α- l-Rhamnosidase: A review

Process Biochemistry 45 (2010) 1226–1235

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Review

␣-l-Rhamnosidase: A review Vinita Yadav, Pramod K. Yadav 1 , Sarita Yadav, K.D.S. Yadav ∗ Department of Chemistry, D.D.U. Gorakhpur University Gorakhpur, Gorakhpur 273009, U.P., India

a r t i c l e

i n f o

Article history: Received 19 January 2010 Received in revised form 13 May 2010 Accepted 21 May 2010 Keywords: ␣-l-Rhamnosidase l-Rhamnose Derhamnosylation Naringin Glycosides Glycosidase

a b s t r a c t ␣-l-Rhamnosidase [E. C. 3.2.1.40] cleaves terminal ␣-l-rhamnose specifically from a large number of natural products. The enzyme has wide occurrence in nature and is reported from animal tissues, plants, yeasts, fungi and bacteria. It is a biotechnologically important enzyme due to its applications in debittering and clearance of citrus fruit juices, enhancement of wine aromas and derhamnosylation of many natural products containing terminal ␣-l-rhamnose to compounds of pharmaceutical interests. Though ␣-l-rhamnosidases have been investigated actively during recent years, there is no recent review on ␣-l-rhamnosidases. An attempt has been made to fill up this gap in this review. It consists of a brief introduction of ␣-l-rhamnosidase which is followed by a critical description of the methods used for assaying the enzyme activity. Purifications, characterizations and properties of ␣-l-rhamnosidases from different sources have been discussed and the available structural and molecular biological studies on the enzyme have been given. Biotechnological applications of this enzyme in different processes have been briefly described. The review concludes with the identification of areas which needs further extensive studies. © 2010 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Assay methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Purification and characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Substrate specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kinetics and mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enzyme inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structural aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular biology aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biotechnological applications of ␣-l-rhamnosidases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction ␣-l-Rhamnosidase [E. C. 3.2.1.40] cleaves terminal ␣-lrhamnose specifically from a large number of natural products which include naringin, rutin, quercitrin, hesperidin, diosgene, ter-

∗ Corresponding author. Tel.: +91 551 2204943; fax: +91 551 2340459. E-mail address: kds [email protected] (K.D.S. Yadav). 1 Present address: Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai 400005, India. 1359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2010.05.025

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penyl glycosides and many other natural glycosides containing terminal ␣-l-rhamnose [1–6] (Reaction Scheme 1). The enzyme has wide occurrence in nature and has been reported from animal tissues, plants, yeasts, fungi and bacteria. This enzyme has turned out to be a biotechnologically important enzyme due to its applications in a variety of processes like debittering of citrus fruit juices [7–13], manufacture of prunin from naringin [14], manufacture of l-rhamnose by hydrolysis of natural glycosides containing terminal l-rhamnose [15], enhancement of wine aromas by enzymatic hydrolysis of terpenyl glycosides containing l-rhamnose [5,16], elimination of

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Reaction Scheme 1. (a) Hydrolysis of naringin to prunin by ␣-l-rhamnosidase. (b) Hydrolysis of prunin to naringenin by ␤-d-glucosidase.

hesperidin crystals from orange juices [3,17], conversion of chloropolysporin B to chloropolysporin C [18], the derhamnosylation of many l-rhamnose containing steroids for example, diosgene, desglucoruscin, ginsenosides-Rg2, etc. whose derhamnosylated products have their clinical importance [4,19–21]. In spite of ␣-l-rhamnosidases being biotechnologically important enzymes, review article on ␣-l-rhamnosidases is not available in the literature. The ␣-l-rhamnosidase activities are associated with debittering enzymes which are commercially known as hesperidinases [20] and naringinases [22]. There are two reviews on naringinase: the one by Chandler and Nicol [23] which covers literature up to 1975 and the other by Puri and Banerjee [24] which covers literature up to 2000. In this article, an attempt has been made to review the recent literature on ␣-l-rhamnosidases. 2. Assay methods A convenient method for assaying the activity of ␣-lrhamnosidase has been a problem before the investigators working on this enzyme since the very beginning. This problem has been solved partly by Romero et al. [22] using synthetic substrate pnitrophenyl-␣-l-rhamnopyranoside and monitoring the liberation of p-nitrophenolate ion spectrophotometrically at 400 nm using molar extinction coefficient value of 21.44 mM−1 cm−1 (Reaction Scheme 2). Though the synthetic substrate, p-nitrophenyl-␣-l-rhamnopyranoside, is commercially available but it is expensive. The method for synthesizing p-nitrophenyl-␣-l-rhamnopyranoside tri-

