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-L-RHAMNOSIDASES: OLD AND NEW INSIGHTS 123

S. avenae 0.091 Avenacoside A and B  −1 4 n.d. Hughes et al., 2004

Bacteria

Bacillus sp.

RhaA

RhaB

0.119

0.282

Naringin, gellan disaccharide

 −1 2−1 3

Rhamnose, Hg

 Cu

 Fe

glucose,

6-deoxyglucose

Hashimoto

et al., 1999, 2003

Bacteroides sp. 0.29 Hesperidin, naringin,

neohesperidin, poncirin,

quercitrin, robinin, rutin,

saikosaponin C

Rhamnose, fucose, saccharic

acid, 1,4-lactone, Pb

p-chloromercuriphenylsulfonic acid

Bokkenheuser

et al., 1987; Jang and

Kim, 1996

C. stercorarium n.d. Hesperidin, naringin  −1 2−1 6Hg

 Cu

 Zn

, SDS,

p-chloromercuribenzoate

Zverlov et al., 2000

S. paucimobilis 1.18 Hesperidin, naringin,

proscillaridin A, quercitrin,

rutin, saikosaponin C,

rhamnosyl-disaccharides

 −1 2−1 3

 −1 4−1 6

1 to aglycon

 Pb

 Cd

 Zn

 Ba

Miake et al., 2000

Thermomicrobia Birgisson et al., 2004

RhmA

RhmB

0.46

0.66

Naringin, hesperidin, rutin

Naringin, hesperidin

 −1 2−1 6

Rhamnose, glucose, ethanol

Rhamnose, glucose, ethanol, Zn

using pNPR as substrate with the exception of S. avenae -L-rhamnosidase K

determined on 26-desglucoavenacoside.

n.d.: not determined

124 MANZANARES ET AL.

Figure 1. Structure of some substrates hydrolysed by RHAs. A: naringin; B: neohesperidin; C:

hesperidin; D: rutin; E: quercitrin; F: avenacoside A. The arrows indicate the possible linkages hydrolysed

by RHAs

activity since RHAs are able to hydrolyse various substrates. These enzymes are

able to cleave both glycosidic bonds and aglycon-saccharide bonds (Fig. 1). As

regards glycosidic bonds, RHAs hydrolyse substrates in which the L-rhamnose

residue is linked to a -glucosidic residue through either i) an -1,2 linkage such as

that present in the flavonoids naringin, neohesperidin and poncirin and in ginseng

saponins, ii) an -1,3 linkage such as that in the gellan disaccharide -L-rham-

(1-3)--D-glc, iii) an -1,4-linkage such as that in oat saponins, or iv) an -1,6

linkage, as found in the flavonoids hesperidin and rutin, in the grape glycosides

geranyl- and 2-phenylethyl-rutinosides, and in the saponin gypenoside-5. RHAs

are also able to hydrolyse substrates in which L-rhamnose is directly linked to

an aglycon, as is the case in the flavonoids quercitrin and robinin, and in the

aryl-rhamnoside -nitrophenyl--L-rhamnopyranoside (pNPR) the model substrate

for assaying rhamnohydrolase activity. The latter has been commonly used for

determining RHA K

values, which can range from 0.119 to 2.9 mM (see Table 2).

values for hesperidin (0.06 mM), rutin (0.13 mM), naringin (0.17 mM) and

quercitrin (0.18 mM) for S. paucimobilis RHA have also been determined (Miake

et al., 2000). A K

value of 7.0 mM for naringin has been described for Penicillium

sp. RHA (Romero et al., 1985).

Differences in aglycon structure may explain the differences observed in the

hydrolysis of glycosides having the same linkages. For instance the reason why

-L-RHAMNOSIDASES: OLD AND NEW INSIGHTS 125

rutin and hesperidin (both of which have an -1,6 linkage) are so differently

hydrolysed by Aspergillus RHAs may be explained by steric hindrance due to the

attachment of the diglycoside to the aglycon molecule via C7 in hesperidin whereas

the attachment is via C3 in rutin. Differences in the hydrolysis of substrates in

which L-rhamnose is directly linked to the aglycon have also been described, and

in general RHAs show a clear preference for pNPR in comparison to quercitrin or

robinin. However, RHAs able to hydrolyse different saponins such as those from

Absidia species (Yu et al., 2002, 2004) and S. avenae (Hughes et al., 2004) hardly

hydrolyse pNPR.

