Studies on production of 6 amino penicillanic acid by using locally isolated different free and immobilized soil microbes

Abstract

Penicillin is the first and still one of the most widely used antibiotic agents, derived from the Penicillum mold. The activity of penicillin is associated with the and#946;-lactam ring within its molecular structure. 6-Amino penicillanic acid (6-APA) is an important commercial intermediate in the industrial production of and#946;-lactam antibiotics such as ampicillin, amoxicillin, cloxacillin, sulbactum. 6-APA can also be ring expanded to 7-amino desacetoxy cephalosporanic acid (7-ADCA) to generate some important orally active cephalosporins such as cephalexin, cephadroxil and cephadrin. Penicillin acylase (EC 3.5.1.11) was discovered in microorganisms that can hydrolyze penicillin G and release 6-APA under mild conditions. newlinePresent study was undertaken to isolate the Soil bacteria from the different locations of Dibrugarh and screened for the penicillin acylase enzyme. Different methods was used for the screening of penicillin acylase in isolated bacteria such as phenol red indicator for the quick determination of penicillin G breakdown, assay of Penicillin acylase activity by flurorescamine reagent, TLC determination of 6-APA, detection of and#946;-lactamase by rapid iodometric test and HPLC assay for the presence of 6-APA. Six isolated bacteria coded as AT-1, AT-2, AT-3, AT-4, AT-5 and AT-6 show the positive result for the presence of penicillin acylase. newlineThe enzyme activity of isolated bacteria was determined by the p-dimethylamino benzaldehyde method and the parameters such as pH, temperature, substrate concentration in the reaction mixture and pH, temperature, effect of inducer in the fermentation media, age of the culture were optimized to get maximum enzyme activity. After optimization of all the parameters AT-3 strain was found to show the maximum enzyme activity. newlineThe bacterial strains AT-1, AT-2, AT-3 and AT-4 were immobilized by using alginate and carrageenan matrix. Enzyme activity was evaluated to determine the effect of pH and temperature on immobilized bacteria and it was found that immobilized cells have better stability as compared to correspondent free cells. The reusability of immobilized cells upto 8 cycles was observed. This is the main advantage of immobilization. The net activity of immobilized cells was high as compared to corresponding free cells. Alginate was found the better matrix as compared to carrageenan. newlineThe results of Morphological and biochemical tests showed that AT-1, AT-3, AT-5 and AT-6 were Pseudomonas sp., AT-2 was Bacillus sp. and AT-4 was E. coli. The 16S rRNA gene sequencing results of Strain AT-1 and AT-3 showed that both of the strains were of Pseudomonas aeruginosa. newline newline