acetate is available in the literature [25], but it requires expert synthetic organic chemists to control the experimental conditions for its preparation and it also is not convenient for biochemists, microbiologists and biotechnologists working on ␣l-rhamnosidase. Naringin is the most commonly used natural substrate for assaying the activity of ␣-l-rhamnosidase using the Davis method [26]. ␣-l-Rhamnosidase cleaves terminal ␣-l-rhamnose of naringin and converts it to prunin as shown in Reaction Scheme 1(a). If ␤-d-glucosidase activity is present along with ␣-l-rhamnosidase activity, prunin is converted into naringenin and glucose as shown in Reaction Scheme 1(b). Naringenin so produced reacts with the Davis reagent [26] to form much less color than naringin. Samples of the reaction mixture taken at intervals show a continuous decrease in color on reacting with the reagent. This makes it possible to follow the course of naringin hydrolysis. The concentration of naringin at different intervals of time could be determined by drawing calibration curve with the known concentrations of naringin. The difficulty with this method is that it measures the disappearance of either naringin or prunin or both and thus it is not specific for the assay of ␣-l-rhamnosidase activity. However, this is the only method which could be performed conveniently. The ␣-l-rhamnosidase activity could also be determined by monitoring the formation of prunin from naringin as shown in Reaction Scheme 1(a) using HPLC as done by Romero et al. [22] but the method needs modification in the light of the method reported by Yusof et al. [27] in the context of determination of naringin contents in citrus fruits.

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Reaction Scheme 2. Hydrolysis of p-nitrophenyl-␣-l-rhamnopyranoside by ␣-l-rhamnosidase.

The ␣-l-rhamnosidase activity could also be assayed by monitoring the concentration of l-rhamnose liberated by the enzymatic hydrolysis of glycosides containing terminal ␣-l-rhamnose using Nelson and Somogyi colorimetric method [28,29]. However, if other glycosidases are present in ␣-l-rhamnosidase enzyme sample, this method cannot be used. Several investigators have separated lrhamnose using TLC and have quantified it for measuring the activity of l-rhamnosidase [2,4] but the method is inconvenient and time consuming. Use of HPLC with carbohydrate column and RI detector is a possibility but has rarely been used [30]. The activity of ␣-l-rhamnosidase can also be assayed by monitoring the amount of rhamnose liberated from the enzymatic hydrolysis of a natural substrate containing terminal ␣-l-rhamnose using rhamnose dehydrogenase [31] and NAD+ . This method is expensive due to the costs of the enzyme and NAD+ . Thus a convenient method for assaying ␣-l-rhamnosidase using a reasonably priced substrate is not available and the research workers in the most of cases have to develop their own method depending on the nature of the problem and the facilities available to them.

3. Sources Isolation of naringinase, an enzyme complex containing ␣-lrhamnosidase and ␤-d-glucosidase activities, has been reported in the literature as early as 1938 from celery seeds [32]. The presence of the same enzyme has been reported in grape fruit leaves [32–34]. The enzyme with the name rhamnodiastase, a mixture of ␣-l-rhamnosidase and ␤-d-glucosidase, has been reported from Rhamnus dahurica [35]. ␣-l-Rhamnosidase has been studied from the seeds of Fagopyrum esculentum [36]. However, ␣-lrhamnosidases from other plant sources have not been reported so far. The ␣-l-rhamnosidases from only two animal sources, viz. Turbo cornutus liver and pig liver have been reported [37,38]. The human intestine Bacteroid JY-6 and Fusabacterium K-60 have been shown to produce ␣-l-rhamnosidase [39,40]. The production of ␣-l-rhamnosidase by thermophilic anaerobic bacterium Clostridium stercorarium has been reported [41]. Some Pseudoalteromonas species and Ralstonia pickettii, which were obtained from the sea water of sub Antarctic environment, show the ␣-lrhamnosidase activities in the low temperature range of −1 to 8 ◦ C [42]. Sphingomonas paucimobilis and Bacillus spGL1 show substantial ␣-l-rhamnosidase activities in a medium containing gellan as a carbon source [43,44]. Corticium rolfsii produces ␣-l-rhamnosidase which is active at low pH [45]. Two new thermostable ␣-lrhamnosidases from the thermophilic bacterium PRI-1686 have been reported by Birgisson et al. [46]. The ␣-l-rhamnosidases from few Lactobacillus species have been reported [47,48]. Some yeasts like Saccharomyces cerevisiae, Hanshula anomala, Debaryomyces ploymorphus show low level of ␣-l-rhamnosidase activities [49]. However, Pichia angusta X349 is a remarkable producer of ␣-l-rhamnosidase [50]. Though some fungal sources are patented and some are kept secrets by the industries, even then ␣-l-rhamnosidases reported

