Polaina J., MacCabe A.P. (ed.). Industrial Enzymes. Structure, Function and Applications
Подождите немного. Документ загружается.
-L-RHAMNOSIDASES: OLD AND NEW INSIGHTS 133
can be successfully used to improve the sensory properties of grapefruit juices.
(Soares and Hotchkiss, 1998; Del Nobile et al., 2003).
Since the demonstration that the aromatic components of certain grape varieties
are present in the grape berry both in free form and also bound to sugars in
the form of glycosides, the usefulness of glycosidases for the release of varietal
aromas from precursor compounds during winemaking has been investigated. The
bound aroma fraction comprises glucosides and disaccharide glycosides such as
6-O--L-arabinofuranosyl--D-glucopyranosides, 6-O--L-rhamnopyranosyl--D-
glucopyranosides and 6-O--D-apiofuranosyl--D-glucopyranosides. Compounds
such as terpenols, terpene diols, 2-phenylethanol, benzyl alcohol and C
13
noriso-
prenoids have been shown to be aglycons of such glycosides. Enzymatic hydrolysis
of the aroma precursor compounds requires two sequential reactions: first, an
RHA, an -L-arabinofuranosidase, or a ß-D-apiosidase cleaves the -1,6 glyco-
sidic linkage; subsequently the aroma/flavour compounds are liberated from the
monoglucosides by the action of a ß-D-glucosidase. Since grape and yeast glycosi-
dases seem to be insufficient to process aromatic precursors completely during
winemaking, the addition of exogenous glycosidases during or after the fermentation
is now common practise in wineries. RHA is a component of these commercial
enzymatic preparations and its key role in aroma release has been established.
Contrary to that which is described for debittering processes, in order to develop
the aromatic potential of a wine to the full all glycosidase activities are essential
(for a review see Günata, 2003). Besides exogenous enzyme addition, it is possible
to achieve increases in the content of volatile compounds during vinification by
using recombinant wine yeast strains expressing such hydrolytic activities. A genet-
ically modified industrial wine yeast strain expressing the A. aculeatus rhaA gene
has been constructed and wines produced in microvinifications conducted using a
combination of this strain together with another strain expressing a ß-D-glucosidase
showed increased content mainly of the aromatic compound linalool (Manzanares
et al., 2003).
Although the use of glycosidases to release flavour compounds from glyco-
sidic precursors was initially examined in wines, fruit juice flavour may also be
enhanced by RHA application given the ubiquity of flavour glycoconjugates in
fruits (Günata, 2003).
Since flavonoid glucosides have been reported to be more bioavailable than
their rutinoside (glucose +rhamnose) counterparts (Hollman et al., 1999), both
A. aculeatus RHAs (RhaA and RhaB) have been used to produce functional
beverages based on potentially increased flavonoid bioavailability (González-Barrio
et al., 2004). Incubation of blackcurrant juice, orange juice and green tea infusion
with either RhaA or RhaB resulted in a decrease in the flavonoid rutinoside content
(anthocyanins in blackcurrant juice, flavanones in orange juice and flavonols in
green tea) and a concomitant increase in flavonoid glucosides.
With respect to the manufacture of food additives, RHA could be used in the
preparation of versatile food additives from biopolymers and in the production of
sweeteners. Biopolymers contribute to food quality as gelling agents, thickeners,
134 MANZANARES ET AL.
stabilizers, lubricants, flocculants and flavour enhancers (Giavasis et al., 2000).
Among the biopolymers, the bacterial exopolysaccharide gellan is principally
composed of a tetrasaccharide repeating unit of one rhamnose, one glucuronic
acid, and two glucose units. Various branching chains are attached to the repeating
unit of the polysaccharide and determine the rheological properties of the polymer
(Jansson and Lindberg, 1983). To date however the application of gellan gum has
largely been limited due to its high viscosity. It has been proposed that enzymatic
treatments, among which RHA would play a key role, could be used to prepare
low-viscosity and low molecular weight gellans having novel physiological and
food-technological functions (Hashimoto et al., 1999). As regards sweeteners,
another potential application of RHA is based on its ability to cleave insoluble
hesperidin (see Fig. 1) to rhamnose and hesperetin glucoside, the latter being a
precursor in sweetener production (Sánchez et al., 1987).
