Molecular and Physiological Studies on Pseudomonas Putida

Abstract

Limonin, a highly oxygenated triterpene derivative, has special significance in citrus industries because of its intense bitterness and adverse affects on citrus juice quality. Its degradation or elimination contributes to juice debittering. The bitterness due to limonin develops gradually in juices after their extraction (an effect known as delayed bitterness) India holds third rank in respect of production of citrus fruits in the world. The commercial cultivation of citrus is concentrated in the northern, eastern and western parts of India. Several methods to control limonin bitterness from citrus products have been proposed. The application of microorganisms as a tool for debittering takes advantage of the existence of isolated enzymes or whole bacterial cells capable of metabolizing limonin. The potential of one such putative microorganism, which is a strain of Pseudomonas putida, was exploited in the present study. The test organism was found to have the ability to utilize limonin by virtue of its metabolism. Therefore development of physiological and molecular insights on this strain of Psudomonas putida may offer a promising solution to problem of debittering. In the present study P.putida strain was observed to be metabolically versatile and is capable of tolerating all concentrations of limonin despite of the fact that limonin exhibits antimicrobial properties Moreover, it can tolerate the toxic effect of acetonitrile to some extent. While utilizing limonin as sole source of carbon for its growth and energy, 64% of limonin gets reduced and final biomass of accumulated was 6.5logCFU/ml units in 36 hours in batch cultures. Studies were undertaken to detect presence of plasmids in the test organism and characterize the role of plasmid in limonin degradation. P.putida harbors a single and large plasmid of size of approximately ~80kb and are present in low copy number. The plasmid belongs to class of nah plasmids that encodes for naphthalene oxidative enzymes. Curing results have established that plasmid do not confer genes for limonin degradation.