Limonoid enzymes

Limonin D-ring lactone hydrolase is the only limonoid enzyme which has been isolated from Citrus and characterized (11). It is of interest to note that this enzyme is extremely heat resistant. It requires 15 min of heating at 100°C to inactivate it completely. Its functional characteristics are very similar to those of the bacterial hydrolase mentioned previously. 

These enzymes attack not only XIV, but other limonoids which contain a furan ring and epoxide, provided that the D-ring is open. The enzymes require sulfhydryl groups and Zn for their catalytic activity. 

Pseudomonas sp. 321-18 (21). Hydrolyzing activity is optimal at pH 8.0, whereas lactonizing activity is optimal at pH 6.0. The enzyme attacks limonoids whose structures differ from I in the vicinity of the A or A -ring. The enzyme does not attack XVI and XVIII. 

Control of Juice Bitterness. A number of advances have been reported in this field since it was last reviewed (3). A commercial application of the cellulose acetate adsorption technique for the removal of limonin from citrus juices was undertaken (49). New sorbent gel forms of cellulose esters for adsorption of limonin were developed (50). Knowledge was gained that limonoids are biosynthesized in citrus leaves and translocated to the fruit (12) and that specific bioregulators can inhibit accumulation of XIV in citrus leaves (15). Additional studies were carried out on the use of neodiosmin to suppress limonin and other types of bitterness (30,51). The influence of extractor and finisher pressures on the level of limonin and naringin in grapefruit juice was reported (34). Also, further studies were conducted on the microbial sources and properties of limonoate dehydrogenase (52), the enzyme that converts XIV to XV and can be used to prevent limonin from forming in freshly expressed citrus juices (53). 

Hasegawa (54) reported on properties of the lactonase. The enzyme purified from grapefruit seeds hydrolyzed limonoids that have the D-ring intact but differ from limonin in the vicinity of the A and A -rings, namely, obacunone, nomilin and ichangin. 

Limonoids Induce protective enzymes Citrus fruits... 

Bitterness, caused by naringin, is also removed by an enzyme, naringinase. Another possibility would be to eliminate one or more of the enzymes in the limonin biosynthetic pathway by using recombinant DNA techniques. Limonin, a triterpenoid, is probably synthesized via the mevalonate pathway, as are the monoterpenoid flavor compounds. It appears that nomilin, a precursor of limonin, is synthesized in the stems and roots of citrus and then the precursor transported to the fruit where it is converted by several enzymes to limonin and other bitter limonoids (46). 

Biological Removal Six species of bacteria, each capable of metabolizing limonoids, have been isolated from soil by enrichment using limonoids as carbon sources. They are Arthrobacter globiformis (34), Pseudomonas 321-18 (35), Arthrobacter globiformis II (36), Bacterium 342-152-1, Corynebacterium fasclans (37) and Aclnetobacter sp. (38). Based on the metabolites and enzymes produced by these species of bacteria, five metabolic pathways of limonoids have been established in bacteria (3)(Fig. 4). 

Limonin and nomilin cause bitter flavor in citrus products. The bitterness is a major problem for the industry. Hasegawa et al. (this volume) have identified three target enzymes involved in limonoid biosynthesis for development of transgenic citrus free of limonin and nomilin bitterness. They are linoleate dehydrogenase, UDP glucose transferase and nomilin deacetylesterase. The isolation of the genes for these enzymes is currently being conducted in order to eventually insert them into cultured citrus cells where they will convert the bitter compounds to non-bitter derivatives. From the cultured cells mature citrus plants will be produced and these plants should produce fhiit free of bitter flavor. 

The enzyme responsible for this conversion has been identified as limonoid UDP-D-glucose transferase (25). Insertion of copies of this gene with altered expression controls, such as constitutive expression, may result in preventing the accumulation of significant levels of any limonoate A-ring lactone at time of harvest. 

Nomilin deacetylase As previously discussed. Citrus ichangensis and its hybrids possess another limonoid biosynthetic pathway from nomilin (3) to deacetylnomilin (12) (26, 27, 31). The conversion of nomilin to deacetylnomilin is catalyzed by an enzyme, nomilin deacetylesterase. The existence of nomilin deacetylase in C. ichangensis has been demonstrated by radioactive tracer work... 

Other possible enzymes. Limonol (9) is a nonbitter limonoid (21). This compound is a minor limonoid and the pathway from limonin to limonol has not been investigated. This enzyme gene may be useful as specific target enzyme gene, but we assume that the activity is very low and it may not be practical to isolate the enzyme from citrus tissues. Deoxylimonin (8) is also a nonbitter limonoid present in Citrus. (20). The conversion of limonin to deoxylimonin requires a multienzyme system. Therefore, it is not practical to utilize this pathway for genetic engineering manipulation. 

This material also contains chromoplast lipids and cytoplasmic materials such as proteins, bioflavanoids, soluble carbohydrates, limonoids and hydrolytic enzymes (5). 

No evidence of limonoid biosynthesis in fruit or seed tissues exists, despite the fact that most of the limonins are found in the seeds of mature fruits. Limonoid synthesis occurs in the leaves and limonoids are transported into the fruits (Maier, 1983). In citrus tissues, the naturally occurring precursor of limonin is a salt of limonoic acid A-ring lactone (60) (Fig. 25.13) in which the A ring is closed and the D ring is open. This tasteless compound is stable only in the salt form (Maier, 1983). In the presence of acid or the enzyme citrus limonoate D-ring hydrolase, the D-ring lacton-izes to form limonin (19). The rate of lactonization is accelerated by pasteurization of the juice. In the fruit, the precursor appears to be located in a compartment of the cell where the pH is neutral or alkaline, probably the cytoplasm (Maier, 1983). 

The antitumor potential of withanolides has been extensively investigated by the researchers all around the world, which led to the identification of diverse properties such as cytotoxicity, cell differentiation induction, cancer chemoprevention, and COX-2 and quinine reductase enzymes inhibition potential of withanolides. Recently, over 80,000 natural and synthetic compoxmds were evaluated by Santagat and coworkers for their anticancer activity, targeting protein homeostasis. They concluded that many active compoxmds were natxxral products belonging to the five classes, limonoids, celastraloids, coUetofiragarones, curvularins, and withanolides [65]. 

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