EVALUATIONOF LINAMARASE ACTIVITIES OF MICROBIAL ISOLATES FROM CASSAVA(MANIHOT ESCULENTA) PEELS

Abstract

The blanched and un-blanched cassava peels were fermented for 96 hours at room temperature 28±2oC. The microorganisms isolated were identified using standard microbiological and biochemical methods. Screening of microbial isolates for linamarase production were done using de Mann Rogosa Sharpe modified medium containing 1% para-nitrophenyl β-D glucoside. Eleven bacterial and five fungal isolates were screened for linamarase production in submerged fermentation. The screened isolates showed variation in their enzyme activities. Linamarase production by the isolates was detected by the development of black colour around the perimeter of injection of the inoculum in the modified de Mann Rogosa Sharpe agar medium after incubation at 30oC. The process parameters were optimized using the best linamarase-producing bacterium. Partial purification of crude extract from Lactobacillus plantarum – the best linamarase producing bacterium was carried out by ammonium sulphate precipitation, dialysis, ion exchange chromatography (DEAE-Saphadex A-50) and gel filtration (Saphadex G-150). Further fractionation of the enzyme on ion exchange with Sephadex G-150 yielded one activity peak. A pH of 6.0 was optimum for purified linamarase activity and relatively stable between 20 and 60 minutes of incubation at pH 7.0, 10.0 and 12.0 (neutral to alkaline medium). The optimum temperature for linamarase activity was 50oC with 85% relative activity. The linamarase was relatively thermostable at 70oC and 80oC between 40 to 60 minutes after which a slight decrease in enzyme activity was observed. The apparent Michaelis-Menten constant (Km) for the hydrolysis of para-nitrophenyl β-D glucoside from Lineweaver-Bulk plot was 2.27 mg/ml, while maximum velocity (Vmax) was 294.12 μmol/min/ml. The incubation of Ca2+ and Mg2+ (metal ions) at 10mM caused the enhancement of enzyme activity, while varied degree of linamarase inhibition was exhibited by Zn2+, Fe2+ and Cu2+. The molecular weight of the purified linamarase was approximately 28kDa. The naturally fermented and mono-culture fermented cassava peels, showed increased crude protein, ash and fat content while carbohydrate content and crude fibre decreased. The mono-culture fermented peels showed significant reduction (p<0.05) in cyanide contents when compared with naturally fermented peels. However, the moisture content of the naturally fermented sample was lower when compared with the inoculated sample. The use of linamarase-producing bacterium in fermentation of cassava peels resulted in degradation of cyanide with increase in protein content. The findings revealed the wide range of linamarase activity exhibited by microbial isolate under the cultural conditions of pH 3.0 to 9.0 and moderate temperature of 30oC to 70oC. However, the bacterium Lactobacillus plantarum could be explored in food industry as a source of enzyme where extreme conditions are required. Therefore, an increase in the nutritive value of the mono-culture fermented cassava peels with linamarase-producing isolate suggest that it could be harnessed as sole based diet in animal feed formulation