Author(s): Sreepad Jois1, Shilpachandru1,Ashajyothi chavan1, RamachandraYarapaLakshmikantha1*and Padmalatha RaiSatwadi2
Water sample microbes were isolated and screened for the potential producers of L-asparaginase using modified M-9 mediumplate method. The single discrete colonies which have exhibited clear pink zone surrounding microbial colonies after 48 hours incubation indicate L-asparaginase producing cultures. This can easily be detected by the change in pH of the medium using phenol red. These colonies (L-Asparaginase positive cultures) were picked up and grown in the modified Czapeck Dox agar. E coliand Pseudomonas aeruginosaare optimised for comparative study and are inoculated in modified M-9 medium as production media. The largest quantity of L-asparaginase was produced when pseudomonasaeruginosawas grown in the modified M-9 medium for 52 hrs. Enzyme activity and specificity was calculated for all the crude and purified samples of the two different organisms. The maximum enzyme activity and specific activity was observed in Pseudomonas aeruginosaby using L-Aspargine as substrate. The activity of the Pseudomonasincreased to 602 U/g as compare to specific activity of L-asparaginase from E coli520 U/g. The molecular weight of the protein was found to be 75KDa. Characterisation of the enzyme showed optimum pH 7.0 for both E coliand Pseudomonas aeruginosa. Optimum temperature was 550C.