4.2. Pharmaceutical Industry
The health-promoting activity of rhamnosides and their derivatives has opened a
broad field for RHA applications since their biological or pharmacological benefits
have been observed to be inversely related to the amount of sugar residues present
in the rhamnoside (Hollman et al., 1999; Chen et al., 2003). Studies investigating
the impact of glycosidase treatments on the enhancement of the biological activities
of rhamnosides are abundant in the literature, and several describe examples where
the action of RHA activity is crucial.
Extracts from the plant Ruscus aculeatus L. are known to possess various
pharmacological properties including anti-inflammatory (Pisvejcová et al., 2003)
and cytostatic (Mimaki et al. , 1998) activities, and are also used in the treatment
of chronic venous insufficiency (Boyle et al., 2003). The steroid saponins ruscin
and ruscoside and their derivatives desglucoruscin, desglucodesrhamnoruscin and
desglucoruscoside are the compounds that possess these properties. Biotechno-
logical production of desglucoruscin and desglucodesrhamnoruscin (Fig. 4A) as
well as new derivatives can be achieved employing an RHA activity (Di Lazzaro
et al., 2001; Acquati and Ponzone, 2000).
Similar studies have been carried out with the triterpenoid saponin ginsenosides
and gypenosides, the physiologically active compounds of some oriental herbs (Yu
et al., 2004). The removal of one rhamnose residue (Fig. 4B) converts gypenoside-5
into ginsenoside Rd that prevents kidney injury by anti-cancer drugs (Yokozawa
and Owada, 1999).
Chloropolysporins A, B and C and their partially deglycosylated derivatives,
new members of the glycopeptide antibiotic family, are active against Gram-
positive bacteria, including clinically isolated methicillin-resistant Staphylococci
and anaerobic enterobacteria. Derhamnosyl derivatives showing stronger activities
than the parent compounds have been enzymatically obtained by treatment with
RHA (Takatsu et al., 1987a,b).
-L-RHAMNOSIDASES: OLD AND NEW INSIGHTS 135
Rha - Glc
Glc - Glc
Glc - Glc
Desglucoruscin
OH
O
Ara
Desglucodesrhamnoruscin
Rha - Ara
R
R
R
R
A
B
O
O
Gypenoside-5
Ginsenoside Rd
O
O
Glc
OH
O
O
O
OH
OH
Figure 4. Chemical structures of desglucoruscin (A) and gypenoside-5 (B) and their RHA conversion
products. Arrows indicate the possible linkages hydrolysed by these enzymes
4.3. Chemical Industry
The application of RHAs in the chemical industry is related to the design
of low-cost processes for the production of valuable compounds. Among these,
L-rhamnose has gained importance in recent years as both a precursor for the indus-
trial production of aromatic compounds and flavours and as a chiral compound
for chemical synthesis. As L-rhamnose is not biosynthesised as a free monomer it
must be liberated from L-rhamnose-containing glycosides or polysaccharides, but
this option is limited by the availability of suitable raw material. In this context,
an integrated microbial/enzymatic process for the production of rhamnolipids and
L-rhamnose from rapeseed oil has been developed (Trummler et al., 2003). The
process is aimed at improving the yield of L-rhamnose rather than rhamnolipids.
This concept combines microbial rhamnolipid production by a Pseudomonas strain,
with simultaneous enzymatic hydrolysis of rhamnolipid products in the same
bioreactor.
Processes for the production of pure anthocyanidin glucosides from blackcurrant
anthocyanidin rutinosides as well as the obtention of an isoquercitrin-enriched
product from rutin by RHA treatments have been recently patented (Matsumoto
et al., 2002; Chang and Muir, 2004).
Due to the importance of rhamnosides, glycosylation catalysed by RHAs has
been suggested as a way to produce pure rhamnosides in a single step. In this
sense, a process has been proposed for the enzymatic synthesis of short chain length
alkyl--L-rhamnosides using rhamnose or naringin as the glycosylation agents and
water soluble alcohols as acceptors (Martearena et al., 2003).
136 MANZANARES ET AL.
5. FUTURE PERSPECTIVES
In comparison to other glycosidases, knowledge of the molecular and structural
characteristics of RHAs as well as an understanding of their biological function
is still scarce. Nevertheless the increasing importance of RHA is reflected in the
number of studies focused on possible applications. Since the first characterisation
of an RHA encoding gene in 2000 more RHA genes are now available, and
the application of DNA recombinant techniques for the overproduction of pure
enzyme preparations and the modification of RHA stability, selectivity or speci-
ficity are now feasible. These techniques will considerably extend the scope of
potential applications and will convert RHAs into an important industrial enzyme
in the near future.