2.2. Structure

Compared with the number of RHAs that have been purified and characterized,

the number of genes known that encode RHAs is much lower. Cloning has been

performed mainly by two methods: i) construction of a library followed by selection

of clones by screening for RHA activity using pNPR or 4-methylumbelliferyl--L-

rhamnoside plate assays, and/or ii) the construction of a library followed by selection

of clones by screening for RHA production with polyclonal antibodies. The first

RHA encoding gene (ramA), isolated from the thermophilic anaerobic bacterium

C stercorarium (Zverlov et al., 2000), consists of 873 or 874 codons and codes for a

protein with a predicted molecular mass of 100 kDa and a global secondary structure,

as detected by circular dichroism spectroscopy, consisting of 27% -helices and

50% -sheets. Soon thereafter the genes rhaA and rhaB encoding two A. aculeatus

RHAs were cloned (Manzanares et al., 2001). Conceptual translation of the cDNAs

corresponding to rhaA and rhaB yields primary structures of 660 and 597 amino

acids (71 and 64 kDa, respectively). However, the N-terminal amino acid sequences

actually determined for RhaA and RhaB correspond to positions 20 and 17, hence

the derived molecular masses of the mature proteins are 69 and 62 kDa.

To date, only eight RHA genes have been cloned and heterologously expressed:

those from C stercorarium and A. aculeatus (Zverlov et al., 2000; Manzanares

et al., 2001), two genes (rhaA and rhaB) from Bacillus sp. (Hashimoto et al., 2003),

two genes (rhmA and rhmB) from Thermomicrobia sp. (Birgisson et al., 2004) and

one gene (rhaM) from

S paucimobilis (Miyata et al., 2005).

The value of genome sequences for the rapid identification of candidate RHA

genes is obvious. Hypothetical proteins of unknown function found by such

analyses are putative novel RHAs. For example, two sequences from Lacto-

bacillus acidophilus and Aspergillus fumigatus exhibited sequence similarities with

the enzymes RamA of C. stercorarium and RhaA of A. aculeatus, respectively.

Whereas electrophoretic and chromatographic detection of RHAs indicate the

presence of only one or two isoenzymes in a given organism, data from genome

sequencing projects suggest the presence of many different isoforms. We have

previously reported (Orejas et al., 1999; Manzanares et al., 2000) the production

of an apparently unique extracellular RHA in A. nidulans with a molecular mass

of 102 kDa. However, in silico analysis of the A. nidulans genome reveals the

126 MANZANARES ET AL.

presence of 8 ORFs encoding hypothetical RHAs ranging from 63 to 253 kDa

(family GH 78 in the CAZY database; de Vries et al., 2005), the functions of which

still have to be demonstrated. The presence of various ‘isoforms’ of RHA within

the filamentous fungi was also suggested by Monti et al. (2004) based on inducer

and substrate specificity data.

To date no three-dimensional structure of RHA has been published.

2.3. Sequence Comparisons

Some micro-organisms have already been reported that produce two RHAs.

Whereas in the case of A. aculeatus the primary structures of RhaA and RhaB are

highly homologous (60% identity) (Manzanares et al., 2001), those produced by

Bacillus sp. strain GL1 and those produced by Thermomicrobia sp. strain PRI-1686

have quite different primary structures (only 23% identity) (Hashimoto et al., 2003;

Birgisson et al., 2004).

Using the FASTA program (Pearson, 1990), RhaA of Bacillus sp. was found

to exhibit significant homology with RamA of C. stercorarium (41% identity in

848 aa overlap) but no homology with either RhaA or RhaB of A. aculeatus

(Hashimoto et al., 2003). Despite the observed similarity between Bacillus RhaA

and C. stercorarium RamA, these enzymes are distinct in their quaternary structures

(pentameric versus homodimeric, see Table 1). By contrast, Bacillus RhaB exhibits

∼24% identity with RhaA and RhaB of A. aculeatus (Hashimoto et al., 2003) but

differs significantly from the fungal enzymes since the A. aculeatus RHAs have

considerably lower molecular masses as calculated by conceptual translation of the

ORFs (see above). Using the BLAST program (Altschul et al., 1997) it has been

shown that Thermomicrobia RhmA and RhmB display greatest similarity to the

isozymes RhaA and RhaB of Bacillus sp.: RhmA has 41% identity with RhaA and

RhmB has 50% identity with RhaB.

The Sphingomonas enzyme RhaM has no similarity to other known RHAs,

and Miyata et al. (2005) have suggested that this protein could be a member of a

new bacterial subfamily within glycoside hydrolase family 78. In BLAST analysis,

RhaM showed 58% identity and 72% homology (in 1113 aa overlaps) with the

hypothetical protein Saro 02001624 of Novosphingobium aromaticivorans. This

hypothetical protein, predicted from complete genome sequence analysis, may thus

also have RHA activity.

