T AYOGU, E AMADI
ammonium sulphate., enzyme, fermentation, medium, stagnant water
T AYOGU, E AMADI. Amylase Production by Rhizopus nigricans Using Mashed Maize. The Internet Journal of Microbiology. 2009 Volume 8 Number 1.
Microbial amylases are distributed among bacteria, protozoa, algae and fungi(1). The fungal amylases occur mainly in the species of
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
Materials And Methods
Collection Of Samples
Stagnant water found around the environment of the Institute of Management and Technology was chosen as the source of the 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
Preparation Of Crude Enzyme
The 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 activity. Enzyme recovery from the fermentation broth was by filtration and the filtrate served as the crude enzyme.
Ammonium Sulphate Precipitation
150ml of the crude amylase sample was brought to 70% ammonium sulphate saturation by adding 22.18g of crystalline ammonium sulphate. This was carefully stirred and left in the refrigerator overnight.
Desalting On Sephadex G- 25
A few grams (15g) of sephadex G – 25 were swollen in sufficient amount of 10mM sodium acetate buffer pH 5.0 containing 1mM EDTA and packed into a column (1.20 x 14cm). The samples were loaded onto different columns and eluted with the same buffer.
Batch Elution With Deae – Sephadex A50
The desalted sample was mixed separately with DEAE- Sephadex 50 resin in 50mM tris buffer pH 7.0. The mixture was stirred and centrifuged at 1500 rpm for 30 mins. The supernatant collected was used for enzyme assay.
The method described by Somogyi (9) was used to determine amylase activity. In this method, one unit of amylase activity was defined as one micromole of reducing sugar (as glucose) under the assay condition while protein concentration was determined by the method of Lowry et al.,(10) using albumin as standard. The above tests were repeated twice and mean values recorded.
Effect Of Substrate Concentration On Growth And Amylase Production
The effect of substrate concentration (mashed maize) on the growth and amylase production was determined.
Results and Discussion
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
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