from fungal sources are abundant in literature [22,23]. Only two commercial preparations of ␣-l-rhamnosidases, naringinase and hesperidinase are available and both are from fungal sources. Hesperidinase is from Aspergillus niger and Penicillium species [20] and naringinase is from Penicillium decumbens [22]. Monti et al. [20] have shown the induction of ␣-l-rhamnosidase production in the fungal strains Acremonium persicinum CCF 1850, Aspergillus aculeatus CCF 108, A. aculeatus CCF 3134, A. aculeatus CCF 3138, A. niger CCIM K2, Aspergillus terreus CCF 3059, Circinella muscae CCF 2417, Emericella nidulans CCF 2912, Eurotium amstelodami CCF 2723, Fusarium oxysporum CCF 906, Mortierella alpina CCF 2514, Mucor circinelloides griseo-cyanus CCIM, Penicillium oxalicum CCF 2430, Rhizopus arrhizus CCF 100, Talaromyces flavus CCF 2686 and Trichoderma harzianum CCF 2687 using l-rhamnose, naringin, rutin, hesperidin as inducers. Shanmugam and Yadav [51] have reported extracellular production of ␣-l-rhamnosidase by Rhizopus nigricans. A number of Aspergillus species have been reported for the production of ␣-l-rhamnosidases [16,52–57]. Feng et al. [58] have reported saponin rhamnosidase from Curvularia lunata. Scaroni et al. [59] have reported some mesophilic fungal strains (viz. Aspergillus flavus, Mucor racemosus, Fusarium sambucinum, Aspergillus kawachii, Penicillium aureatiogriseum, Trichoderma longibrachiatum, Fusarium solani) for the production of ␣-l-rhamnosidases. Hughes et al. [60] have characterized an ␣-lrhamnosidase from the fungal pathogen of oat leaf, Stagonospora avenae which specifically hydrolyses a saponin avenacoside. The different sources of ␣-l-rhamnosidases are summarized in Table 1 along with some properties of the enzymes from different sources. It is obvious from the table that only fungal and bacterial sources of the enzyme have been explored to some extent. Other sources of ␣-l-rhamnosidases have rarely been studied and need extensive studies.

4. Purification and characterization The ␣-l-rhamnosidase from the liver of T. cornutus, a marine gastropod, has been purified to homogeneity using column chromatography with CM cellulose and Sephadex G-150, heat treatment, freezing and thawing in acidic pH [37]. The purification of ␣-l-rhamnosidase from pig liver involved extraction of the enzyme by homogenizing pig liver with buffer, fractional precipitation with ammonium sulphate, dialysis and ion exchange chromatography on DEAE-cellulose [38]. The purification of ␣-lrhamnosidase from the seeds of F. esculentum involved extraction of the crude enzyme, fractionation by ammonium sulphate precipitation and chromatography on columns of Sephadex G-75, DEAE-Sephadex and Ultrogel AcA-44 and has been found to be pure according to the criteria of discgel electrophoresis [61]. The ␣-l-rhamnosidase from the human intestinal bacterium Bacteroides JY-6 has been purified by disrupting the bacterial cells suspended in 20 mM phosphate buffer of pH 7 by ultrasonicator, fractionating the resultant extract by ammonium sulphate precipitation, column chromatography on DEAE-cellulose, SilicaPAE, Sephacryl S-300, and hydroxyapatite, respectively [39]. The ␣-l-rhamnosidase from another human intestinal bacterium

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Table 1 Sources of ␣-l-rhamnosidases along with some properties of the enzyme. S. no.

Source

pH optima

Temperature optima (◦ C)

Fagopyrum esculentum Turbo cornutus Pig Liver Bacteroid JY-6 Fusabacterium K-60 Clostridium stercorarium Pseudoalteromonas species Ralstonia pickettii Bacillus sp GL1 PRI-1686 (RhmA & RhmB)

– 2.8 7 7 5.5 7.5 6.0 – 7 7.9, 5–6.9

– – 42 –

11.

Lactobacillus plantarum NCC 245 (RhaB1 and RhaB2 )

7 and 5

50 and 60

12. 13. 14. 15. 16. 17. 18.

Lactobacillus plantarum Lactobacillus acidophilus Corticium rolfsii Pseudomonas paucimobilis FP2001 Pichia angusta X349 Rhizopus nigricans Aspergillus aculeatus (RhaA and RhaB) Aspergillus aculeatus (pnp-rhamnohydrolase and RG-rhamnohydrolase) Aspergillus nidulans Aspergillus terreus Aspergillus flavus Mucor racemosus Fusarium sambucinum Aspergillus kawachii Penicillium aureatiogriseum Trichoderma longibrachiatum Fusarium solani Curvularia lunata Absidia sp. Aspergillus niger Aspergillus kawachii

7 6 2 7.8 6 6.5 4.5–5.0

– – – 45 40 60–80 –

5.5 and 4

60

4.5–6 5.5 6.5 5.5–6.5 5.5–6.5 4.5 – 4.5–5.5 6.5 4 5 4 4

60 60 50 55–60 55–60 60 60 60 – 50 40 50 50

20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. a b c

pI

Reference

70,000 – 47,000b 120,000b , 240,000a 41,000b , 170,000a

3.7 – – 4.2 5.2

– – 100,000b , c 104,000b , 210,000a and 107,000b , 210,000a 73,000b , 155,000c , a and 57,000b , 100,000c , a – – – 112,000b , a 88,000b , 90,000c – 92,000b and 85,000b 87,000b and 84,000b

– –

[61] [37] [38] [39] [40] [41] [42] [42] [44] [46]

a

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

19.

