Identification and characterization of cellular locus of limonin biotransforming enzyme in Pseudomonas putida

Abstract

Limonin is a highly oxygenated triterpene derivative of class of limonoids which causes delayed bitterness in citrus. Pseudomonas putida (P. putida) is able to reduce 47% and 63% of limonin from juice serum extract and standard limonin, respectively. Biochemical studies with P. putida indicate that probably two metabolic pathways viz. 17-dehydrolimonoid and deoxylimonoid pathway exists in the test organism. Experimental results indicate that the enzyme limonoate dehydrogenase which is found to be localized in periplasmic space of P. putida plays a major role in conversion of limonin to 17-dehydrolimonoate A-ring lactone. Enzymatic studies have shown a 72% reduction in limonin. The experimental results show 9 folds reduction in limonin content in presence of NAD as cofactor. The molecular weight of one of its polypeptide is found to be 66 kDa. The optimum pH and temperature for enzyme activity is 8.5 and 30°C, respectively.

The Chemistry and Pharmacology of Citrus Limonoids

“…The compounds 13-epi-24,25,26,27,30-pentanordammarane and 13-epi-24,25,26,27,30-pentanorlanostane may be considered compound 16's closest parent hydrides (cf. the dammarane (17) and lanostane (18) structures in Figure 2). may be considered compound 16's closest parent hydrides (cf.…”

“…may be considered compound 16's closest parent hydrides (cf. the dammarane (17) and lanostane (18) The term "protolimonoid" [37,41] to indicate reference molecular entities like 16 should be discouraged since properly called protolimonoids (also named protomeliacins or melianes [42]) are (a) naturally occurring compounds; (b) bear an intact (eventually oxygenated) dammarane-like C-17 side chain [43]; and (c) seem to be biogenetically related to limonoids as biochemical precursors [42][43][44], while the synthetic compound 16 [41] that bears a furan ring at position C-17 has not been detected in the plant kingdom.…”

“…Thus, early studies on CLs focused on enhancing the commercial value of fruit juices by employing debittering methods that reduce the content of these secondary metabolites [1]. In the last decade, delayed bitterness has continued to attract interest from several research groups and a number of papers have been dedicated to studies on mechanisms underlying delayed bitterness [10][11][12][13][14], possible ways of reducing [15][16][17][18] or modulating [19][20][21][22][23] the formation of bitter limonoids [1] in citrus fruits and juice products. On the other hand, evaluation of biological activities of CLs have disclosed great potential for these phytochemicals that furnished the rational basis of traditional medicinal uses of CL-containing folk remedies [24][25][26][27][28][29] as well as modern nutraceutical products [29,30].…”

Modeling of permeabilization process in Pseudomonas putida G7 for enhanced limonin bioconversion

“…Upon optimization of the significant variables for permeabilization of P. putida G7, the surface plots helped in the better understanding of the interactions of the significant variables and thus enabled the determination of the optimal level of each variable for maximizing limonin bioconversion. The slightly lower rates of limonin bioconversion by permeabilized cells observed while treating citrus juice may be attributed to the components of the juice (like essential oils) leading to a possible loss of enzyme activity (Verma et al 2010).…”

“…Our earlier studies reported the ability of a Pseudomonas putida G7 to biotransform limonin (Ghosh et al 2006) by virtue of a periplasmically located dehydrogenase which converts limonin to nonbitter derivatives (Verma et al 2010). Recently, we reported that permeabilization could improve the limonin bioconversion potential of P. putida (Malik et al 2011).…”