Production of Chitinase by Trichoderma Virens Ukm1 from Colloidal Chitin and Shrimp Waste

Shrimp waste being the main waste from marine industry is a source of surface pollution in coastal areas consisting of mainly protein, calcium carbonate and chitin. Chitin, the second most abundant biopolymer is a -(1,4)-linked N-acetyl-Dglucosamine (GluNac) heterogeneous polymer that has versatile biological and agrochemical applications. Chitinase a glycosyl hydrolase is produced constitutively as isozymes in fungus for de novo chitin metabolism. Chitin chains are converted into chitooligosaccharides and GluNac reducing sugars by chitinase with specific modes of action at the reducing ends. In this study, shrimp waste was pretreated with chemical and physicochemical methods to determine the best pretreatment before fermentation with a locally isolated fungus, Trichoderma virens UKM1. Experiments in shake flasks and 2 L stirred tank reactor (STR) demonstrated sun dried ground shrimp waste as the best pretreatment, 1 x 106 spores/mL as the best total spore concentration and fermentation pH control at pH 6.0 as the most effective for chitinase production. Subsequent optimisation in 2 L STR showed that fermentation at 200 rpm and 0.33 vvm gave the highest chitinase productivity of 4.1 U/L/h and 5.97 U/L/h, respectively. Microbial chitin bioconversion employing optimal conditions in medium with colloidal chitin and medium with sun dried ground shrimp waste as the sole carbon source showed an increase of 7.25 fold and 1.57 fold in chitinase activity, respectively from shake flasks culture to 2 L STR. The respiration rate (Qo2X) during the highest chitinase productivity was 3.864 mg of DO g-1 of fungal biomass h-1 while the specific respiration rate (Qo2) was 20.337 mg of DO g-1 of fungal biomass h-1 and the maximum specific growth rate, μmax was 0.0078 h-1 with the corresponding doubling time, td of 88.85 hours. Concentration and partial purification of crude chitinase showed that ammonium sulphate precipitation at 80% saturation gave highest chitinase activity in line with the results of enzymatic chitin bioconversion from DNS chitinase assay and HPLC analysis.