Molecular weight

60 40 – 50 70

102,000b 89,000b , 97,000a – – – – – – – 66,000b 53,000b 168,000a 90,000b

–

[48]

7.1 4.9 –

[47] [47] [45] [62] [50] [51] [54] [63]

5 – – – – – – – – –

[30] [64] [59] [59] [59] [59] [59] [59] [59] [65] [21] [66] [57]

Molecular weight determined by gel filtration. Molecular weight determined by SDS-PAGE. Molecular weight determined by native-PAGE.

Fusobacterium K-60 has been purified using a method which involved disruption of bacteria by ultrasonicator, fractionation of the protein by ammonium sulphate precipitation and column chromatography on Butyl-Toyopearl, hydroxyapatite, Sephacryl S300 and Q-Sepharose [40]. Hashimoto et al. [44] have purified ␣-l-rhamnosidase of Bacillus sp. GL1 by extracting the enzyme after disrupting the cells by ultrasonication, ammonium sulphate precipitation and column chromatography on DEAE-Sepharose CL6B, Butyl-Toyopearl 650M, Sephacryl S-200HR and QAE-Sephadex A-25. Miake et al. [62] have purified and characterize an intracellular ␣-l-rhamnosidase from Pseudomonas paucimobilis FP2001. The thermo stable ␣-l-rhamnosidase Ram A of C. stercorarium has been purified and characterized by Zverlov et al. [41]. Beekwilder et al. [47] have purified ␣-l-rhamnosidases from Lactobacillus plantarum and Lactobacillus acidophilus. Ávila et al. [48] have over expressed two ␣-l-rhamnosidases genes from L. plantarum NCC 245 in Escherichia coli and have purified it. So far only one ␣-l-rhamnosidase from a yeast source, i.e. P. angusta X349 has been purified to homogeneity using ammonium sulphate precipitation and column chromatography on concanvalin A-Sepharose, DEAE Bio Gel A agarose, Rhamnose-Sepharose 6B and hydroxyapatite [50]. Roitner et al. [6] have tried to characterize ␣-l-rhamnosidase and ␤-d-glucosidase activities from the commercial preparation of naringinase of A. niger origin. By gel filtration, the enzyme complex can be separated into various oligopeptides which are

multiples of the smallest active subunit with molecular mass of 95 kDa. The oligomers show either both enzymatic activities or mere rhamnosidase activity. Protein fractions with glucosidase activity could not be isolated. However, in fractions with rhamnosidase activity only, the glucosidase activity can be restored by immobilization of the enzyme. The glucosidase activity was related to the concentration of protein in the solution, which disappears in very dilute solution where as rhamnosidase was still active. This observation needs further investigation with other ␣-l-rhamnosidase activities. Two ␣-l-rhamnosidases with different substrate specificities have been isolated from a commercial preparation produced by A. aculeatus by Mutter et al. [63]. The first was active towards p-nitrophenyl-␣-l-rhamnopyranoside, naringin and hesperidin. The second ␣-l-rhamnosidase was active towards rhamnogalacturonan (RG fragments) releasing rhamnose. Soria et al. [64] have purified ␣-l-rhamnosidase from the culture filtrate of A. terreus CECT-2663 grown on medium containing either rhamnose or naringin as a carbon source. The purification procedure included ammonium sulphate precipitation, ion exchange chromatography on DEAE-Sepharose CL-6B, gel filtration on Sephadex-G 200. They were successful in separating ␤-glucosidase activity from ␣-l-rhamnosidase activity when rhamnose was inducer. ␣-l-Rhamnosidase from Aspergillus nidulans CECT 2544 has been purified from the culture filtrate of the fungal strain grown on l-rhamnose as the sole carbon source by the combination of batch adsorption on DEAE A-50, two steps of Hi