ACKNOWLEDGEMENTS
Work in the authors’ laboratories was supported by CICYT grants AGL2002-
01906, AGL2004-00978 and AGL2005-02542 (Spanish Ministry of Science and
Education-FEDER).
REFERENCES
Acquati, W. and Ponzone, C. (2000) Preparation of desglucodesrhamnoruscin by hydrolysis of Ruscus
aculeatus steroid glycosides by fermentation in the presence of Aspergillus niger. Patent number
WO200073489-A.
Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. (1997)
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic
Acid Res. 25, 3389–3402.
Bader, G., Wray, V., Just, U. and Hiller, K. (1998) Enzymatic hydrolysis of the cytotoxic glycoside
virgaureasaponin 1. Phytochem. 49, 153–156.
Bae, E.-A., Shin, J.-E. and Kim, D.-H. (2005) Metabolism of ginsenoside Re by human intestinal
microflora and its estrogenic effect. Biol. Pharm. Bull. 28, 1903–1908.
Bar-Peled, M., Lewinsohn, E., Fluhr, R. and Gressel, J. (1991) UDP-rhamnose-flavanone-7-O-glucoside-
2”-O-rhamnosyltransferase-purification and characterization of an enzyme catalysing the production
of bitter compounds in citrus. J. Biol. Chem. 226, 20953–20959.
Barker, S.A., Somers, P.J. and Stacey, M. (1965) Arrangement of the L-rhamnose units in Diplococcus
pneumoniae type II polysaccharide. Carbohydr. Res. 1, 106–115.
Benavente-García, O., Castillo, J., Marín, F.R., Ortuño, A. and Del Río, J.A. (1997) Uses and properties
of Citrus flavonoids. J. Agric. Food Chem. 45, 4505–4515.
Birgisson, H., Hreggvidsson, G.O., Fridjónsson, O.H., Mort, A., Kristjánsson, J.K. and Mattiasson, B.
(2004) Two new thermostable -L-rhamnosidases from a novel thermophilic bacterium. Enzyme
Microb. Technol. 34, 561–571.
Bokkenheuser, V.D., Shackleton, C.H.L. and Winter, J. (1987) Hydrolysis of dietary flavonoid glycosides
by strains of intestinal Bacteroides from humans. Biochem. J. 248, 953–956.
Bourbouze, R., Percheron, F. and Courtois, J.E. (1976) -L-Rhamnosidase from Fagopyrum esculentum:
purification and some properties. Eur. J. Biochem. 63, 331–337.
Boyle, P., Diehm, C. and Roberston, C. (2003) Meta-analysis of clinical trials of Cyclo 3 Fort in the
treatment of chronic venous insufficiency. Int. Angiol. 22, 250–262.
-L-RHAMNOSIDASES: OLD AND NEW INSIGHTS 137
Chang, P.R. and Muir, A. (2004) Isoquercitrin-enriched composition prepared by incubating solution
having rutin after adding naringinase, terminating the incubation, the proportion of isoquercitrin in
composition being controlled by adjusting duration of incubation. Patent Number WO2004027074-A2.
Chen, Y.-C., Shen, S.-C. and Lin, H.-Y. (2003) Rutinoside at C7 attenuates the apoptosis-inducing
activity of flavonoids. Biochem. Pharmacol. 66, 1139–1150.
Chua, J.E.H., Manning, P.A. and Morona, R. (1999) The Shigella flexneri bacteriophage Sf6 tailspike
protein (TSP)/endorhamnosidase is related to the bacteriophage P22 TSP and has a motif common to
exo- and endoglycanases, and C-5 epimerases. Microbiol. 145, 1649–1659.
Coutinho, P.M. and Henrissat, B. (1999) Carbohydrate-active Enzymes server at URL http://afmb.
cnrs-mrs.fr/CAZY/.
Davies, G. and Henrissat, B. (1995) Structures and mechanisms of glycosyl hydrolases. Structure 3,
853–859.
Del Nobile, M.A., Piergiovanni, L., Buonocore, G.G., Fava, P., Puglisi, M.L. and Nicolais, L. (2003)
Naringinase immobilization in polymeric films intended for food packaging applications. J. Food Sci.
68, 2046–2049.
Deng, L., Kasper, D.L., Krick, T.P. and Wessels, M.R. (2000) Characterization of the linkage between
the type III capsular polysaccharide and the bacterial cell wall of group B Streptococcus. J. Biol.
Chem. 275, 7497–7504.