In order to locate putative catalytic sites in RHAs, an amino acid sequence

alignment using the ClustalW program (Thompson et al., 1994; Higgings et al.,

1996) was performed with those members of the GH78 family for which the

corresponding encoding genes have been functionally characterised (Fig. 2). Since

glycosyl hydrolases require the presence of two or more carboxylic acid moieties

for their function, the aligned sequences were examined for conserved Asp and

Glu residues. Two fully conserved and four well conserved carboxylic amino acid

residues were found in the alignment, all of which are possible candidates for

playing an important role in 

RHA catalytic function. Site-directed mutagenesis

-L-RHAMNOSIDASES: OLD AND NEW INSIGHTS 127

RhmA-Thermomicrobia 1 -----------------------------------------------MLRIDRVKVERSR

RhaA-Bacillus 1 ---------------------------------------------MAEIEVYGLSCNNRS

RamA-C.stercorarium 1 -----------------------------------------------MMRVYNLKTNRIK

RhmB-Thermomicrobia 1 ---MQWQASWIWLEGEPSPRNDWVCFRKSFELDRSASPLEEAKLSITADSRYVLYVNGQL

RhaB-Bacillus 1 MAGRNWNASWIWGGQEESPRNEWRCFRGSFDAP--ASVEGPAMLHITADSRYVLFVNGEQ

RhaA-A.aculeatus ------------------------------------------------------------

RhaB-A.aculeatus ------------------------------------------------------------

RhmA-Thermomicrobia 14 DGLGLGTGRPRLCWRVETDIRDWRQAAYEVELYDGS-----GQLVGSTGRVESGESVWVA

RhaA-Bacillus 16 NPMGIGDKAPRLGWKLRSERRGVVQEAYRIQVAEDASFE--GGLAWDTERMAGGRSVAVP

RamA-C.stercorarium 14 NPMGFVINKPKLSWLVESDT-AKHQVAAQVEISADIN-F--ENIIFDSGKRTDIDSISYS

RhmB-Thermomicrobia 58 VGRGPVRSWPFEQSYDTYDLRHLLHPG-RNCLAVLVTHFGVSTFSYVRGRGGLLAQLELS

RhaB-Bacillus 59 VGRGPVRSWPKEQFYDSYDIGGQLRPGVRNTIAVLVLHFGVSNFYYLRGRGGLIAEIEA-

RhaA-A.aculeatus ------------------------------------------------------------

RhaB-A.aculeatus ------------------------------------------------------------

RhmA-Thermomicrobia 69 WPFEALGSRQRAGVRVRVWGEDGSESDWSDLQWLEVGLLARDDWQG-AFITPDWEEDTSV

RhaA-Bacillus 74 YEGEPLRSRTRYFYRVKVWGNRGQESEWSAAAYFETALLSVEEWAGAEWISHTLPANEEG

RamA-C.stercorarium 70 PQVE-LKPRTRYYWRVRVWGDDGSE-AVSEAAWFETSKMD-EPWKA-KWITPDFDPS---

RhmB-Thermomicrobia 117 SGDDRTTIGTDGSWKVHRHLGYSRRTTRISPQQGFVEQLDARAWSS-EWKDLMYDDSGWE

RhaB-Bacillus 118 --DGRTLAATDAAWRTERLGGQRSNSPRMACQQGFGEVIDARELAE-DWALPAFDDGGWA

RhaA-A.aculeatus 20 -----------VPFEDYILAPQSRTLNPSLVYQVNGTVDNPEALVG--------------

RhaB-A.aculeatus 17 ---------ARVPYREYILAPSSRVIVPASVRQVNGSVTNAAGLTG--------------

RhmA-Thermomicrobia 128 ANPCPYLRKTFSLPGGVRRARLYVTGLGVYEVELNGQR-----------VGDHVLSPGWT

RhaA-Bacillus 134 HEPIAYLRKSFRLDRPVASARIYATALGMYRLFVNGEA-----------ADDTHLNPGWT

RamA-C.stercorarium 123 VHPVVFT--DFSIERDVADARAYVCGLGLYEMSVNGEK-----------TGDEYLAPGLV

RhmB-Thermomicrobia 176 DAMIVGPVGTPPWEQLVPRDIPFLTEEVLHPTRVVSLHSTVPPKIAVAVDMRAIMMPDSA

RhaB-Bacillus 175 QARSIGPAGTAPWTSLVPRDIPFLTEEKLYPASIQSLSRVKAPKYAAALDLRNQMVPESV

RhaA-A.aculeatus 55 ---------------------LTTHQTTLHGKS---------------------------

RhaB-A.aculeatus 54 ---------------------SSLGTAVFHGVS---------------------------

RhmA-Thermomicrobia 177 SYRHRLLYETFDVTGLLREGDNCLGAILGDGWYRGRLGFGG-------------------

RhaA-Bacillus 183 SYGHRVQYQTYDVTALLTEGANALGLMLGNGWYRGRLGWQQ-------------------

RamA-C.stercorarium 170 AYDKWIPYQTYDITSQLKKGINTAEFLLGNGWYKGRYGLNR-------------------

