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CHAPTER 8

-L-RHAMNOSIDASES: OLD AND NEW INSIGHTS

PALOMA MANZANARES

1∗

, SALVADOR VALLÉS

, DANIEL RAMÓN

12

AND MARGARITA OREJAS

Departamento de Biotecnología de Alimentos, Instituto de Agroquímica y Tecnología de Alimentos,

CSIC, Paterna, Valencia, Spain and

Departamento de Medicina Preventiva y Salud Pública, Ciencias de los Alimentos, Toxicología y

Medicina Legal, Universitat de València, Valencia, Spain

∗

pmanz@iata.csic.es

1. INTRODUCTION

L-Rhamnose is a component of plant cell wall pectic polysaccharides (Mutter

et al., 1994; Ridley et al., 2001), glycoproteins (Haruko and Haruko, 1999) and

secondary metabolites such as anthocyanins (Renault et al., 1997), flavonoids

(Bar-Peled et al., 1991) and triterpenoids (Friedman and McDonald, 1997). It has

also been found in bacterial heteropolysaccharides (Hashimoto and Murata, 1998),

rhamnolipids (Ochsner et al., 1994) and in the repeating units of the O-antigen

structure of the lipopolysaccharide component of bacterial outer membranes (Chua

et al., 1999). Some rhamnosides are important bioactive compounds, e.g. cytotoxic

saponins (Bader et al., 1998; Yu et al., 2002), antifungal plant glycoalkaloids

(Oda et al., 2002) and bacterial virulence factors (Deng et al., 2000). In plants

L-rhamnose-containing terpenyl glycosides are important aroma precursors (Günata

et al., 1985) and may also play a protective role against the toxicity of free

aglycons; L-rhamnose-containing flavonoid glycosides have antioxidant and anti-

inflammatory activities (Benavente-García et al., 1997).

-L-rhamnosidases (EC 3.2.1.40) and -L-rhamnosidases (EC 3.2.1.43) catalyse

the hydrolysis of terminal, non-reducing L-rhamnose residues in - and

-L-rhamnosides respectively. In contrast, endorhamnosidases (EC 3.2.1.-) act by

cleaving specific linkages between internal rhamnose residues in rhamnosides.

-L-rhamnosidases have been found in many micro-organisms and in some plant

and animal tissues (see below), whereas -L-rhamnosidase has only been described

117

J. Polaina and A.P. MacCabe (eds.), Industrial Enzymes, 117–140.

118 MANZANARES ET AL.

in Klebsiella aerogenes (Barker et al., 1965). Endorhamnosidases seem to be

restricted to bacteriophages (Steinbacher et al., 1994; Chua et al., 1999). This

chapter will mainly focus on the -L-rhamnosidases (RHAs) as a group of

hydrolytic enzymes having crucial biological functions and important potential

biotechnological applications.

In 1991 a classification of glycoside hydrolases based on amino acid

sequence similarities was introduced (Henrissat, 1991). This classification, which

is regularly updated, now comprises more than 100 sequence-based families

(URL://http://afmb.cnrs-mrs.fr/CAZY/). The RHAs, with the exception of that of

Sphingomonas paucimobilis that has recently been assigned to family 106, belong to

family 78 (Coutinho and Henrissat, 1999). Currently, 61 different putative RHAs

from bacteria, yeasts and moulds are known. Genome-sequencing projects, partic-

ularly those focussing on bacterial, fungal and plant genomes, are beginning to

generate large numbers of potential RHA sequences, and a very recent search of

the NCBI database (http://www.ncbi.nlm.nih.gov) yielded more than 100 entries

for putative RHA encoding genes. Endorhamnosidases, with the exception of that

of bacteriophage Sf6 which has not been classified, are placed in family 90.

RHAs are inverting glycoside hydrolases. Enzymatic hydrolysis of the glyco-

sidic bond takes place via general acid catalysis that requires two critical residues:

a proton donor and a nucleophile/base (Davies and Henrissat, 1995). These residues

are as yet unidentified in RHAs. In general, inverting glycosyl hydrolases (GH)

typically employ two side chain carboxylates (supplied by Asp or Glu) in the active

site to effect catalysis.

RHAs have recently been the focus of several research initiatives because of

their key roles in fundamental biological processes (e.g. detoxification mechanisms,

symbiosis) and utility in biotechnological applications (e.g. elucidation of the struc-

tures of biologically important glycosides, biomass conversion, beverage quality

enhancement and the manufacture of hydrolysis products from natural glycosides).