Di Lazzaro, A., Morana, A., Schiraldi, C., Martino, A., Ponzone, C. and De Rosa, M. (2001) An
enzymatic process for the production of the pharmacologically active glycoside desglucodesrhamnor-
uscin from Ruscus aculeatus L.J. Mol. Catal. B.-Enzym. 11, 307–314.
Friedman, M. and McDonald, G.M. (1997) Potato glycoalkaloids: chemistry, analysis, safety, and plant
physiology. Crit. Rev. Plant Sci. 16, 55–132.
Gallego, M.V., Piñaga, F., Ramón, D. and Vallés, S. (1996) Production and characterization of an
Aspergillus terreus -L-rhamnosidase of oenological interest. Z. Lebensm.-Unters.-Forsch. 203,
522–527.
Gallego, M.V., Piñaga, F., Ramón, D. and Vallés, S. (2001) Purification and characterization of an
Aspergillus terreus -L-rhamnosidase of interest in winemaking. J. Food Sci. 66, 204–209.
Giavasis, I., Harvey, L.M. and McNeil, B. (2000) Gellan gum. Crit. Rev. Biotech. 20, 177–211.
Grimaldi, A., Bartowsky, E. and Jiranek, V. (2005) A survey of glycosidase activities of commercial
wine strains of Oenococcus oeni. Int. J. Food Microbiol. 105, 233–244.
González-Barrio, R., Trindade, L.M., Manzanares, P., de Graaff, L. H., Tomás-Barberán, A. and
Espín, J.C. (2004) Production of bioavailable flavonoid glucosides in fruit juices and green tea by use
of fungal -L-rhamnosidases. J. Agric. Food Chem. 52, 6136–6142.
Günata, Z. (2003) Flavor enhancement in fruit juices and derived beverages by exogenous glycosidases
and consequences of the use of enzyme preparations. In Handbook of Food Enzymology.
Whitaker, J.R., Voragen, A.G.J. and Wong, D.W.S. eds. Marcel Dekker Inc., New York, pp 303–330.
Günata, Y.Z., Bayonove, C.L., Baumes, R.L. and Cordonnier, R.E. (1985) The aroma of grapes.
I. Extraction and determination of free and glycosidically bound fractions of some grape aroma
components. J. Chromatogr. 331, 83–90.
Haruko, U. and Haruko, O. (1999) Glycobiology of the plant glycoprotein epitope: structure, immuno-
genicity and allergenicity of plant glycotopes. Trends Glycosci. Glycotech. 11, 413–428.
Hashimoto, W., Miyake, O., Nankai, H. and Murata, K. (2003) Molecular identification of an
-L-rhamnosidase from Bacillus sp. strain GL1 as an enzyme involved in complete metabolism of
gellan. Arch. Biochem. Biophys. 15, 235–244.
Hashimoto, W. and Murata, K. (1998) -L-Rhamnosidase of Sphingomonas sp. R1 producing an unusual
exopolysaccharide of sphingan. Biosci. Biotechnol. Biochem. 62, 1068–1074.
Hashimoto, W., Nankai, H., Sato, N., Kawai, S. and Murata, K. (1999) Characterization of
-L-rhamnosidase of Bacillus sp. GL1 responsible for the complete depolymerization of gellan. Arch.
Biochem. Biophys. 368, 56–60.
Henrissat, B. (1991) A classification of glycosyl hydrolases based on amino acid similarities. Biochem.
J. 280, 309–316.
138 MANZANARES ET AL.
Higgings, D., Thompson, J. and Gibson, T. (1996) Using CLUSTAL for multiple sequence alignments.
Methods Enzymol. 266, 383–402.
Hollman, P.C., Bijsman, M.N., van Gameren, Y., Cnossen, E.P., de Vries, J.P. and Katan, M.B. (1999)
The sugar moiety is a major determinant of the absorption of dietary flavonoid glycosides in man.
Free Radical Res. 31, 569–573.
Hughes, H.B., Morrissey, J.P. and Osbourn, A.E. (2004) Characterisation of the saponin hydrolysing
enzyme avenacoside--L-rhamnosidase from the fungal pathogen of cereals, Stagonospora avenae.
Eur. J. Plant. Pathol. 110, 421–427.
Jang, I.-S. and Kim, D.-H. (1996) Purification and characterization of -L-rhamnosidase from
Bacteroides JY-6, a human intestinal bacterium. Biol. Pharm. Bull. 19, 1546–1549.
Jansson, P.-E. and Lindberg, B. (1983) Structural studies of gellan gum, an extracellular polysaccharide
elaborated by Pseudomona elodea. Carbohydr. Res. 124, 135–139.