RhmB-Thermomicrobia 236 DHAEQVQYAGFLATILRTDGEGTARLLLSKPWVGDGIAASINGQVYGAELMSRTPTGREL

RhaB-Bacillus 235 NHANPVSYCGYVATILTLETSGVVTLGFPTGVRGSGVWVDG---VLQTEWTGVQPE-RYY

RhaA-A.aculeatus 67 SVTYDFGRNIAGIVSLDVTSVSSKAAQLGVTFTESSLWISS-------------------

RhaB-A.aculeatus 66 SVTYDFGKNVAGIVSLTVGSSSSPSAFLGVTFSESSLWASS-------------------

RhmA-Thermomicrobia 218 GRRNLYGERLALLAQLEVELEDGSRQVVVTDGSWRAHR------GPILESGIYDGEVYDA

RhaA-Bacillus 224 ASK--YGDTRAALVWLHVRYADGTESVVGSDGSWLGSD------GALRWSELYDGEHYDA

RamA-C.stercorarium 211 KQPFRYGNEFALICEIHITYQDGTADVIYTDTSWKARK------SKVIDSGIYDGEIYDD

RhmB-Thermomicrobia 296 EVELSAGDNLLLVYVCGSDHADPLRLALDSDLGLELVSPTGGE-SAFVAIGPLASRVVRN

RhaB-Bacillus 291 SLNLAAGEHLVLVDITSSDHGGSSHFAIDSEAAFTLRSPAGDNGVPLATIGTFDQSEYID

RhaA-A.aculeatus 108 ------------------EACDATSDAGLDSPLWFSVG----------------------

RhaB-A.aculeatus 107 ------------------EACDATGNSGLDAPLWFPVG----------------------

RhmA-Thermomicrobia 272 RLEMPGWSTPEYDDSEWAGTRELGWPTESLE--------------------------PLE

RhaA-Bacillus 276 RLARKDWSLPAFDDSEWGNVSPYAHPKTALV--------------------------AQE

RamA-C.stercorarium 265 ----------TFCDDAVYPVRIADLDVNKLE--------------------------PRR

RhmB-Thermomicrobia 355 FDFSQPLEYDETAVRRISSCAS-VADLRAWSHLPRSVPPELVSPADVFTLCTWPRQRTEL

RhaB-Bacillus 351 HRPGRRMQTDHPDYRALPEAAPTAAALEAFASWVKPFEPSLYTEENVFGSNVWRTLAERR

RhaA-A.aculeatus ------------------------------------------------------------

RhaB-A.aculeatus ------------------------------------------------------------

RhmA-Thermomicrobia 306 V-PARRTQEVAP------REILRSFSGKTIVDFGQNLVGRVRLRVSGPRGQRVRLRHAEV

RhaA-Bacillus 310 SEPVRIVDTIVS------VSVWTTPSGETLLDMGQNMVGWVRFTVRAEAGTVVTVRHAEV

RamA-C.stercorarium 289 SPGIKIKERIKP------AEIIRTPEGETVIDMGQNMVGWLEFTNRAPKGAEIMLQFGEV

RhmB-Thermomicrobia 414 TTGKELEAMVFPSKDPGLVPILRAGDTELVLDFGQEVSGYLFLDVEASEGTLIDLYGFEF

RhaB-Bacillus 411 AVPRSVLNAILPVPEPGVLPVFEDGDCELVIDLGAERSGFIGFELEAPAGTIIDAYGVEY

RhaA-A.aculeatus 128 -----------------------HGPGTYGADKKHLRGAFRYLTLVNNSTATISLDGLSI

RhaB-A.aculeatus 127 -----------------------QKAGTYTPDSKYVRGGFRYLTVVSNTSATIPLNSLHI

* : :

Figure 2. (Continued)

128 MANZANARES ET AL.

RhmA-Thermomicrobia 359 LE-GGELCTRTLRTARATDEYVLRGDGEEEWEPRFTFHGFRYVEVEGWPGELRAEDLVAV

RhaA-Bacillus 364 LDKHGNFYTGNLRSAKQTVAYVCSGEGEETFEPTFSFQGFRYVKIEGIPPEQVPGRFVGC

RamA-C.stercorarium 343 LQ-DGNFYRDNLRTAKCEFHYISDGK-VKKVRPHFTFYGFRYVKLTKWEGEVNPEDFTGL

RhmB-Thermomicrobia 474 ME---DDYRQDTVGLDNTLRYTCREGRQHYVSPQRRGLRYLMLTVREARAPLRVHGVGVV