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Load 16/10 Q Sepharose FF column, Hi Load 26/S Sepharose FF column and finally by gel filtration on Superose 12 HR 10/30 column [30]. Manzanares et al. [54] have purified and characterized two different ␣-l-rhamnosidases RhaA and RhaB from the culture filtrate of A. aculeatus grown on hesperidin using cation exchange and gel filtration chromatography. Purification and characterization of ginsenoside ␣-l-rhamnosidase from fungus Absidia sp. (EECDL-39) has been reported by Yu et al. [21]. The purification involved removal of mycelia from the culture filtrate, ammonium sulphate precipitation and fractionation on Bioscale Q-2 column of BioRad. Feng et al. [65] have purified and characterized a saponin active rhamnosidase from C. lunata using ammonium sulphate precipitation, gel filtration, cation and anion exchange chromatography. Purification and characterization of naringinase from A. niger MTCC 1344 has been reported by Puri and Kalra [66]. The purification involved concentration of the culture filtrate by ultrafiltration, precipitation by ammonium sulphate, ion exchange chromatography on Q Sepharose and gel filtration on Sephadex G-200. Koseki et al. [57] have purified an ␣-l-rhamnosidase from the culture filtrate of A. kawachii grown on l-rhamnose as a sole carbon source using fractional precipitation by ammonium sulphate, HPLC on ion exchange and gel filtration columns. It was found to be thermostable and retained its more than 80% activity at 60 ◦ C for 1 h. The physicochemical properties of the purified ␣-lrhamnosidase are summarized in Table 1. The reported ␣-l-rhamnosidases have pH optima in the range 2.0–8.0. The ␣-l-rhamnosidases having pH optima above pH 8.0 have not been reported. The temperature optima of the reported ␣-lrhamnosidases are in the range of 40–80 ◦ C though one bacterial

␣-l-rhamnosidase active at 4 ◦ C is reported [42]. The relative molecular masses of the reported ␣-l-rhamnosidases are in the range 53.0–240.0 kDa though in some cases oligomeric forms of the enzyme having relative molecular mass as high as 500 kDa have been reported [77].

5. Substrate specificity The structure of commonly used substrates is given in Fig. 1 and the substrate specificities of ␣-l-rhamnosidases purified from different sources are summarized in Table 2, where the available Km values using p-nitrophenyl-␣-l-rhamnoside, naringin, hesperidin, rutin, quercitrin, poncirin, saikosaponin C, proscillaridin A and neohesperidin are given. The substrate specificities of the purified ␣-l-rhamnosidases towards ␣-1, 2; ␣-1, 3; ␣-1, 4 and ␣-1, 6 glucosidic linkages are also mentioned wherever available. The Km values for p-nitrophenyl-␣-l-rhamnoside are in the range 0.057–2.8 mM, for naringin 0.021–1.9 mM, for hesperidin 0.02–1.33 mM, for rutin 0.028–1.44 mM, for quercitrin 0.077–0.89 mM and for poncirin 0.02–0.93 mM. Majority of ␣-lrhamnosidases are active on ␣-1, 2 glucosidic linkages whereas the number of ␣-l-rhamnosidases active on ␣-1, 6 linkages comes second. There are some ␣-l-rhamnosidases active on ␣-1, 4 linkage but ␣-l-rhamnosidases active on other glycosidic linkages are rare. The glucoamylase from C. lunata having steroidal saponin rhamnosidase activity hydrolyses a large number of spirostanoside and furostanoside (the interested readers are referred to Ref. [56]).

Fig. 1. Structure of some commonly used substrates.

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Table 2 Substrate specificity of ␣-l-rhamnosidases. S. no.

Organism

Substrate

Type of linkage

Km value (mM)

Specific activity

Reference

1.

Fagopyrum esculentum

p-Nitrophenyl-␣-l-rhamnoside 6-O-alpha-l-rhamnosyl-d-glucopyranose

␣-1 ␣-1,4

0.33 2.2

– –

[61] [61]

2.

Bacteroides JY-6

p-Nitrophenyl-␣-l-rhamnoside Neohesperidin Naringin Poncirin Hesperidin Rutin Saikosaponin C

␣-1 ␣-1,2 ␣-1,2 ␣-1,2 ␣-1,6 ␣-1,6 ␣-1,4

0.29 0.82 0.89 0.93 1.33 1.44 1.6

162.57 190.21 242.67 174.60 109.31 88.12 2.93

[39] [39] [39] [39] [39] [39] [39]

3.

Pseudomonas paucimobilis FP2001

Hesperidin Proscillaridin A Rutin Naringin Saikosaponin C Quercitrin p-Nitrophenyl-␣-l-rhamnoside

␣-1,6 ␣-1 ␣-1,6 ␣-1,2 ␣-1,4 ␣-1 ␣-1

0.06 0.07 0.13 0.17 0.88 0.89 1.18

0.12 1.48 0.17 0.18 2.52 11.20 92.40

[62] [62] [62] [62] [62] [62] [62]

4.

Fusobacterium K-6

p-Nitrophenyl-␣-l-rhamnoside Quercitrin Hesperidin Naringin Poncirin Rutin

␣-1 ␣-1 ␣-1,6 ␣-1,2 ␣-1,2 ␣-1,6

0.057 0.077 0.022 0.021 0.020 0.028

3.40 5.00 0.52 0.34 0.35 0.07

[40] [40] [40] [40] [40] [40]

5.

Clostridium stercorarium

p-Nitrophenyl-␣-l-rhamnoside Naringin Hesperidin

␣-1 ␣-1,2 ␣-1,6

– – –

82 1.5 0.46

[41] [41] [41]

6.

Bacillus spGL1 (RhaA and RhaB)

p-Nitrophenyl-␣-l-rhamnoside Naringin Gellan

␣-1 ␣-1,2 ␣-1,3

0.119, 0.282 – –

104, 79.4 – –

[77] [77] [77]

7.

