affecting the availability of nutrients or directly by action on the cell surfaces. Another important environmental factor is the incubation temperature, which is important to the production of proteases by microorganisms. Higher temperature is found to have some adverse effects on metabolic activities of microorganisms producing proteolytic enzymes (Tunga, 1995). However, some microorganisms produce heat stable proteases which are active at higher temperatures. The thermal stability of the enzymes may be due to the presence of some metal ions or adaptability to carry out their biological activity at higher temperature (Al-Shehri, 2004; Haq et al., 2006).

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).

The purpose of this study was to isolate, produce and purify protease from Aspergillus flavus, and to characterize some properties of the isolated enzymes using wheat bran as a substrate under SSF.

Protease

Proteases, proteinases, or peptidases are enzymes that are essential for all life forms (Barrett et al., 1998).They are essential for the synthesis of all proteins, controlling protein composition, size, shape, turnover and ultimate destruction. Their actions are exquisitely selective, each protease being responsible for splitting very specific sequences of amino acids under a preferred set of environmental conditions. There are over 500 human proteases, accounting for 2% of human genes and many proteases occur in every plant, insect, marine organism and in all infectious organisms that cause disease.

CHAPTER THREE

METHODOLOGY

Cellulosic material

In our preliminary studies, various agro wastes were used as a carbon source, and, hence, it could reduce the cost of enzyme production, which is collected in dried form from cattle shop, Coimbatore. Substrates, like wheat bran, cotton seed, rice bran, rice straw and sugarcane bagasse, were screened for enzyme production, in which wheat bran showed higher protease production, so it was used for further studies.

Organism and inoculum preparation

Fungal strains were isolated from soil of sugarcane field Coimbatore, India by serial dilution plate method (Waksman, 1922). Fungus were isolated from 10^-3 – 10^-4 dilutions by plating into Potato Dextrose Agar (PDA) medium. Isolated fungal cultures were screened for protease enzyme production. The organisms were identified using lactophenol cotton blue mounting method (Konemann et al., 1997). The isolated culture (Aspergillus flavus) was purified by routine sub-culturing and stored at 4^oC for further use.

CHAPTER FOUR

Result, Data presentation and Analysis

Results

Enzyme production by microorganisms is greatly influenced by media components, especially carbon and nitrogen sources, and physical factors such as temperature, pH, incubation time and inoculum density. It is important to produce the enzyme in inexpensive and optimized media on a large scale for the process to be commercially viable; hence the studies on the influence of various physico-chemical parameters such as incubation periods, inoculum size, temperature, pH, carbon, and nitrogen sources. Agricultural byproducts rich in cellulosic biomass can be exploited as cheap raw material for the industrially important enzymes and chemicals (Bigelow and Wyman, 2004). The fermentation medium was inoculated with the fungal strain and incubated for various time intervals (1-8 days). The enzyme production was gradually increased with the passage of time and highest enzyme activity (49.3 U mL^-1) was obtained on the 7th day of incubation (Fig.1). It was also observed that prolonged incubation decreased the enzyme activity. However the growth of the microorganism was not significantly affected.

CHAPTER FIVE

Conclusion

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.

V_max and K_m 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 V_max of 60.0 U/mg of protein and K_m value of 0.6mg/ml. Matta et al. (1994) has reported proteases with lower K_m values with casein substrate from Bacillus alcalophilus and Pseudomonas species, which showed K_m values of 0.4 and 2.5 mg/ml, respectively. A slightly higher K_m 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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