RhaB-Bacillus 471 MR---EGYTQHTYGLDNTFRYICREGRQSYVSPVRRGFRYLFLTVRGNSAPVKLHEIYIR

RhaA-A.aculeatus 165 NY-----TAAPTQDLRGYKGYFHSSDELINRIWYAGAYTLQLCTIDPTTGDS--------

RhaB-A.aculeatus 164 TF-----TAAPDQDLQAYQGWFHSNDELLNEIWYAGAYTNQLCTIDPTYGS---------

RhmA-Thermomicrobia 418 VCHSDMERIGWFGCSDPLVERLHENVVWSMRGNFLHIPTDCPQRDERLGWTGDIQVFSPA

RhaA-Bacillus 424 VLTSDLQTAGRFRCSDPMINKLADNIVWGQIGNFVDVPTDCPQRDERLGWTGDAQAFVRA

RamA-C.stercorarium 401 VLYSDLERTGNITTDNSLVNRLFLNALWSQKGNFLDVPTDCPQRDERMGWTGDAQVFSGA

RhmB-Thermomicrobia 531 QSTYPVSQVGTFRCSDPLLNDIWEISRLTTKLCMEDTFVDCPAYEQTF-WVGDSRNEALT

RhaB-Bacillus 528 QSTYPVAEQGSFRCSDALLNATWEISRHTTRLCMEDTFVDCPSYEQVF-WVGDSRNEALV

RhaA-A.aculeatus 212 -----LIWLGVISSSDNITLPQTDSWWNNYTITNGSTTLVDGAKRDRLVWPGDMSIAIES

RhaB-A.aculeatus 210 ------------ASSETISTSGLNYWYNNLTIANGTSTVTDGAKRDRAVWPGDMSISLES

RhmA-Thermomicrobia 478 ACFIYDASGFLTSWLRDVALDQDESGA-VPFVVPNALGGQVIPAAAWGDAAVIVPWVLYQ

RhaA-Bacillus 484 STYNRNVQSFFAKWLRDLAADQQPDGG-VPHVVPDVSIAGAN-SSAWGDAAAIVPWVLYE

RamA-C.stercorarium 461 AAFNMDVFAFFGKYLYDLKQEQKARGGNVPVVVP-AHDVKQNGACGWGDAAVIIPWNMYL

RhmB-Thermomicrobia 590 AYYLFGAEELVRRCLRLVPGSRR----YTPLYMDQVPSGWVSVIPNWTFLWVMACREYYE

RhaB-Bacillus 587 NYYVFGETEIVERCLNLVPGSAD----ETPLYLDQVPSAWSSVIPNWTFFWILACREYAA

RhaA-A.aculeatus 267 AAVSTADLESVRTALESLFVLQKA----NGQLPYAGRPFLDIVSFTYHLHSLIGASSYYQ

RhaB-A.aculeatus 258 IAVSTNDLYSVRMGLEALLALQSS----EGQLPWGGKPFNIDVSYTYHLHSLIGMSFLYR

* : :

RhmA-Thermomicrobia 537 RYGDAGVLEAQWPSMRAWVDCIKTIAGPA---RLWNKGFQFGDWLDPAAPPDNPAAARTD

RhaA-Bacillus 542 RYGDDRVLREQYASMKGWVEYIRAQGESE---YLWDTGFHFGDWLGLDAK-ENSYIGATP

RamA-C.stercorarium 520 HYGDVSILEQQYKSMKGWVDYIKSKDDAAGGRRLWLNDFHYGDCVSLDVEDPFNRFGGTE

RhmB-Thermomicrobia 646 RTGDLAFVQDIWPDIQYTLDHYLQHINDDG--LLEISAWNLLDWAPIDQP-------NSG

RhaB-Bacillus 643 HTGNEAFAARIWPAVKHTLTHYLEHIDDSG--LLNMAGWNLLDWAPIDQP-------NEG

RhaA-A.aculeatus 323 YTGDRSWITRYWGQYKKGLQWALSSLDNSGLANITASADWLRFGMGGHNI------EANA

RhaB-A.aculeatus 314 FSGDKVWLSNYWGQYSKGVEWAVRSVADG-------------------------------

: :

RhmA-Thermomicrobia 594 PYIVASAYFARSAEIVGLSAQVLGMQDMAEEYLGLASEVREAFNREYVTPNGRVVSDAQT

RhaA-Bacillus 598 RELIATAFYAHSTDLLAKSADVLGYADEAVKYAELRDNVARAFRAEYVTPSGRLASPTQT

RamA-C.stercorarium 580 HAYLASAFYSYSAGIVSKAAKILNKKEDAEYYRKLSEEVKNAIRKEYFTPTGRLAVNTQT

RhmB-Thermomicrobia 697 VVTHQNCFFVRALKDADELGQSAG-DETAGRYAERARELAAAINTHLWSDEHKAYIDSIH

RhaB-Bacillus 694 IVTHQNLFLVKALRDSRALAAAAGATEEADAFAARADLLAETINAVLWDEEKRAYIDCIH

RhaA-A.aculeatus 377 ILYYVLNDAISLASSLDDRANVGNWSTAASKIKAAANARLWDAQNSLYRDNETTTLHPQD

RhaB-A.aculeatus 343 -------------------------------VKSAANQLLWDDQAGLYRDNQTTELHPQD

