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  • Characterization of Halomonas sp. Strain H11 -Glucosidase Activatedby Monovalent Cations and Its Application for Efficient Synthesis of-D-Glucosylglycerol

    Teruyo Ojima,a Wataru Saburi,a,b Takeshi Yamamoto,a and Toshiaki Kudoc

    Nihon Shokuhin Kako Co., Ltd., Shizuoka, Japana; Research Faculty of Agriculture, Hokkaido University, Sapporo, Japanb; and Graduate School of Fisheries Science andEnvironmental Studies, Nagasaki University, Nagasaki, Japanc

    An -glucosidase (HaG) with the following unique properties was isolated from Halomonas sp. strain H11: (i) high transglu-cosylation activity, (ii) activation by monovalent cations, and (iii) very narrow substrate specificity. The molecular mass of thepurified HaG was estimated to be 58 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). HaGshowed high hydrolytic activities toward maltose, sucrose, and p-nitrophenyl -D-glucoside (pNPG) but to almost no other di-saccharides or malto-oligosaccharides higher than trisaccharides. HaG showed optimum activity to maltose at 30C and pH 6.5.Monovalent cations such as K, Rb, Cs, and NH4

    increased the enzymatic activity to 2- to 9-fold of the original activity.These ions shifted the activity-pH profile to the alkaline side. The optimum temperature rose to 40C in the presence of 10 mMNH4

    , although temperature stability was not affected. The apparent Km and kcat values for maltose and pNPG were significantlyimproved by monovalent cations. Surprisingly, kcat/Km for pNPG increased 372- to 969-fold in their presence. HaG used somealcohols as acceptor substrates in transglucosylation and was useful for efficient synthesis of -D-glucosylglycerol. The efficiencyof the production level was superior to that of the previously reported enzyme Aspergillus niger -glucosidase in terms of smallamounts of by-products. Sequence analysis of HaG revealed that it was classified in glycoside hydrolase family 13. Its amino acidsequence showed high identities, 60%, 58%, 57%, and 56%, to Xanthomonas campestris WU-9701 -glucosidase, Xanthomonascampestris pv. raphani 756C oligo-1,6-glucosidase, Pseudomonas stutzeri DSM 4166 oligo-1,6-glucosidase, and Agrobacteriumtumefaciens F2 -glucosidase, respectively.

    -Glucosidase (EC, -D-glucoside glucohydrolase) isa ubiquitous enzyme widely distributed in microorganisms,plants, and animals. It catalyzes liberation of -glucose from thenonreducing ends of -glucosides such as malto-oligosaccharidesand sucrose. Its substrate specificity is significantly diverse, de-pending on the enzyme origins, and the enzymes are classified intothree groups on the basis of their substrate specificities (10).Group I enzymes are more active toward heterogeneous sub-strates, represented by sucrose and synthetic glycosides such asp-nitrophenyl -D-glucoside (pNPG) and phenyl -D-glucoside,than toward homogeneous substrates such as maltose. Group IIenzymes, known as maltases, prefer homogeneous substrates toheterogeneous substrates. Like group II enzymes, group III en-zymes mainly hydrolyze homogeneous substrates but have highactivity toward long-chain substrates like glycogen and solublestarch.

    According to the sequence-based classification of glycoside hy-drolase (GH), -glucosidases are generally classified into two fam-ilies, GH families 13 (subfamily 30) and 31 (9). Enzymes frombacteria, some yeast such as Saccharomyces cerevisiae, and insectsare mainly classified into the former family, and those from plants,animals, some yeast such as Schizosaccharomyces pombe, and fungiare in the latter.

    Enzymes of both families catalyze hydrolysis of -glucosides, withnet retention of the anomeric configuration, through a double dis-placement mechanism, where two acidic amino acid residues act as acatalytic nucleophile and a general acid/base catalyst (31). The generalacid/base catalyst donates a proton to the glucosidic oxygen, and thecatalytic nucleophile simultaneously attacks the anomeric carbon toform a -glucosyl enzyme intermediate. Consequently, the general

    acid/base catalyst activates a water molecule as a general base catalystto stimulate nucleophilic attack of the water molecule at the anomericcarbon of the glucosyl-enzyme intermediate. Transglucosylation canoccur if a free hydroxy group of a sugar or alcohol, rather than water,attacks nucleophilically.

    In the food and pharmaceutical industries, -glucosidases playessential roles in the production of isomalto-oligosaccharides,nigero-oligosaccharides (48, 57), and some glucosides such as-D-glucosyl vitamin E (33) and -D-glucosylglycerol (GG)(50). Glucosylation of nonsugar compounds is a useful techniquefor improving their physicochemical properties, such as stabilityand water solubility (26, 33, 47, 56).

