Amylase Production by Rhizopus nigricans Using Mashed Maize

摘要

Rhizopus nigricans isolated from stagnant water was studied as a source of amylase using boiled mashed maize (yellow variety) as sole source of carbon, while ammonium sulphate was used as nitrogen source. Concentrations of mashed maize and ammonium sulphate were 50g/l and 20g/l respectively. These levels were optimal for growth and amylase production at 30oC and pH 6.0. Partial purification of the enzyme by salting, desalting and elution using DEAE-Sephadex A 50 were carried out to increase enzyme activity. Amylase activities using some native starches as test substrates showed a maximum of 7.0U/L for rice starch, followed by potato starch 6.5U/L. Mashed maize, therefore, could serve as sole source of carbon for amylase production by Rhizopus nigricans. 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 SamplesStagnant water found around the environment of the Institute of Management and Technology was chosen as the source of the isolation of Rhizopus nigricans. 10ml of the stagnant water samples were collected in sterile bottles and taken to the laboratory for analysis.Isolation Of The starch medium was produced by the method described by Higashiara and Okada(8) with slight modification. An increased quantity of ammonium sulphate was added to the medium and subsequently used for the isolation of Rhizopus. Pure culture of the isolate was obtained after several cycles of growth. Identification and characterization of the Rhizopus species was based on morphological characteristics of plate and slide cultures. Prepared samples were observed under the light microscope and identified. Positive cultures were screened for amylase production using various concentrations of the mashed maize and ammonium sulphate, as sole sources of carbon and nitrogen respectively and sugar production was tested by the method of Somogyi(9). Preparation Of Crude EnzymeThe medium composition for amylase production contained 50g/L boiled mashed maize (yellow variety) and 2.0g/L ammonium sulphate (NH4)2 SO4 as nitrogen source, adjusted to pH 6.0 and sterilized by autoclaving. Fermentation was carried out in 500ml flasks with a working volume of 250ml at 30oC in a rotary shaker at 100rpm for 72h. The fermentation was started with pure culture 10% (v/v) inoculum (107 spores/ml) of 24h growth suspension of the organism, obtained from the starch medium.Periodical analysis of samples taken was carried out for amylase acti