PRODUCTION OF L –ASPARAGINASE USING MARINE ACTINOMYCETES AND ITS EFFECTS ON ACRYLAMIDE CONTENT OF PASTRY FOODS.

ABSTRACT

 Pastry products are the most important staple foods in most countries and cultures. They are a valuable source of nutrients in our diet providing us with most of our food calories and approximately half of our protein requirements. Enzymes such as fungal alpha- amylases have been used in bread making for decades. However, rapid advances in biotechnology have made a number of exciting new enzymes available for the baking industry. The importance of enzyme has increased as consumers demand more natural products free of chemical additives. In baking, L- asparaginase is used to reduce the formation of acrylamide. The aim of this study was to use marine actinomycetes isolates for the production of L-asparaginase and to study its effects on acrylamide content of pastry foods.

 Marine sediments, soil and water samples were collected from 10 m depth from   Lagos lagoon and Atlantic Ocean. Specifically soil samples were collected from the sea shore and   sea depth samples were collected using sterile universal containers. Isolation and enumeration of actinomycetes were   carried out by pour plate method using starch casein agar. Pure cultures of marine actinomycetes were obtained by using the streak plate method. The isolates were screened for L-asparaginase production using rapid plate method and secondary screening was by submerged fermentation.  Production of L-asparaginase was carried out in Erlenmeyer flask containing Czapek Dox broth supplemented with L-asparagine (1g/L), as production medium for 7 days at 120 rpm and at 28 ºC. A 5 % inoculum was used containing 2.5 x10⁵  spores/ml. Enzyme activity was determined by Nesslerization. Optimization of L – asparaginase was carried out by varying different concentration of pH, glucose and nitrogen source. Ammomium sulphate was used to precipitate crude protein.  Dialysis, ion exchange chromatography and gel filtration (size exclusion) chromatography were used to carried out the purification. Quantification was carried out  by calculating the total protein and total crude enzyme at each step of purification and characterization was determined by the maximium velocity, Michealis Menten (Km) value, half life and SDS PAGE. The effect of L-asparaginase on pastries was determined by varying the concentrations of the enzyme before exposing the dough to heat.