ABSTRACT
Endoglucanase (EC3.2.1.4) from sorghum (S. bi-color) and millet (Pennisetum typhoides & Digitaria exilis) malts were purified to homogeneity through the methods of ammonium sulphate precipitation and gel filtration. Molecular mass of 35 KDa and 41 KDa were determined by SDS-PAGE. The purified enzymes catalyzed the hydrolysis of carboxy-methylcellulose with optimum activity at pH of 4.8, 5.0, 6.0, and temperature of 60ºC, 60ºC and 70ºC for Digitaria exilis, S. bi-color and Pennisetum typhoides respectively. More than 90% activity was retained in S. bi-color and Pennisetum typhoides and 73% activity in Digitaria exilis after 1.0 hour pre-incubation at 60ºC. Km values of 0.11, 0.09, 0.20 mM and Vmax 17.53, 15.0 and 11.10 U/mg/min were obtained for S. bi-color, Pennisetum typhoides and Digitaria exilis respectively. Co2+ inhibited endoglucanase activity whereas Ca2+, Ba2+, and Zn2+ enhanced enzyme activity. The enzyme was inactivated by glucose, a major end product of cellulose hydrolysis. Results indicate that endoglucanase of S. bi-color and Pennisetum typhoides are more suitable for malting and a blend of the two will produce high quality malt.
ABSTRACT
Aims: To investigate possible use of Glycosylphosphatidylinositol-specific phospholipase C (GPIPLC) as a target protein for the development of vaccine against Trypanosoma brucei brucei infection was investigated. Study Design: GPI-PLC from T. brucei brucei was purified, characterized and the protein was used as antigen in raising antibody against the parasite Place and Duration: Department of Biochemistry, Ahmadu Bello University Zaria-Nigeria, between September 2011 and October 2012 Methodology: GPI-PLC was isolated from T. brucei brucei and purified by ammonium sulphate precipitation, gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). The GPI-PLC was further used to raise antisera in rabbits, which was subsequently used to immunize rats for 14 and 21 days pre-infection to investigate the possible use of T. b. brucei GPI-PLC as target protein in vaccine production against T. b. brucei infection. Results: An overall yield of 48.76% and purification fold of 10.86 were recorded after gel filtration. The result from SDS-PAGE showed the enzyme to be a 39.585 kDa protein with optimum temperature, optimum pH and activation energy to be 35°C, 8.1 and 19.494 kJ/ mol respectively. The Vmax and Km values were 6.67 × 10-3 μmol/hr and 2.67 × 10-3 μM respectively when 212.5 μg of enzyme was used in the reaction mixture. Immunization with anti GPI-PLC for 14 and 21 days pre-infection significantly lowered the Packed Cell Volume (PCV). Result for the time course of parasitemia following infection with 7.9 x 105 Cells/ml showed a decrease in parasitemia level, thus leading to lowering of mortality rates in Groups immunized with GPI-PLC for 14 and 21 days pre-infection by 20% and 40% respectively relative to Group infected but not treated. Conclusion: These results suggest that GPI-PLC as a target protein significantly reduced the progression of the T. b. brucei infection.