OPTIMIZING ALPHA AMYLASE PRODUCTION FROM LOCALLY ISOLATED ASPERGILLUS SPECIES USING SELECTED AGRO WASTES AS SUBSTRATES

ABSTRACT

α-Amylase are enzymes that are used in the degradation of starch. This study focuses on optimizing the production of α-Amylase using locally isolated Aspergillus japonicus in defined and undefined medium.

Five grams of cocoyam waste, sweet potato waste, wheat waste and plantain peel were utilized as substrates during fermentation for the production of α-Amylase using 1 ml spore suspension as inoculum. The fermentation media contained in g/l (0.8 NaCl, 0.8 KCl, 0.1 CaCl₂, 2.0 Na₂HPO₄, 0.1 FeSO₄, 8.0 Fructose, 2.0 NH₄Cl).  Temperature, pH, sugar content and amylase activity of the culture filtrates were monitored after every 48 hours. For optimization, mycelia was used as inoculum and different inoculum sizes (5%, 10% and 15%) were used for fermentation in a defined medium containing starch as substrate. The effect of urea as nitrogen source on α -Amylase activity was monitored. Different concentrations (2g and 5g) of cocoyam waste and sweet potato waste were also utilized for optimization using 10% inoculum. The effect of temperature and pH on amylase was determined.

Two grams (2g) of sweet potato waste with NH₄Cl as nitrogen source, using 10% inoculum gave the highest yield of α-Amylase after 96 hours. The optimum pH and temperature for α -amylase production were pH 5 and 50 ^oC respectively.

The ability of the amylase to act within an acidic pH suggests that it is stable within a wide range of acidic pH (2 – 6) and its ability to withstand relatively high temperature (40 ^o C – 60 ^o C) above the optimum growth temperature  of A. japonicus suggests that it is thermo stable. The crude α-Amylase produced from A. japonicus retained 67% of its activity at 60^oC.α-Amylase from this fungus has the potential to be utilized for various biotechnological processes. Agro industrial wastes which are often carelessly discarded into the environment causing health hazards can be utilized as cheap and readily available substrate for the production of alpha amylase hence, it is practicable to rid our environment of these hazardous wastes.  

Keywords:α-Amylase, Aspergillus japonicus, Optimization, Agro industrial wastes,    Environment

CHAPTER ONE

INTRODUCTION

1.1       Background to the Study

Amylases are enzymes that are well known for their applications in starch, food, brewing, distilling, textile, paper and pharmaceutical industries (Gupta et al., 2003; Krishna et al., 2011; Pandey et al., 2000). They are currently utilized in various fields e.g. brewing industries, medicinal, analytical chemistry and food processing (Anto et al., 2006; Chimata et al., 2010; Nimkar et al., 2010). This wide range of applications is the reason for the industrial production of amylase (Khan & Yadav, 2011). Amylases are one of the most important and well-known enzymes that can hydrolyse starch or glycogen (Krishna et al., 2011). They hydrolyse α 1-4 glycosidic bonds of glycogen, amylopectin and other related compounds (Lehninger, 1982). It can be produced by submerged fermentation or solid state fermentation (Egas et al., 1998; Khan & Yadav, 2011; Krishna, 2011). The enzyme is one of the mostly sought after, as it has huge importance in biotechnology; comprising a group of industrial enzymes that controls about 25% of the total enzyme market of the world (Rajagopalam & Krishnan, 2008; Reddy et al., 2003).

Amylases are a group of hydrolases that split the O-glycosidic bonds present in starch thereby breaking starch into simple units (Alva et al., 2007; Crabb & Mitchinson, 1997). They have been reported to be produced by microbial, plant and animal sources, although amylase produced by microorganims has been reported to be most effective (Khan & Yadav, 2011). A wide range of microorganisms, such as bacteria and fungi are utilized in the industrial production of amylases (Krishna et al., 2011). The use of microbes for the production of amylases is economical because microorganisms can be easily manipulated to produce metabolites e.g. enzymes (Aiyer, 2005). However, fungi are preferred over bacteria for enzyme production because of their filamentous nature, which helps in its penetration through solid substrate (Ramachandran et al., 2004).

The synthetic media utilized for the production of amylases are costly and this poses a major challenge to researchers especially in developing countries. Hence, researches are now focused on methods to reduce production cost (Khan & Yadav, 2011). Wastes from agro based industries have been reported to be good and readily available substrates for the cost effective production of α-Amylase (Kirankumar et al., 2011; Pandey et al., 2000). Agrarian nations possess inexhaustible supply of wastes annually generated from their breweries, rice mills, yam flour, plantain and banana chips producing outfits, processing units, and other small industries (Adeniran & Abiose, 2009).