    GG, a collective term for 2-O--D-glucosylglycerol (2-O-GG), (2R)-1-O--D-glucosylglycerol [(2R)-1-O-GG], and(2S)-1-O--D-glucosylglycerol [(2S-1-O-GG], is a glucosylatedcompound observed in some osmotolerant bacteria and plants(18) and is also found in traditional Japanese liquors producedfrom rice (49). GG has been reported to have some beneficialfunctions as a moisturizing agent in cosmetics (58) and potentialas a health food material and therapeutic agent because it stimu-

    Received 10 November 2011 Accepted 23 December 2011

    Published ahead of print 6 January 2012

    Address correspondence to Teruyo Ojima,

    Supplemental material for this article may be found at

    Copyright 2012, American Society for Microbiology. All Rights Reserved.


    1836 0099-2240/12/$12.00 Applied and Environmental Microbiology p. 18361845

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  • lates insulin-like growth factor behavior (17) and has antitumoreffects (37).

    GG can be synthesized enzymatically from malto-oligosaccharidesand glycerol with fungal -glucosidase (50) or bacterial cyclodex-trin glucanotransferase (EC. (35). However, the produc-tion yield has been restricted because accumulation of by-products such as malto-oligosaccharides and di- or higherglucosylated glycerols was unavoidable as a result of the nature ofthe enzyme. In contrast, sucrose phosphorylase (EC. hasbeen reported to regioselectively produce 2-O-GG from sucroseand glycerol in high yield (15). Although sucrose phosphorylase isan attractive biocatalyst for glucosides (29), not all types of gluco-sides can be synthesized using it, and research on its use for theproduction of various kinds of glucosides is still under develop-ment (1).

    In this work, we focused on -glucosidase as a biocatalystfor the production of glucosides because its substrate, malto-oligosaccharides, can be obtained by degradation of starch at lowcost. We screened -glucosidases showing high transglucosyla-tion activity, and an enzyme with the desired properties was suc-cessfully obtained from a halophilic marine bacterium, Halomo-nas sp. strain H11, isolated from the surface of coral. In addition toits high transglucosylation activity, the enzyme has several uniqueproperties: first, significant stimulation by monovalent cationssuch as NH4

    , K, Rb, and Cs, and second, very narrow spec-ificity for substrate chain length and glucosidic bonds. In this ar-ticle, the enzymatic properties and applications of -glucosidasefrom Halomonas sp. H11 (HaG) are described.

    MATERIALS AND METHODSIsolation and identification of bacterial strains. Bacterial strains fromcoral, collected in the sea near Okinawa, Japan, were propagated with aculture medium consisting of 1% (wt/vol) sodium arginate (Wako PureChemical Industries, Osaka, Japan), 0.5% (wt/vol) peptone (Becton Dick-inson, Franklin Lakes, NJ), 0.5% (wt/vol) yeast extract (Becton Dickin-son), 0.1% (wt/vol) KH2PO4 (Kanto Chemical, Tokyo, Japan), 0.02%(wt/vol) MgSO4 7H2O (Kanto Chemical), and 10% (wt/vol) NaCl; thepH was adjusted to 10 with separately sterilized 1% (wt/vol) Na2CO3(Kanto Chemical). The culture medium was incubated at 30C for 2weeks, and a single colony was isolated from the resulting culture fluid onthe same medium containing 2% (wt/vol) agar. Chromosomal DNA ofthe obtained bacterium was extracted using an Instragene matrix (Bio-Rad, Hercules, CA) and used as the template in PCR. Primestar HS DNApolymerase (Takara Bio, Shiga, Japan) and a primer set of 9F and 1510R(34) were used to amplify a region of 16S rRNA (see Table S1 in thesupplemental material). A BigDye Terminator (version 3.1) cycle se-quencing kit (Applied Biosystems, Foster City, CA) and primers 9F, 785F,802R, and 1510R (34) were used in sequence analysis (see Table S1 in thesupplemental material). An automatic DNA sequencer, the ABI Prism3130xl genetic analyzer system (Applied Biosystems), and the softwareChromaspro (version 1.4; Technelysium Pty., Tewantin, Australia) wereused. Sequence similarity searches and phylogenetic classifications wereperformed using the software Aporon (version 2.0; Techno Suruga Lab-oratory, Shizuoka, Japan), the database Aporon DB-BA (version 6.0;Techno Suruga Laboratory), GenBank, the DNA Data Bank of Japan(DDBJ), and the EMBL Nucleotide Sequence Database. Cells grown onMarine Broth 2216 (Becton Dickinson) containing 2% (wt/vol) agar wereused for the following tests. Morphological observations were carried outusing an optical microscope, BX50F4 (Olympus, Tokyo, Japan). Cellswere stained with Feyber G Nissui (Nissui Seiyaku, Tokyo, Japan) in aGram-staining test. Other physiological tests were performed by themethods described by Barrow and Feltham (6) and using a kit, API 20NE(bioMrieux, Lyon, France).

    Purification of -glucosidase from Halomonas sp. strain H11. Ha-lomonas sp. H11 was cultured aerobically at 37C for 24 h