Kaji, A. and Ichimi, T. (1973) -L-Rhamnosidase activity in culture filtrate of Corticium rolfsii. Enzymic
activity at low pH. Agr. Biol. Chem. 37, 431–432.
Kim, J.H., Curtis-Long, M.J., Seo, W.D., Lee, J.H., Lee, B.W., Yoon, Y.J., Kang, K.Y. and Park, K.H.
(2005) -L-Rhamnosidase inhibitory activities of polyhydroxylated pyrrolidine. Bioorg. Med. Chem.
Lett. 15, 4282–4285.
Kumar, S., Tamura, K. and Nei, N. (2004) MEGA3: Integrated software for molecular evolutionary
genetics analysis and sequence alignment. Brief Bioinform. 5, 150–63.
Kurosawa, Y., Ikeda, K. and Egami, F. (1973) -L-Rhamnosidases of the liver of Turbo cornutus and
Aspergillus niger. J. Biochem. 73, 31–37.
Manzanares, P., de Graaff, L.H. and Visser, J. (1997) Purification and characterization of an
-L-rhamnosidase from Aspergillus niger. FEMS Microbiol. Lett. 157, 279–283.
Manzanares, P., Orejas, M., Gil, J.V., de Graaff, L.H., Visser, J. and Ramón, D. (2003)
Construction of a genetically modified wine yeast strain expressing the Aspergillus aculeatus
rhaA gene, encoding an -L-rhamnosidase of enological interest. Appl. Environ. Microbiol. 69,
7558–7562.
Manzanares, P., Orejas, M., Ibáñez, E., Vallés, S. and Ramón, D. (2000) Purification and characterization
of an -L-rhamnosidase from Aspergillus nidulans. Lett. Appl. Microbiol. 31, 198–202.
Manzanares, P., van den Broeck, H., de Graaff, L.H. and Visser, J. (2001) Purification and charac-
terization of two different -L-rhamnosidases, RhaA and RhaB, from Aspergillus aculeatus. Appl.
Environ. Microbiol. 67, 2230–2234.
Martearena, M.R., Blanco, S. and Ellenrieder, G. (2003) Synthesis of alkyl--L-rhamnosides by water
soluble alcohols enzymatic glycosylation. Bioresource Technol. 90, 297–303.
Matsumoto, H., Hanamura, S. and Hirayama, M. (2002) Enzymatic production of purified anthocyanins
and crystalline products from natural resources, for use in (functional) foods, beverages, drugs and
cosmetics. Patent Number WO200222847-A.
McMahon, H., Zoecklein, B.W., Fugelsang, K. and Jasinski, Y. (1999) Quantification of glycosidase
activity in selected yeasts and lactic acid bacteria. J. Ind. Microbiol. Biotechnol. 23, 198–203.
Miake, F., Murata, K., Kuroiwa, A., Kumamoto, T., Kuroda, S., Terasawa, T., Tone, H. and Watanabe,
K. (1995) Characterization of Pseudomonas paucimobilis FP2001 which forms flagella depending
upon the presence of rhamnose in liquid medium. Microbiol. Immunol. 39, 437–442.
Miake, F., Satho, T., Takesue, H., Yanagida, F., Kashige, N. and Watanabe, K. (2000) Purification and
characterization of intracellular -L-rhamnosidase from Pseudomonas paucimobilis FP2001. Arch.
Microbiol. 173, 65–70.
Miklosy, E. and Polos, V. (1995) Yeasts with -D-glucosidase activity: properties and posible aplication
in winemaking processes. Acta Alimentaria 24, 167–180.
Mimaki, Y., Kuroda, M., Kameyama, A., Yokosuka, A. and Sashida, Y. (1998) New steroidal constituents
of the underground parts of Ruscus aculeatus and their cytostatic activity on HL-60 cells. Chem.
Pharm. Bull. 46, 298–303.
Miyata, T., Kashige, N., Satho, T., Yamaguchi, T., Aso, Y. and Miake F. (2005) Cloning, sequence
analysis, and expression of the gene encoding Sphingomonas paucimobilis FP2001 -L-rhamnosidase.
Curr. Microbiol. 51, 105–109.
-L-RHAMNOSIDASES: OLD AND NEW INSIGHTS 139
Monti, D., Pisvejcová, A., Kren, V., Lama, M. and Riva, S. (2004) Generation of an -L-rhamnosidase
library and its application for the selective derhamnosylation of natural products. Biotechnol. Bioeng.
