Isolation and Characterization of Chitinase Producing Bacteria from Soil and Optimization of Conditions for Enzyme Production

Abstract

Chitinases are enzymes that break down the β-(1→4) -glycoside bond of N-acetyl d-glucosamine in chitin to produce mono- and oligomers. Chitin is the second most abundant biopolymer in nature. The main aim of this thesis was to isolate and characterize bacterial strains from the Hulauka and Taltile river banck soils, and optimization of the conditions for the enzyme production. The soil samples were collected from the study site and serially diluted and spread plated on colloidal chitin media. The isolated bacterial strains were characterized morphologically and biochemically. Ten isolates were selected from 29 total chitinolytic strains for chitinase production based on the zone of clearance on colloidal chitin agar, those ten strains were submitted to MALID-TOF MS and eight species were identified at species level as Rhodococcus marinonascens HSI1, Aeromonas media HSII1, Lilliottia amnigena HSII2, No peaks found HSII3, Pseudomonas congelans HSII4, Ligilactobacillus salivarius HSIII1, Lactobacillus amylovorus HSIII2, Stenotrophomonas maltophilia TSI1 and Pseudomonas putida TSIII3 based on MALID-TOF Biotyper analysis. Furthermore, those eight identified species were optimized at different parameters to select the best conditions for enzyme production. However, optimization of temperature, pH and incubation time in all isolates were showed the highest chitinase enzyme production at 45oC, in pH 7 and 96hr. The effect of carbon source; glucose and lactose were enhanced the production of chitinase enzyme and sucrose were slightly reduced catinase activity. The addition of nitrogen source; yeast extract, beef extract and peptone significantly reduced chetinase enzyme activity. The results of this thesis revealed that bacterial species with high chitinase activities were present in the soils collected from Huluka and Taltale River bank which will be used for mass production of chitinase enzyme for its application in various sector.