RhamA

α-L-Rhamnosidase (E.C. 3.2.1.40) specifically cleaves terminal α-L-rhamnose from a large number of natural products. The enzyme has wide occurrence in nature and widely exists in animal tissues, plants, yeasts, fungi and bacteria. Because of its application in the debittering and clearance of citrus juice, the enhancement of wine aroma and the derhamnosylation of many natural products containing terminal α-L-rhamnose, it is an important enzyme of biotechnological significance.

Sources

In plants, an enzyme complex named naringinase, containing α-L-rhamnosidase and β-D-glucosidase activities, has been isolated from celery seeds and grape fruit leaves. The enzyme with the name rhamnodiastase, a mixture of α-L-rhamnosidase and β-D-glucosidase, has been reported from Rhamnus dahurica. α-L-Rhamnosidase has been studied from the seeds of Fagopyrum esculentum. α-L-Rhamnosidase comes from only two animal sources, namely Turbo cornutus liver and pig liver.

The human intestine Bacteroid JY-6 and Fusabacterium K-60 have been shown to produce α-L-rhamnosidase. The production of α-L-rhamnosidase by thermophilic anaerobic bacterium Clostridium stercorarium has been reported. Some Pseudoalteromonas species and Ralstonia pickettii, which were obtained from the sea water of sub Antarctic environment, show the α-L-rhamnosidase activities in the low temperature range of −1 to 8 °C. Sphingomonas paucimobilis and Bacillus spGL1 show substantial α-L-rhamnosidase activities in amedium containing gellan as a carbon source. Corticium rolfsii produces α-L-rhamnosidase which is active at low pH. Some yeasts like Saccharomyces cerevisiae, Hanshula anomala, Debaryomyces ploymorphus show low level of α-L-rhamnosidase activities. However, Pichia angusta X349 is a remarkable producer of α-L-rhamnosidase.

Structure

The molecular mass of the enzyme is 106 kDa and it contains 956 amino acid residues. The enzyme consists of five domains designated as N, D1, D2, A and C in order of N-terminal to C-terminal. Domains N, D1, D2 and C are β-sandwiched structure whereas domain A is an (α/α) 6-barrel structure. Rhamnose binds to the deep cleft of (α/α) 6-barrel domain. Several negatively charged residues such as Asp567, Glu572, Asp579 and Glu841 interact with rhamnose and RhaB mutants of these residues drastically reduced the enzyme activity indicating that these residues are crucial for the enzyme catalysis and the substrate binding.

Figure 1. Structure of the homodimer structure of α-L-Rhamnosidase. (Yadav V, et al. 2010)

Inhibitors

Several investigators have reported inhibition of α-L-rhamnosidase by L-rhamnose, glucose, citric acid and several metal ions. However interest in synthesizing the α-L-rhamnosidase inhibitors has emerged due to the finding that certain compounds of this type have displayed activity against the human immunodeficiency virus and it is thought that activity of such compounds may lie in their ability to inhibit glucosidase impairing processing of viral glycoprotein. There are a few research papers aimed at synthesizing potent α-L-rhamnosidase inhibitors which may also inhibit the activities of other glucosidases and may have potential in pharmaceutical industries.

Application

Bitterness of citrus fruit juices is due to naringin and limonin. The bitterness due to naringin could be removed by treating the juice with α-L-rhamnosidase. The α-L-rhamnosidase hydrolyses naringin to prunin and α-L-rhamnose. The bitterness of prunin is only one third that of naringin. A number of processes for debittering of citrus fruit juices based on α-L-rhamnosidase are patented and numerous immobilized α-L-rhamnosidase preparations for the debittering of citrus fruit juices have been published. The crystallization of soluble hesperidin in the canned mandarin orange juice causes the turbidity to the juice. The hesperidinase enzyme containing α-L-rhamnosidase activity is used to prevent the turbidity of canned orange juice. The α-L-rhamnosidase treated hesperidin and hesperidin glycosides are highly soluble in water and are free from crystal precipitation even when stored for long period of time.

The volatile components such as linalool, geraniol, nerol, citronellol and α-terpeniol are responsible for the aroma of wines. However, most of them are present in the grape skin as odourless diglycosides of terpenes, viz. α-L-arabinofuranosyl-β-D-glucopyranosides, α-L-rhamnopyranosyl-β-D-glucopyranosides which on the two sequential hydrolysis release volatile terpenol. The immobilized α-L-rhamnosidase along with β-glucosidase and α-arabinosidase has been used for the aroma enhancement in wine. The α-L-rhamnosidase from P. angusta X349 shows high tolerance towards glucose and ethanol indicating that the enzyme could be used for wine making process.

α-L-Rhamnosidase can be used in the preparation of many drugs and drug precursors. α-L-Rhamnosidase hydrolyses the diosgene (a saponin) to produce α-L-rhamnose and diosgenin which is used in the synthesis of clinically useful steroid drugs such as progesterone. α-L-Rhamnosidase produced by C. lunata can remove L-rhamnose from a number of steroidal saponins. Quercitin is a flavanol which is obtained by the derhamnosylation of quercitrin. It exhibits antioxidative, anticarcinogenic, anti-inflammatory, antiaggregatory and vasodeilating effects. The anticarcinogenic activity of hesperitin which is obtained by the action of α-L-rhamnosidase on hesperidin has been shown in the laboratory animals. Quercitin-3-glucoside a derhamnosylated product of rutin has been reported to be antioxidant. The ginsenoside-Rh1 obtained by the removal of α-L -rhamnose from ginsenosides-Rg2 exhibits anticancer activity. The glycopeptide antibiotic chlorosporin C is obtained by the derhamnosylation of chlorosporin B. Prunin, the derhamnosylated product of naringin, has anti-inflammatory and variable activity against DNA/RNA viruses.

Reference

1. Yadav V, Yadav P K, Yadav S, et al. α-L-Rhamnosidase: a review. Process Biochemistry, 2010, 45(8): 1226-1235.