87, 763–771.
Morrissey, J.P., Wubben, J.P. and Osbourn, A.E. (2000) Stagonospora avenae secretes multiple enzymes
that hydrolyse oat leaf saponins. Mol. Plant Microbe Interact. 13, 1041–1052.
Mutter, M., Beldman, G., Schols, H.A. and Voragen, A.G.J. (1994) Rhamnogalacturonan
-L-rhamnopyranohydrolase. A novel enzyme specific for the terminal nonreducing rhamnosyl unit
in rhamnogalacturonan regions of pectin. Plant Physiol. 106, 241–250.
Norouzian, D., Hosseinzadeh, A., Inanlou, D.N. and Moazami, N. (1999) Various techniques used
to immobilize naringinase produced by Penicillium decumbens PTCC 5248. World J. Microbiol.
Biotechnol. 15, 501–502.
Ochsner, U.A., Fiechter, A. and Reiser, J. (1994) Isolation, characterization, and expression in
Escherichia coli of the Pseudomonas aeruginosa rhlAB genes encoding a rhamnosyltransferase
involved in rhamnolipid biosurfactant synthesis. J. Biol. Chem. 31, 19787–19795.
Oda, Y., Saito, K., Ohara-Takada, A. and Mori, M. (2002) Hydrolysis of the potato glycoalkaloid
-chaconine by filamentous fungi. J. Biosci. Bioeng. 94, 321–325.
Orejas, M., Ibáñez, E. and Ramón, D. (1999) The filamentous fungus Aspergillus nidulans produces an
-L-rhamnosidase of potential oenological interest. Lett. Appl. Microbiol. 28, 383–388.
Osbourn, A.E. (1996a) Saponins and plant defense-a soap history. Trends Plant Sci. 1, 4–9.
Osbourn, A.E. (1996b) Preformed antimicrobial compounds and plant defense against fungal attack.
Plant Cell 8, 1821–1831.
Pearson, W.R. (1990) Rapid and sensitive sequence comparison with FASTP and FASTA. Methods
Enzymol. 183, 63–98.
Pisvejcová, A., Monti, D., Sedmera, P., Tarabiono, C., Lama, M., Riva, S. and Kren, V. (2003)
-L-rhamnosidases and their use in selective trimming of natural compounds. 12th European Carbo-
hydrate Symposium. Grenoble, France, July 6–11. PC022.
Prakash, S., Singhal, R.S. and Kulkarni, P.R. (2002) Enzymic debittering of Indian grapefruit (Citrus
paradis) juice. J. Sci. Food Agric. 82, 394–397.
Puri, M., Kaur, H. and Kennedy, J.F. (2005) Covalent immobilization of naringinase for the transfor-
mation of a flavonoid. J. Chem. Technol. Biotechnol. 80, 1160–1165.
Puri, M., Marwaha, S.S., Kothari, R.M. and Kennedy, J.F. (1996) Biochemical basis of bitterness in
citrus fruit juices and biotech approaches for debbitering. Crit. Rev. Biotechnol. 16, 145–155.
Qian, S., Yu, H., Zhang, C., Lu, M., Wang, H. and Jin, F. (2005) Purification and characterization of
dioscin--L-rhamnosidase from pig liver. Chem. Pharm. Bull. 53, 911–914.
Renault, J.-H., Thepenier, P., Zeches-Hanrot, M., Le Men-Olivier, L., Durand, A., Foucault, A. and
Margraft, R. (1997) Preparative separation of anthocyanins by gradient elution centrifugal partition
chromatography. J. Chromatogr. A 763, 345–352.
Ridley, B.L., O’Neill, M.A. and Mohnen, D. (2001) Pectins: structure, biosynthesis and oligogalacturonide-
related signalling. Phytochem. 57, 929–967.
Rodríguez, M.E., Lopes, C.A., van Broock, M., Vallés, S., Ramón, D. and Caballero, A.C. (2004)
Screening and typing of patagonian wine yeasts for glycosidase activities. J. Appl. Microbiol. 96, 84–95.
Romero, C., Manjón, A., Bastida, J. and Iborra, J.L. (1985) A method for assaying the rhamnosidase
activity of naringinase. Anal. Biochem. 149, 566–571.
Sánchez, M.A., Romero, C., Manjón, A. and Iborra, J.L. (1987) Activity of soluble and immobilized
hesperidinase on insoluble hesperidin. Biotechnol. Lett. 9, 871–874.