Pichia angusta X349

Naringin Rutin Hesperidin Quercitrin

␣-1,2 ␣-1,6 ␣-1,6 ␣-1

– – – –

– – – –

[50] [50] [50] [50]

8. 9. 10. 11.

Absidia sp. Aspergillus niger Aspergillus aculeatus (RhaA and RhaB) Stagonospora avenae

20(S)-Ginsenoside, 20(R)-ginsenoside Naringin p-Nitrophenyl-␣-l-rhamnoside Avenacoside-␣-l-rhamnoside

␣-1,2, ␣-1,2 ␣-1,2 ␣-1 ␣-1,4

– 1.9 0.3, 2.8 0.091

– 21 24, 14 –

[21] [66] [54] [60]

6. Kinetics and mechanism Kinetics and mechanism of ␣-l-rhamnosidase catalyzed reactions have rarely been studied [41,63]. Zverlov et al. [41] have determined the type of enzymatic mechanism of RamA by analyzing the hydrolytic products of p-nitrophenyl-␣-lrhamnopyranoside by the 1 HNMR spectra of the products. The kinetic behaviour has clearly indicated an inverting mechanism of hydrolysis in which ␤-rhamnose was formed from the ␣-rhamnoside via a single displacement mechanism but was spontaneously converted to the ␣-form by mutarotation. The ␣l-rhamnosidase from A. aculeatus also acts as an inverting enzyme [67]. However, further research work on kinetics and mechanisms of reactions catalyzed by ␣-l-rhamnosidases from different sources are required to understand the molecular mechanism of catalysis by this enzyme. 7. Enzyme inhibitors Several investigators have reported inhibition of ␣-lrhamnosidase by l-rhamnose, glucose, citric acid and several metal ions [30,39,50,55,66]. However interest in synthesizing the ␣-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 ␣-l-rhamnosidase inhibitors

which may also inhibit the activities of other glucosidases and may have potential in pharmaceutical industries [68–75]. Researchers in this area also are in early stage and need extensive efforts.

8. Structural aspects The crystal structure of only one ␣-l-rhamnosidase, RhaB from the Bacillus sp. GL1, is available at 1.9 A´˚ resolution [76]. The molecular mass of the enzyme is 106 kDa and it contains 956 amino acid residues. The over all structure is shown in Fig. 2 and consists of five domains designated as N, D1, D2, A and C in order of N-terminal to C-terminal. The secondary elements are shown in Fig. 2(c). Domains N, D1, D2 and C are ␤-sandwiched structure whereas domain A is an (␣/␣) 6-barrel structure. Tertiary structures similar to RhaB have been searched in the protein Data Bank by DALI. It has been found that RhaB shares the significant structural similarity with chitobiose phosphorylase ChBP from Vibrio proteolyticus and maltose phosphorylase MaIP from the Lactobacillus brevis though the primary structure of RhaB is significantly different from the primary structure of these two enzymes. The structure of rhamnose bound RhaB has also been determined at 2.1 A´˚ resolution. Rhamnose binds to the deep cleft of (␣/␣) 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. There is a scientific need to crystallize ␣-l-rhamnosidases

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Fig. 2. Overall structure of RhaB. (a) Forward view of the homodimer structure. (b) Side view of the structure. (c) Topology diagram of RhaB. Cylinder, ␣-helices; arrows, ␤-strands; gray ball, calcium ion; green stick and red ball, glycerol. Red, domain N; blue, domain D1; green, domain D2; yellow, domain A; cyan, domain C [76]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

from other sources and to solve their crystal structures to obtain extensive structural variations in ␣-l-rhamnosidases. 9. Molecular biology aspects Only a few reports on the isolation, cloning and over expression of the gene coding for ␣-l-rhamnosidase are available [41,47,48,54,57,77,78]. The ram A, which belongs to the new type of glycoside hydrolase family, represents the first cloned ␣-l-rhamnosidase gene and it was obtained from the anaerobe thermophilic bacterium [41]. Two genes rhaA and rhaB from A. aculeatus encoding the ␣-l-rhamnosidases RhaA and RhaB respectively were cloned by using polyclonal antibodies [54]. The genes rhaA and rhaB from Bacillus sp. GL1 encoding two different ␣-lrhamnosidases were cloned in E. coli and over expressed. The RhaA of Bacillus sp. GL1 shows 41% sequence identity with RamA of C. stercorarium, 23% identity with RhaB of Bacillus sp. GL1, while RhaB shows only 20% identity with RamA and 24% identity with RhaA and RhaB of A. aculeatus. Only RhaB was produced when Bacillus sp. GL1 cell were grown in gellan medium. The gene ramA, rhaA and rhaB are categorised into family GH 78 [76]. The cloning and expression of gene rhaM encoding ␣-l-rhamnosidase of Sphingomonas paucimibilis FP2001 was done in E. coli. The RhaM protein

