No abstract

In an attempt to enhance the industrial production of α-amylases in the tropics, sterile fresh bread was inoculated with spore suspensions of Penicillium citrinum at 25 o C. Extracellular α-amylases were produced and subjected to partial purification by ammonium sulphate precipitation and dialysis. Further purification by gel filtration and ion-exchange chromatography was engaged. The molecular weights of the α-amylase fractions obtained and estimated by gel filtration using Sephadex G-100 were approximately 56,234 Daltons, 53,089 Daltons and 11,885 Daltons. The apparent Michalis-Menten constant (K m) values for the hydrolysis of starch by the purified α-amylase fractions were approximately 8.3 mg/ml, 10 mg/ml and 7.14 mg/ml respectively. Optimum activities were at 30 o C for one of the fractions and 35 o C for the other two fractions and were at pH 5.5 and pH 6.0. The activities of the α-amylase fractions produced by the fungus were stimulated at varying degrees by NaCl, KCl, CaCl 2 and MgCl 2 but inhibited by Ethylene Diamine Tetraacetic Acid (EDTA), mercuric chloride (HgCl 2) and 2,4-dinitrophenol (DNP). The α-amylase fractions were sensitive to heat, losing all their activities within twenty minutes of heating at 80 o C. The industrial production of α-amylases should be encouraged in the tropics using bread as a cheap source of substrate.

Lignocellulose ethanol promises to be the cheapest form of fuel, however, the drawback in the production is in the pretreatment process to remove lignin and the efficient hydrolysis of free cellulose. This research work is designed to delignify sugarcane bagasse, hydrolyze and ferment it with immobilized cellulase from the snail gut isolates and yeast respectively. The biomass were pretreated with Ca(OH) and then placed in the 2 0 0 0 0 0 0 water-bath with temperature of 20 C, 40 C, 60 C, 80 C,100 C and 120 C. The pretreated 0 biomass was hydrolysed with free and immobilized cellulase at 50 C for 5-48hrs. The activity, optimum pH, optimum temperature, substrate concentration profile and kinetic parameters, V and K of cellulase were also determined. The optimum pH for free and max m immobilized cellulase ranged from 4.0-5.5 and optimum temperature was recorded at 0 0 45 C and 55 C for free and immobilized cellulase respectively. The effect of temperature on both free and immobilized cellulases showed that immobilized cellulase has higher resistance to temperature than the free cellulase. Also the yield of glucose (40mg/ml) was higher with immobilized enzyme after 24hrs. The results obtained has also shown that immobilized cellulase has a higher Km when compared with free cellulase The maximum reaction rate (V) obtained from Michaelis Menten plots was lower for max immobilized cellulase than for the free enzyme. Higher value of V for free enzyme max indicated that the enzyme converted more substrate to product per unit time upon saturation with substrate. The biomass was fermented for 48hrs with immobilized Saccharomyces cerevisiae and the results showed the ethanol yield of 31.75% at 24hrs and 70.84% at 48hrs. The initial glucose concentration was 40mg/ml and this significantly reduced to 6.21mg/ml after 24hrs and 1.25mg/ml after 48hrs of the fermentation process. These results showed a proportional increase in ethanol yield against a depleting concentration of glucose which is being used up in the fermentation reaction revealing the maximum efficiency of the immobilized yeast cells. In this study, it has shown that the entrapped cellulase cells produced high levels of reducing sugars in hydrolysis compared with their native counterparts and immobilized yeast cells also gave a high yield of ethanol. The immobilization process therefore obtained more thermostable biocatalysts with increased productivity which is more economical for biofuel production.