Scaroni, E., Cuevas, C., Carrillo, L. and Ellenrieder, G. (2002) Hydrolytic properties of crude
-L-rhamnosidases produced by several wild strains of mesophilic fungi. Lett. Appl. Microbiol. 34,
461–465.
Soares, N.F.F. and Hotchkiss, J.H. (1998) Naringinase immobilization in packaging films for reducing
naringin concentration in grapefruit juice. J. Food Sci. 63, 61–65.
Steinbacher, S., Seckler, R., Miller, S., Steipe, B., Huber, R. and Reinemer, P. (1994) Crystal
structure of P22 tailspike protein: interdigitated subunits in a thermostable trimer. Science 265,
383–386.
140 MANZANARES ET AL.
Suzuki, H. (1962) Hydrolysis of flavonoid glycosides by enzymes: rhamnodiastase from Rhamnus and
other sources. Arch. Biochem. Biophys. 99, 476–483.
Takatsu, T., Takahashi, S., Takamatsu, Y., Shioiri, T., Iwado, S. and Haneishi, T. (1987a) Chloropoly-
sporins A, B and C, novel glycopeptide antibiotics from Faenia interjecta p.nov. IV. Partially
deglycosylated derivatives. J. Antibiot. (Tokyo) 40, 941–945.
Takatsu, T., Katayama, T., Nakajima, M., Takahashi, S., Haneishi, T., Magaribuchi, T. and Tajima, M.
(1987b) Chloropolysporins A, B and C, novel glycopeptide antibiotics from Faenia interjecta p.nov.
V. Comparative studies of the biological properties. J. Antibiot. (Tokyo) 40, 946–952.
Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of
progressive multiple sequence alignment through sequence weighting, position-specific gap penalties
and weight matrix choice. Nucleic Acids Res. 22, 4673–4680.
Trummler, K., Effenberger, F. and Syldatk, C. (2003) An integrated microbial/enzymatic process for
production of rhamnolipids and L-(+)-rhamnose from rapeseed oil with Pseudomonas sp. DSM 2847.
Eur. J. Lipid Sci. Technol. 105, 563–571.
de Vries, R.P., van Grieken, C., vanKuyk, P.A. and Wösten, H.A. B. (2005) The value of genome
sequences in the rapid identification of novel genes encoding specific plant cell wall degrading
enzymes. Curr. Genom. 6, 157–187.
Yanai, T. and Sato, M. (2000) Purification and characterization of an -L-rhamnosidase from Pichia
angusta X349. Biosci. Biotechnol. Biochem. 64, 2179–2185.
Yokozawa, T. and Owada, S. (1999) Effects of ginsenoside Rd in cephaloride-induced renal disorder.
Nephron 81, 200–207.
Young, N.M., Johnston, R.A.Z. and Richards, J.C. (1989) Purification of the -L-rhamnosidase of
Penicillium decumbens and characteristics of two glycopeptide components. Carbohydr. Res. 191,
53–62.
Yu, H., Gong, J., Zhang, C. and Jin, F. (2002) Purification and characterization of ginsenoside--L-
rhamnosidase. Chem. Pharm. Bull. 50, 175–178.
Yu, H., Liu, H., Zhang, C., Tan, D., Lu, M. and Jin, F. (2004) Purification and characterization of
gypenoside--L-rhamnosidase hydrolysing gypenoside-5 into ginsenoside Rd. Process Biochem. 39,
861–867.
Zverlov, V.V., Hertel, C., Bronnenmeier, K., Hroch, A., Kellermann, J. and Schwarz, W.H. (2000)
The thermostable -L-rhamnosidase RamA of Clostridium stercorarium: biochemical characterization
and primary structure of a bacterial -L-rhamnoside hydrolase, a new type of inverting glycoside
hydrolase. Mol. Microbiol. 35, 173–179.
CHAPTER 9
APPLICATION OF GLYCOSIDASES
AND TRANSGLYCOSIDASES IN THE SYNTHESIS
OF OLIGOSACCHARIDES
FRANCISCO J. PLOU, ARÁNZAZU GÓMEZ DE SEGURA
AND ANTONIO BALLESTEROS
∗
Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, CSIC, Cantoblanco,
Madrid, Spain
∗
a.ballesteros@icp.csic.es
1. INTRODUCTION
It is generally accepted that an oligosaccharide is a carbohydrate consisting of 2–10
monosaccharide residues linked by O-glycosidic bonds (Eggleston and Cote, 2003;
McNaught, 1997). The development of efficient and scalable processes for the
synthesis of oligosaccharides is of considerable interest to the food and pharmaceu-
tical industries (Kren and Thiem, 1997; Macmillan and Daines, 2003).