RhmA-Thermomicrobia 654 AYSLAIGFALLPTQEQRQHAGERLAELVRAEGYKIGTG-----FVGTPLICDALCATGHH

RhaA-Bacillus 658 AYAVALMFDLLEEG-TRQQAADRLAKLIEESGTQLTTG-----FVGTPYLCHVLTRFGRA

RamA-C.stercorarium 640 AYVIALYMDLVPDE-WKERVAFELRKKLKETKYHLRTG-----FLGTPYLCRVLSEYGSN

RhmB-Thermomicrobia 756 ADSTRSSVISMQTQVVALLTGVAEGDRAEVVRSHIASPPAGWVQIGSPFMSFFLYEAMVR

RhaB-Bacillus 754 ADGRRSDVYSMQTQVVAYLCGVAQGEREAVIEGYLSSPPPAFVQIGSPFMSFFYYEALEK

RhaA-A.aculeatus 437 GNAWAIKANLTLSSNQSEAISSALAARWGPYGAPAPEAG----STVSPFIGGFELQAHYL

RhaB-A.aculeatus 372 GNAWAVKSNLTLSGSQNRAISQALKARWGRYGAPAPEAG----ATISPFIGGFEIQSHFL

RhmA-Thermomicrobia 709 DVAYRLLMSRECPSWLYPVTMGATTIWERWDSLRPDGSVNPG-----EMTSFNHYALGAV

RhaA-Bacillus 712 DLAYKLLERREYPSWLYPVVKGATTIWEHWDGIKPDGSFWSD-----DMNSYNHYAYGAI

RamA-C.stercorarium 694 DIAYRLLTNTDYPGWLYPVTMGATTIWERWNSMLPDGKVSDT-----GMNSFNHYSYGSI

RhmB-Thermomicrobia 816 QGMYAQMLEDIRQKYGLMLEHGATTCWETFPGALGAR----------YTRSHCHAWSAAP

RhaB-Bacillus 814 AGRQTLMLDDIRRNYGQMLRYDATTCWEMYPNFAENRSNPDM-----LTRSHCHAWSAAP

RhaA-A.aculeatus 493 ANEPDRALDLLRLQWGFMLDDPRMTNSTFIEGYSTDGSLAYAPYRNTPRVSHAHGWSTGP

RhaB-A.aculeatus 428 ANQPDVALDMIRLQWGFMLRDPRMTQSTLIEGYSTDGSIHYAPYANDARISHAHGWSTGP

RhmA-Thermomicrobia 764 ADWLHRVVGGLA--PAEPGYRKLRIQPVPGGGLSYARARHVTPYGTAECSWRTE-GGEIE

RhaA-Bacillus 767 GDWLFGSVAGIDTEASAPGYKRIRVRPVPGGSLKFAEATLESPYGEIRSAWRRRPDGGID

RamA-C.stercorarium 749 VEWIYRNAAGIQPVEDAPGFRRFRLKPQPHYLLKSLDAEFLSPAEKIISRWNINENGSVS

RhmB-Thermomicrobia 866 GYFLGAYVLGVR--PGGPGWHRVIVAPQPCDLAWARGSVPLPRGDRVDVSWRREGQ-KLL

RhaB-Bacillus 869 GYFLGSSILGVK--RGADGWRTVDIAPQPCDLTWAEGVVPLPQGGHIAVSWEFVSAGKLK

RhaA-A.aculeatus 553 TSALTHYTAGLR--LTGPAGSTWLFKPQPGNLTEVQAGFETQLGLFATQYQKSATGTFQQ

RhaB-A.aculeatus 488 TYALTAYAAGLQ--LLGPAGNSWLIAPQPGGLTSIDCGFATALGVFSVVFERDSVGRYNS

Figure 2. (Continued)

-L-RHAMNOSIDASES: OLD AND NEW INSIGHTS 129

RhmA-Thermomicrobia 821 VRVVVPPNTSAQVVLPGSGREVEVGSGEHVWRYAFEAHRYPPVTLDTPLKEILEDAEAWE

RhaA-Bacillus 827 YEFEVPANAAANVLLPDASKANAMES-----------------------QQPIDAAEGIS

RamA-C.stercorarium 809 FYFRIPFNTTAELVLPDTEVQDWKEF-------------------------MSLNVVNIL

RhmB-Thermomicrobia 923 LRVERPQEVELEVVPPEE---------------------------------YELELDERV

RhaB-Bacillus 927 LRIEAPEDIEVNVTLPEG---------------------------------IEGEVTQVK

RhaA-A.aculeatus 611 LTFTAPNGTSGSVEIEGATG-----------------------------QLISKRGQAVK

RhaB-A.aculeatus 546 FSFGAPTGTTGRIELPGVRG-----------------------------TLVSTTGQRVQ

