Biochemical Studies on Limonin Biotransformation by Pseudomonas Putida

Abstract

Limonin is a highly oxygenated triterpenoid derivative that is intensely bitter. It comprises of a furan ring and an epoxide group belongs to the class of limonoids. Intact fruit tissues do not contain limonin instead contain non-bitter precursor of limonin, limonoate A-ring lactone (LARL). LARL is found to be endogenously present in membranous sacs. When these sacs get ruptured during juice processing, the LARL encounters the net acidic pH of the juice, which gradually catalyzes closure of the ring to form limonin. This process is known as delayed bitterness. It is an important economic problem in commercial citrus juice production. It not only lowers the quality but also its commercial value. This renders a significant negative economic impact to the citrus industry. To reduce the bitterness in citrus juices below the threshold level for consumers’ acceptability, a number of physicochemical treatments have been devised. A number of column and batch methods using adsorbent and ion exchange resins have been developed to remove the bitter principle i,e limonin from a variety of citrus juices. These methods include adsorptive debittering using polyamides, treatment of juices with ethylene, debittering by passage through Polystyrene-DVB resins and debittering by β-Cyclodextrin. Although these processes effectively remove the bitter constituents from excessively bitter juices, they are of limited use commercially. The delayed bitterness can be overcome either by conversion of bitter limonin to non-bitter compounds or by preventing the formation of limonin precursors through genetic engineering. However, the genetic engineering methods suffer from technical limitations, cannot solve the problem of delayed bitterness. Moreover, the prevention of limonin precursor formation will prevent the formation of other limonoids along with limonin, which are quality constituents of citrus fruits and their juices. This may alter the sensorial properties of citrus juices there by affecting the consumer acceptability.Microorganisms are proven to be versatile biocatalysts by virtue of their metabolic potential. They possess several inducible and constitutive enzymes to carry out biotransformation either by using complex hydrocarbons or limonoids directly as carbon source or via co-metabolism. The bacterial species, that are known so far as limonin metabolizing microorganisms, each one of them are capable of utilizing limonin as carbon source. They are Arthrobacter globiformis, Pseudomonas 321-18 and Bacterium 324-152-1. These species have known to metabolize limonin via 17-dehydrolimonoid and deoxylimonoid pathways. Other isolates are Acinetobacter sp. (degrades limonin via deoxylimonoid pathway), Corynebacterium facians (metabolize limonin via 17-dehydrolimonoid and a trans-19-hydroxyobacunoic acid pathway) and Rhodococcus facians.