2. BIOCHEMICAL PROPERTIES AND STRUCTURE

2.1. Sources and Biochemical Characteristics of -L-Rhamnosidases

Although micro-organisms are the main sources of RHAs, these enzymes have

also been found in animal tissues such as the liver of the marine gastropod

Turbo cornutus (Kurosawa et al., 1973) and pig (Qian et al., 2005), and plants

such as Rhamnus daurica (Suzuki, 1962) and Fagopyrum esculentum (Bourbouze

et al., 1976). Given the sparseness of reports on animal and plant RHAs, data

presented hereafter are mainly focused on microbial enzymes.

RHAs seem to be common in filamentous fungi as revealed by recent screenings

that have identified different strains of Acremonium, Aspergillus, Circinella,

Eurotium, Fusarium, Mortierella, Mucor, Penicillium, Rhizopus, Talaromyces and

Trichoderma as RHA producers (Scaroni et al., 2002; Monti et al., 2004).

The presence of RHA activity in phytopathogenic fungi was first described

-L-RHAMNOSIDASES: OLD AND NEW INSIGHTS 119

in Corticium rolfsii (Kaji and Ichimi, 1973) and more recently in Stagonospora

avenae, a cereal pathogen (Morrissey et al., 2000; Hughes et al., 2004). In

addition, highly specific RHAs from Absidia sp. (Yu et al., 2002, 2004) and

Plectosphaerella cucumerina (Oda et al., 2002) which are able to degrade different

saponins have been purified and characterized. Nevertheless, biochemical charac-

terization of fungal RHAs has been carried out mainly on enzymes purified from

culture filtrates and on commercial enzyme preparations from Aspergillus species

such as A. terreus (Gallego et al., 1996; 2001), A. nidulans (Orejas et al., 1999;

Manzanares et al., 2000), A. niger (Kurosawa et al., 1973; Manzanares et al., 1997)

and A. aculetaus (Mutter et al., 1994; Manzanares et al., 2001, 2003), and also

on commercial preparations of Penicillium species (Romero et al., 1985; Young

et al., 1989).

Regarding yeasts, low levels of RHA activity have been found in screenings

performed on oenological yeast strains (Miklosy and Polos, 1995; McMahon

et al., 1999; Rodríguez et al., 2004), and so far one intracellular RHA has been

purified and characterized from Pichia angusta (Yanai and Sato, 2000).

RHA-producing bacteria have been described in human faecal flora belonging

to the genus Bacteroides (Bokkenheuser et al., 1987; Jang and Kim, 1996), in a

Bacillus sp. and in S. paucimobilis (originally designated Pseudomonas paucimo-

bilis) strains isolated from soil (Hashimoto et al., 1999, 2003; Miake et al., 2000;

Miyata et al., 2005), as well as in the thermophilic bacteria Clostridium sterco-

rarium (Zverlov et al., 2000) and Thermomicrobia sp. (Birgisson et al.

, 2004).

RHA activity has also recently been described in wine strains of Oenococcus oeni

(Grimaldi et al., 2005).

RHAs from various microbial sources have been purified and some of the

general properties of these enzymes are summarized in Table 1. Data presented for

fungal proteins correspond to RHAs purified from wild strains whereas the charac-

terizations of Bacillus sp., C. stercorarium and Thermomicrobia sp. enzymes were

carried out on recombinant counterparts. The main difference found between fungal

and bacterial enzymes concerns their optimal pH. With the exception of P. angusta

RHA, the fungal enzymes show acidic pH optima compared to bacterial RHAs

for which neutral and alkaline pH optima have been found. This characteristic

suggests different potential applications for fungal and bacterial enzymes, making

fungal RHAs more suitable for use in processes operating at low pH such as

winemaking (Manzanares et al., 2003) and citrus juice processing (Puri et al., 1996).

The bacterial enzymes would be useful in processes requiring good activity in basic

solutions, such as the production of L-rhamnose by hesperidin hydrolysis given that

the solubility of this flavonoid glycoside increases at high pH (Scaroni et al., 2002).

Differences in RHA cellular location have been also found, suggesting different

in vivo roles for fungal and bacterial RHAs.