Oligosaccharides are quite complex molecules: 3 different hexopyranose moieties
yield up to 720 trisaccharides. For the preparation of structurally well-defined
oligosaccharides, the stereo- and regio-specificities of enzymes are very attractive
properties compared to chemical processes that require complex protection and de-
protection steps. At present, enzymatic processes are preferred in industry for the
production of most commercial oligosaccharides.
The synthesis of glycosidic bonds in vivo is performed by glycosyltransferases
(EC 2.4.) (Ichikawa et al., 1992). These enzymes catalyse the transfer of a glycosyl
donor to an acceptor molecule forming a new glycosidic bond regio- and stereo-
specifically. According to the nature of the sugar residue being transferred, glyco-
syltransferases are divided into hexosyltransferases (EC 2.4.1.), pentosyltransferases
(EC 2.4.2.), and those transferring other glycosyl groups (EC 2.4.99.). Depending
on the nature of the donor molecule, glycosyltransferases are classified into three
main mechanistic groups: (1) Leloir-type glycosyltransferases, which require sugar
nucleotides (e.g. UDP-glucosyltransferases); (2) non-Leloir glycosyltransferases,
which use sugar-1-phosphates (e.g. phosphorylases); and (3) transglycosidases,
141
J. Polaina and A.P. MacCabe (eds.), Industrial Enzymes, 141–157.
© 2007 Springer.
142 PLOU ET AL.
which employ non-activated sugars such as sucrose, lactose or starch. A distinctive
feature of transglycosidases, compared with Leloir and non-Leloir glycosyltrans-
ferases, is that they also display some hydrolytic activity that can be regarded as a
transfer of a glycosyl group from the donor to water.
In addition to glycosyltransferases, under appropriate conditions glycosidases
(glycoside hydrolases, EC 3.2) can also be used for in vitro synthesis of oligosac-
charides. Glycosidases catalyse the hydrolysis of glycosidic bonds in oligo- and
polysaccharides with a high degree of stereospecificity. It is noteworthy that, in
terms of reaction mechanism, transglycosidases and glycosidases belong to the same
group. In fact, transglycosidases and glycosidases are grouped in the ‘glycoside
hydrolase family (GH family)’ in the Henrissat classification, which is based on
amino acid sequence comparisons (Coutinho and Henrissat, 1999). The GH family
comprises more than 2500 enzymes.
Several reviews on oligosaccharide synthesis by glycosyltransferases of the
Leloir and non-Leloir types have been published recently (Daines et al., 2004;
Hamilton, 2004). The main problems associated with such syntheses are (1) the
requirement for sugar nucleotides or sugar phosphates as substrates, (2) the
inhibitory effect of the nucleotide phosphate released, and (3) the limited avail-
ability of these enzymes. Nevertheless, continuous progress in the study of these
enzymes, the cloning of new variants, and the application of molecular evolution
and site-directed mutagenesis for better performance is improving their potential
for use in oligosaccharide synthesis (Planas and Faijes, 2002). In this chapter we
will focus on the use of transglycosidases and glycosidases for the synthesis of
oligosaccharides because they both employ the same type of substrate and share
the same mechanism of glycosylation (Sanz-Aparicio et al., 1998).
2. SYNTHESIS OF OLIGOSACCHARIDES BY GLYCOSIDASES
Glycosidases are classified as being either retaining (e.g. -galactosidase) or
inverting (e.g. trehalase) because enzymatic hydrolysis of glycoside bonds can
proceed with the net retention or net inversion of the anomeric configuration, respec-
tively. Glycosidases are widely employed for oligosaccharide synthesis because,
under appropriate conditions, the normal hydrolytic reaction can be reversed towards
glycosidic bond synthesis (Ajisaka and Yamamoto, 2002; Scigelova et al., 1999).
Most glycosidases used for synthetic purposes are retaining glycosidases, and
in particular exo-glycosidases. The glycosyl donor can be a monosaccharide, an
oligosaccharide or an activated glycoside (Thiem, 1995). Glycosidase-catalysed
oligosaccharide synthesis can be controlled by thermodynamic and kinetic factors
as explained below.
2.1. Thermodynamic Synthesis (Reverse Hydrolysis)
In thermodynamically controlled synthesis (i.e. the reaction takes place until it
reaches equilibrium), the reaction can proceed between two monosaccharides