RhmA-Thermomicrobia 881 VLTRHFPEVASMPPRRLERIGTIRDLAASVVAFNERVGRLERELQALSRERS

RhaA-Bacillus 864 VVT----------------------ETAEGLAFVVGSGTYRFTVK-------

RamA-C.stercorarium 844 ILT------------------CLKDLTRKCTEQILQCGKFMKMRGQKKL---

RhmB-Thermomicrobia 950 RQTTQ-----------------------------------------------

RhaB-Bacillus 954 YMS-------------------------------------------------

RhaA-A.aculeatus 642 LVN--------------------------GKARGLQGGTWTLKGL-------

RhaB-A.aculeatus 577 LVN--------------------------GTASGLRGGKWKLIESAD-----

Figure 2. (Continued) Multiple sequence alignment of some members of glycosyl hydrolase family

GH78. The alignment was performed using ClustalW (v 1.82) (Thompson et al., 1994) and shading

was performed with Box-shade. The sequences aligned are listed below followed by their GenBank

accession numbers in parentheses. RamA of C. stercorarium (AJ238748), RhaA (AF284761) and RhaB

(AF284762) from A. aculeatus, RhaA (AB046705) and RhaB (AB046706) from Bacillus sp. GL1, and

RhmA (AY505013) and RhmB (AY505014) from Thermomicrobia sp. PRI-1686. In the case of the

A. aculeatus proteins, the putative signal peptides were not included. Identical residues are depicted on a

black background whereas similar residues are shown on a grey background. Conserved carboxylic

amino acid residues and hydrophobic amino acids are marked with * and : respectively

of putative key residues involved in catalysis will indicate their in vivo relevance

and potentially provide a means to modify enzyme characteristics. A phylogenetic

tree (Fig. 3) based on primary sequence homology was also constructed for the

enzymes analysed in Fig. 2 along with RhaM of S. paucimobilis. The pattern

obtained suggests that these enzymes evolved from a common ancestor into at least

3 distinct clusters.

RhaAacu

RhaBacu

RhmBThe

RhaBBac

RhmAThe

RhaABac

RamAClos

RhaMSph

100

0.2

Figure 3. Phylogenetic analysis of sequence similarities between RHAs. MEGA 3 software (Kumar

et al., 2004) was used to carry out the analysis of those RHAs included in Fig. 2 as well as the mature

form of S. paucimobilis RhaM (GenBank accession no. AB080801). Bootstrap values are adjacent to

each internal node, representing the percentages of 1000 bootstrap replicates. The scale represents amino

acid replacements per residue

130 MANZANARES ET AL.

3. BIOLOGICAL FUNCTION

Neither the reason for the redundancy of the genes encoding RHAs nor the in

vivo function of RHAs are completely understood. Speculation on the biological

role of these enzymes is mainly based on studies of the catalytic properties and

substrate specificities of purified RHAs (Table 2). Recently, the design of potent

RHA inhibitors has been suggested as a tool to investigate the biological function

of the enzyme (Kim et al., 2005).

The low specific activities found on the natural substrates tested and the fact

that most of the RHAs characterized show a clear preference for pNPR has led

some authors to suggest that the preferred natural substrate for these glycosidases

is still unknown (Zverlov et al., 2000; Manzanares et al., 2001). Nevertheless, and

as described below, several in vivo functions including heteropolysaccharide and

flavonoid metabolism as well as detoxification processes have been described.

It seems clear that at least RhaB from Bacillus sp. GL1 is indispensably

required for the complete metabolism of gellan and gellan-related polysaccharides

(sphingans), heteropolysaccharides produced by different species belonging to the

genera Sphingomonas (Hashimoto et al., 1999, 2003). Given the co-isolation of

Bacillus sp. GL1 and Sphingomonas sp. R1 from soil samples, it has been suggested

that these micro-organisms could be symbiotic and that due to its gellan degrading

ability Bacillus sp. GL1 would be able to survive in a capsule-like sphingan biofilm

formed by Sphingomonas sp. R1. The capsule would thus function as a barrier to

inhibit diffusion of the low molecular weight depolymerization products necessary

for their growth.

It has also been suggested that RHA may be involved in flagellum formation

based on results showing that S. paucimobilis RHA production is induced by

L-rhamnose and that only in the presence of this sugar does S. paucimobilis form

flagella (Miake et al., 1995).

Glycosidases produced by the human intestinal microflora are known to

participate in the degradation of dietary flavonoids (Bokkenheuser et al., 1987;

Jang and Kim, 1996), the first step being hydrolysis to yield the aglycon. This

conversion probably takes place in the lower part of the ileum and the caecum.

The aglycon molecules are then either further bacterially metabolised or absorbed

into the enterohepatic system. Studies on the metabolism of some ginseng glyco-

sides have suggested a key role for RHAs and -glucosidases in the manifestation

of the pharmacological properties of ginseng such as its oestrogenic effect (Bae

et al., 2005).