Table 2 shows the catalytic properties of some purified microbial RHAs. Studies

of substrate specificity and the observed inhibition of activity by L-rhamnose clearly

demonstrate the specificity of RHAs for rhamnopyranoside residues. By contrast,

the type of linkage present at C1 of -L-rhamnose has only a minor effect on

120 MANZANARES ET AL.

Table 1. General properties of some purified microbial -L-rhamnosidases

Micro-organism Original strain or

overexpression host

Inducer pH optimum Temp.

optimum

(



Molecular

weight (kDa)

Native form pI Cellular location Reference

Fungi

Absidia sp. Original strain

Absidia sp. 39

Absidia sp. 90

Ginseng

Gynostemma

pentaphyllum

n.d.

Extracellular

et al., 2002, Yu

et al., 2004

A. aculeatus Original strain Hesperidin 4.5–5

4.5–5

n.d.

92 (RhaA)

85 (RhaB)

n.d.

6.2

5.2–5.9

Extracellular

Manzanares

et al., 2001, 2003

A. nidulans Original strain Rhamnose 4.5–6 60 102

n.d. 5 Extracellular Manzanares

et al., 2000

A. niger Original strain Hesperidin 4.5 65 85

n.d. 4.5–5.2 Commercial prep Kurosawa et al., 1973;

Manzanares et al., 1997

A. terreus Original strain Rhamnose 4 44 96 n.d. 4.6 Extracellular Gallego

et al., 2001

Penicillium sp. Original strain Naringin 3.5 57 90

n.d. n.d. Commercial prep Romero et al., 1985; Young

et al., 1989

-L-RHAMNOSIDASES: OLD AND NEW INSIGHTS 121

P. angusta Original strain Rhamnose 6 40 90 Monomer 4.9 Intracellular Yanai and Sato, 2000

S. avenae Original strain Starch n.d. n.d. 110 n.d. n.d. Extracellular Hughes et al., 2004

Bacteria

Bacillus sp.

Original strain

E. coli

Gellan 6.5–7

6.5–7

98 (RhaA)

106 (RhaB)

Pentamer

Monomer

n.d.

Intracellular

Hashimoto

et al., 2003

Bacteroides sp. Original strain 7 120

Dimer 4.2 Intracellular Jang and Kim, 1996

C. stercorarium

E. coli 7.5 60 95 (RamA)

Dimer n.d. Intracellular Zverlov

et al., 2000

S. paucimobilis Original strain Rhamnose 7.8 45 112 (RhaM) Monomer 7.1 Intracellular Miake et al., 2000

Thermomicrobia

E. coli 7.9

5–6.9

104 (RhmA)

107 (RhmB)

Dimer

4.6

4.5

Intracellular

Birgisson et al., 2005

n. d.: not determined.

Molecular weight of glycosylated protein.

Data from recombinant enzymes.

Monomeric form molecular weight.

122 MANZANARES ET AL.

Table 2. Catalytic properties of some microbial purified -L-rhamnosidases

Micro-

organism

(mM)

Substrate specificity Linkage

hydrolysed

Inhibitors Reference

Fungi

Absidia sp.

Absidia sp. 39

Absidia sp. 90

n.d.

Ginsenoside Rg2

Gipenoside-5

 −1 2

 −1 6

n.d.

Yu et al., 2002, 2004

A. aculeatus

RhaA 2.8 Hesperidin, naringin, rutin,

monoterpenyl-

glycosides

 −1 2−1 6 Rhamnose

Manzanares

et al., 2001, 2003

RhaB 0.30 Hesperidin, naringin, rutin  −1 2−1 6 Rhamnose

A. nidulans 0.27 Hesperidin, naringin, rutin  −1 2−1 6 Rhamnose, Mg

Manzanares et al., 2000

A. niger 2.9 Hesperidin, naringin, rutin

geranyl--D-rutinoside

2-phenylethyl--D-

rutinoside

 −1 2−1 6 Rhamnose Manzanares et al., 1997

A. terreus 0.17 Naringin, rutin  −1 2−1 6 Rhamnose, ethanol, glucose,

,Cd

Gallego et al., 2001

Penicillium

sp

1.52 Naringin  −1 2 Rhamnose, glucose Romero et al., 1985

P. angusta n.d. Hesperidin, naringin,

quercitrin, rutin, aroma

precursors from grape juice

 −1 2−1 6

1 to aglycon

Rhamnose, Cu

 Hg

p-chloromercuribenzoate

Yanai and Sato, 2000