PRODUCTION OF PROTEASE BY ASPERGILLUS FLAVUS IN SOLID STATE FERMENTATION

ABSTRACT

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 that Aspergillus flavus was able to produce extracellular protease enzymes important in the decomposition of protein materials.

TABLE OF CONTENT

CHAPTER ONE

INTRODUCTION

1.1          Background of the study

1.2          Aim of the study

1.3       Objectives of the study

CHAPTER TWO

LITERATURE REVIEW

2.1          Microorganisms associated with protease productions

 2.1.1     Fungi as source of commercial enzymes

2.2          Enzymes

2.2.1    Protease

2.2.1.1  Classification of proteases

2.2.1.2             Functions and mechanisms of proteases

2.2.1.2.1 Catalysis

2.2.1.2.2  Specificity

2.2.1.2.3  Degradation and autolysis

2.2.2   Uses of protease

2.2.2.1             Industrial uses

2.3       Protein

CHAPTER THREE

3.0                                           MATERIALS AND METHODS

3.1       Study Area

3.2       Materials Used

3.2.1      Sterilization of material used

3.3       Samples Collection

3.4       Preparations and sterilization of media.

3.5          Isolation of Aspergillus flavus fungi

3.6          Preparation of pure cultures of fungal isolates

3.7       Identification of fungal isolates

3.8       Determination of protease potentiality of fungal isolates

3.8.1    Extraction of protease enzyme

3.8.2    Assay of protease enzyme

3.9       Statistical analysis

                                                         CHAPTER FOUR

4.0                                                       RESULTS

4.1       Identifications of         fungal isolates

4.2       Protease activity of the isolated Aspergillus flavus from spoilt bread

CHAPTER FIVE

5.0                               DISCUSSION AND CONCLUSION

5.1       Discussion

5.2       Conclusion

REFERENCES

APPENDIX  1

APPENDIX  2

CHAPTER ONE

1.0            INTRODUCTION

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 determinethe proteolytic potential of the isolated fungi using basal medium.

·        To determine the quantity of the protease enzyme produced by the isolated fungi using spectrophotometer.

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