The RHAs of phytopathogenic fungi seem to be involved in overcoming

saponin-mediated plant defences. Plant saponins are glycosylated compounds that

can repress the growth of fungi (Osbourn, 1996a). Successful plant pathogens

avoid the antifungal properties of saponins by modifying their membrane compo-

sition and/or by detoxifying saponins via hydrolytic removal of their sugar

moieties (Osbourn, 1996b). The RHA of S. avenae, a fungus able to infect oat

leaves (Morrissey et al., 2000), is involved in the latter strategy. In response to

pathogen attack, biologically inactive plant avenacosides saponins are converted

-L-RHAMNOSIDASES: OLD AND NEW INSIGHTS 131

into 26-desglucoavenacosides which possess antifungal activity. These molecules

are comprised of a steroidal backbone linked to a branched sugar chain consisting

of one -L-rhamnose and two or three -D-glucose residues. Isolates of the fungus

that are pathogenic to oats are capable of sequentially hydrolysing these sugar

residues. Degradation is initiated by removal of the L-rhamnose which abolishes

the antifungal activity. A similar function has been described for the RHAs

of several filamentous fungi (Cladosporium cladosporioides, Penicillium sp. and

Plectosphaerella cucumerina) which are able to grow on potato sprouts despite

the accumulation by the latter of the antifungal -chaconine (Oda et al., 2002).

The hydrolysis of one of the two L-rhamnose residues in -chaconine seems to be

the first step in the latter’s detoxification.

4. POTENTIAL INDUSTRIAL APPLICATIONS

OF -L-RHAMNOSIDASES

Nowadays enzyme technology presents an alternative to chemical processes,

reducing both energy and material consumption and minimizing the generation of

wastes and emissions. In this context, RHAs have been demonstrated to be of

biotechnological utility with possible industrial applications. These applications,

directed to the food, pharmaceutical and chemical industries, are mainly based on

RHA hydrolytic activity although some applications based on synthetic activity

have also been described (Table 3).

4.1. Food Industry

The main use of RHA in the food industry is for beverage quality enhancement

(debittering, liberation of aromas and bioactive compounds) and the production of

food additives.

Biotechnological approaches for the debittering of citrus juices are focused on

the development of enzymes capable of hydrolysing naringin and limonin, the

two major contributors to bitterness in processed citrus juices. RHA is involved

together with -D-glucosidase in the stepwise hydrolysis of naringin (see Fig. 1).

Both activities, collectively termed ‘naringinase’, work sequentially. RHA splits

naringin into rhamnose and prunin, and ß-D-glucosidase splits prunin into glucose

and naringenin. Naringenin bitterness is only one third that of naringin, and prunin

is less bitter than naringenin. In fact, only the first hydrolysing activity, RHA,

is essential. The feasibility of the enzymatic approach to debittering has been

shown with both soluble and immobilized naringinase (Norouzian et al., 1999;

Prakash et al., 2002; Puri et al., 2005). Active packaging has also been described

as an alternative method to reduce the naringin content of citrus juices during

storage and transport by means of direct interaction with the product. The system

uses an ‘active’ film comprising a cross-linked matrix in which naringinase is

completely immobilized. Various data suggest that the active package developed

132 MANZANARES ET AL.

Table 3. Possible industrial applications of -L-rhamnosidases in the food, pharmaceutical and chemical industries

Application Reference

Food industry

Beverages Debittering of citrus juice by hydrolysis of naringin either

directly or by active packaging

Soares and Hotchkiss, 1998; Norouzian

et al., 1999; Prakash et al., 2002; Del Nobile

et al., 2003; Puri et al., 2005

Improvement of wine aroma by hydrolysis of aromatic

precursors

Günata, 2003; Manzanares et al., 2003

Enhancement of fruit juice functional properties by increasing

flavonoid bioavailability

Hollman et al., 1999; González-Barrio

et al., 2004

Additives Production of natural sweetener precursor by hydrolysis of

hesperidin

Sánchez et al., 1987

Obtention of new ingredients from biopolymers to enhance

food rheological properties

Jansson and Lindberg, 1983; Hashimoto

et al., 1999; Giavasis et al., 2000

Pharmaceutical

industry

Production of compounds with enhanced pharmacological

properties by enzymatic hydrolysis of different rhamnosides

Mimaki et al., 1998; Acquati and

Ponzone, 2000; Di Lazzaro et al., 2001; Boyle

et al., 2003; Pisvejcová et al., 2003; Yu

et al., 2004

Obtention of novel glycopeptide antibiotics Takatsu et al., 1987a,b

Chemical industry Design of low cost biotechnological processes for the

production of pure compounds

Matsumoto et al., 2002; Trummler et al., 2003;

Chang and Muir, 2004

Production of L-rhamnose as a precursor for industrial use or

as a chiral compound for chemical synthesis

Martearena et al., 2003