showed no significant homology to other ␣-l-rhamnosidases of glycoside hydrolase family 78 [78]. Mutants of RhaB of Bacillus sp. GL1 substituting Asp-567, Glu-572, Asp-579 and Glu-841 with Asn, Gln, Asn and Gln respectively using site directed mutagenesis have been prepared and found to be of reduced enzymatic activity indicating that above amino acids residues are crucial for enzymatic catalysis [76]. The two ␣-l-rhamnosidase genes, rhmA and rhmB were identified in a partially sequenced genome of the bacterium PRI-1686. Whole genes were recovered by amplifying flanking sequences with single specific primers and non-specific walking primers. The recovered genes were then cloned into E. coli [46]. Two putative rhamnosidase genes, ram1LP and ram2LP were identified in the genome of L. plantarum and one rhamnosidase gene ramALA was identified in L. acidophilus genome [47]. The two ␣-l-rhamnosidases genes rhaB1 and rhaB2 were identified in operon rhaP2B2P1B1, was repressed by glucose and induced by lrhamnose, showing regulation at transcription level [48]. Koseki et al. [57] have reported that A. kawachii ␣-l-rhamnosidase encoding gene (Ak-rhaA) encodes for a 655 amino acid protein. The protein possesses 13 potential N-glycosylation recognition sites and exhibits 75% of sequence identity with the ␣-l-rhamnosidases belonging to the glycoside hydrolase family 78 from A. aculeatus and with hypothetical Aspergillus oryzae and Aspergillus fumigatus

V. Yadav et al. / Process Biochemistry 45 (2010) 1226–1235

proteins. So far no attempt has been made to improve the catalytic efficiency of ␣-l-rhamnosidase using the technique of directed evolution or site directed mutagenesis.

10. Biotechnological applications of ␣-l-rhamnosidases Bitterness of citrus fruit juices is due to naringin (4 , 5, 7-trihydroxy flavonone 7-rhamnoglucoside) and limonin. The bitterness due to naringin could be removed by treating the juice with ␣-l-rhamnosidase. The ␣-l-rhamnosidase hydrolyses naringin to prunin and ␣-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 ␣-l-rhamnosidase are patented [79–82] and numerous immobilized ␣-l-rhamnosidase preparations for the debittering of citrus fruit juices have been published [7–13,83–92]. The crystallization of soluble hesperidin in the canned mandarin orange juice causes the turbidity to the juice [93]. The hesperidinase enzyme containing ␣-l-rhamnosidase activity is used to prevent the turbidity of canned orange juice [3]. The ␣-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 [17]. The volatile components such as linalool, geraniol, nerol, citronellol and ␣-terpeniol are responsible for the aroma of wines. However, most of them are present in the grape skin as odourless diglycosides of terpenes, viz. ␣-l-arabinofuranosyl-␤d-glucopyranosides, ␣-l-rhamnopyranosyl-␤-d-glucopyranosides which on the two sequential hydrolysis release volatile terpenol. The immobilized ␣-l-rhamnosidase along with ␤-glucosidase and ␣-arabinosidase has been used for the aroma enhancement in wine [5]. Spagma et al. [16] have shown that purified ␣-l-rhamnosidase from A. niger increases the aroma of model wine solution containing aromatic precursors extracted from muscato grapes. Manzanares et al. [94] have shown that rhaA gene of A. aculeatus that codes ␣l-rhamnosidase when expressed in industrial wine strains along with ␤-d-glucosidase activity, a significant increase in ␣-terpeniol, nerol and linalool were observed in wine indicating the importance of ␣-l-rhamnosidase in aroma the enhancement of wine. Orejas et al. [55] have shown that ␣-l-rhamnosidase activity is only slightly affected by glucose and SO2 and partly inhibited by ethanol indicating a potential use of this enzyme in wine aroma release. The ␣-l-rhamnosidase from P. angusta X349 shows high tolerance towards glucose and ethanol indicating that the enzyme could be used for wine making process [50]. Rhamnose plays the role of chiral intermediate in the organic synthesis of pharmaceutically important agents and plant protective agents. The ␣-l-rhamnosidase cleaves the l-rhamnose from the glycosides that contain terminal l-rhamnose [15,95]. Thus ␣-lrhamnosidase has potential in the manufacture of l-rhamnose. ␣-l-Rhamnosidase can be used in the preparation of many drugs and drug precursors. ␣-l-Rhamnosidase hydrolyses the diosgene (a saponin) to produce ␣-l-rhamnose and diosgenin which is used in the synthesis of clinically useful steroid drugs such as progesterone [19]. ␣-l-Rhamnosidase produced by C. lunata can remove l-rhamnose from a number of steroidal saponins [58]. 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 ␣-l-rhamnosidase on hesperidin has been shown in the laboratory animals [96]. Quercitin-3-glucoside a derhamnosylated product of rutin has been reported to be antioxidant [97]. The ginsenoside-Rh1 obtained by the removal of ␣-l-rhamnose from ginsenosides-Rg2 exhibits anticancer activity [21]. The glycopeptide antibiotic chlorosporin C is obtained by the derhamnosylation of chlorosporin B [18]. Prunin,

