Introduction

Microbial amylases are distributed among bacteria, protozoa, algae and fungi(1). The fungal amylases occur mainly in the species of Aspergillus and Rhizopus (2). The use of these microorganisms in the production of enzymes on an industrial scale has obvious advantages, which include the fast rate of multiplication, diversity of enzymes present and the possibility of genetic manipulation(3) . Microbial amylases are used extensively in industrial practice. The alpha-amylases from Bacillus species are used routinely in the brewing, textile and paper industries(4) . In the brewing industries, amylase facilitates the use of high levels of adjuncts such as barley, maize and sorghum(4). It hydrolyses starch to a product of suitable viscosity for coating the surface of paper and in the textile industry it is used for designing purposes(5).

Usually amylase production from fungi has been carried out using well defined chemical media by submerged fermentation (SMF) and solid state fermentation (SSF) in recent times(6). The economics of enzyme production using inexpensive raw materials can make an industrial enzyme process competitive(7) . Maize grains are produced abundantly in the Eastern part of Nigeria especially during the cropping season between April and September. Their high content of starch has prompted the present attempt to use grains as a medium for amylase production from Rhizopus nigricans. The aim of this study therefore, was to explore the use of local and readily available material (mashed maize) to produce amylase from Rhizopus nigricans.

Materials And Methods

Collection Of Samples

Isolation Of

Preparation Of Crude Enzyme

Ammonium Sulphate Precipitation

Desalting On Sephadex G- 25

Batch Elution With Deae – Sephadex A50

Enzyme Assay

Effect Of Substrate Concentration On Growth And Amylase Production

Results and Discussion

Rhizopus nigricans produced a significant quantity of amylase in a very simple medium of boiled mashed maize, as sole source of carbon and ammonium sulphate as the only nitrogen source. Typical growth and enzyme production profiles in the medium showed that amylase production was highest in the mashed maize concentration of 50g/L, while the biomass and protein concentration was 70g/L and 45g/L respectively (Table 1).

Although the higher the mashed maize concentration, the higher the starch concentration, amylase production did not follow that pattern. It can be seen from table 1, also, that beyond 50g/L concentration, amylase production declined. However, protein levels increased slightly with increased mashed maize concentration, up to a maximum value of 70g/L concentration of mashed maize. Similar patterns have been reported(4) during amylase production using Bacillus subtilis. In this study, amylase production occurred at the end of the exponential phase and entering stationary phase. Protein concentration increased in the medium with the secretion of amylase into the medium.

The effect of different concentrations of the ammonium sulphate in the fermentation medium on the growth and enzyme production and hence protein concentration is shown in table 2, while table 3 shows the maximum activities of the amylase, on starches from different cereals grains. The result of this work shows that large scale production of microbial enzyme (amylase) especially from Rhizopus nigricans could be possible using a very cheap local raw material like mashed maize as carbon source. This growth medium if developed, could replace more expensive chemical media.

Figure 1

Table 1: Effect of Substrate concentration on growth and amylase production

Figure 2

Table 2 : Effect of ammonium sulphate concentration on growth of and amylase production at mashed maize concentration of 50g/L

Figure 3

Table 3 : Maximum activity of amylase produced by (growth on mashed maize) on some starch sources