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the derhamnosylated product of naringin, has anti-inflammatory and variable activity against DNA/RNA viruses [98]. The enzyme ␣-l-rhamnosidase is used in the structural determination of polysaccharides and glycosides [99–101]. The ␣l-rhamnosidase has been used to produce functional beverages (viz.: Black currant juice, orange juice and green tea infusion) that have increased amount of potentially bioavailable flavonoid glucosides [102]. 11. Conclusion Though ␣-l-rhamnosidases have been studied for more than seven decades, its structural, functional and molecular biology aspects have not been investigated extensively as done in cases of other enzymes [103]. For example, crystal structure of only one ␣-l-rhamnosidase has been determined [76]. Rare attempts have been made to understand the mechanism of the enzyme catalyzed reaction on molecular level [41]. The attempts to use site directed mutagenesis for understanding the molecular mechanism of the catalysis have rarely been made [76]. No attempt has been made to improve the catalytic efficiency of this enzyme using directed evolution techniques [103–105]. The studies in the above directions on ␣-l-rhamnosidases from different sources are needed to understand the function of this biotechnologically important enzyme. Acknowledgements One of the authors, V.Y. is thankful to the Department of Chemistry, D.D.U. University Gorakhpur for the award UGC-DSA research fellowship. S.Y. is thankful to DST, New Delhi for the award of a Women Scientist A fellowship. KDSY acknowledges the support of CSIR New Delhi in the form of Emeritus Scientist. References [1] Habelt K, Pittner F. A rapid method for the determination of naringin, prunin, and naringin applied to the assay of naringinase. Anal Biochem 1983;134:393–7. [2] Victor DB, Cedric HLS, Jeanette W. Hydrolysis of dietary flavonoid glycosides by strains of intestinal bacteroides from humans. Biochem J 1987;248:953–6. [3] Yoshinobu T, Takashi K, Takahisa N, Hiroshi T, Shigetaka O. Prevention of hesperidin crystal formation in canned mandarin orange syrup and clarified orange juice by hesperidin glycosides. Food Sci Technol Int 1995;1:29–33. [4] Feng B, Ma B, Kang L, Xiong C, Wang S. The microbiological transformation of steroidal saponins by Curvularia lunata. Tetrahedron 2005;61:11758–63. [5] Caldini C, Bonomi F, Pifferi PG, Lanzarini G, Galante YM. Kinetic and immobilization studies on the fungal glycosidases for the aroma enhancement in wine. Enzyme Microb Technol 1994;16:286–91. [6] Roitner M, Schalkhammer T, Pittner F. Characterization of naringinase from Aspergillus niger. Monatsh Chem 1984;115:1255–67. [7] Gray GM, Olson AC. Hydrolysis of high levels of naringin in grape juice using a hollow fiber naringinase reactor. J Agric Food Chem 1981;29(6):1299–301. [8] Manjon A, Bastida J, Romero C, Jimeno A, Iborra JL. Immobilization of naringinase on glycophase-coated porous glass. Biotechnol Lett 1985;7(7):477–82. [9] Tsen HY, Tsai SY, Yu GK. Fiber entrapment of naringinase from Penicillium sp. and application to fruit juice debittering. J Ferment Bioeng 1989;67(3):186–9. [10] Tsen HY, Yu GK. Limonin and naringin removal from grapefruit juice with naringinase entrapped in cellulose triacetate fibers. J Food Sci 1991;56(1):31–5. [11] Yadav S, Yadav KDS. Secretion of a-l-rhamnosidases by Aspergillus terreus and its role debittering of orange juice. J Sci Ind Res 2000;59:1032–7. [12] Prakash S, Singhal RS, Kulkarni PR. Enzymic debittering of Indian grapefruit (Citrus paradisi) juice. J Sci Food Agric 2002;82:394–7. [13] Busto MD, Meza V, Mateos NP. Immobilization of naringinase from Aspergillus niger CECT 2088 in poly (vinyl alcohol) cryogels for the debittering of juices. Food Chem 2007;104:1177–82. [14] Roitner M, Schalkhammer T, Pittner F. Preparation of prunin with the help of immobilized naringinase pretreated with alkaline buffer. Appl Biochem Biotechnol 1984;9:483–8. [15] Cheetham PSJ, Quail MA. Process for preparing l-rhamnose. US Patent 5,077,206; 1991. [16] Spagma G, Barbagallo RN, Martino A, Pifferi PG. A simple method of purifying glycosidase: a-l-rhamnopyranosidases from Aspergillus niger to increase the aroma of Moscato wine. Enzyme Microb Technol 2000;27:522–30.

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