Graduate Institute of Agricultural Biotechnology1, and Department of Plant Pathology2, National Chung Hsing University, 250 Kuo-Kuang Rd, Taichung, Taiwan40227
Author for correspondence: Menghsiao Meng. Tel: +886 4 22840328. Fax: +886 4 22853527. e-mail: mhmeng{at}dragon.nchu.edu.tw
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ABSTRACT |
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Keywords: endo-1,3-ß-glucanase, ß-1,3-glucanase, laminarinase, carbohydrate-binding module
Abbreviations: CBM, carbohydrate-binding module; GHF, glucosyl hydrolase family; LC/MS, liquid chromatography/mass spectrometry
The GenBank accession number for the sequence reported in this paper is AF21741.
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INTRODUCTION |
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The genes encoding endo-1,3-ß-glucanase have been cloned and sequenced from a variety of plants (Bucciaglia & Smith, 1994 ; Chang et al., 1993
; Chye & Cheung, 1995
; de Loose et al., 1988
; Hudspeth et al., 1996
; Oh & Yang, 1995
) and bacteria and archaea such as Bacillus circulans (Okada et al., 1995
; Yahata et al., 1990
), Oerskovia xanthineolytica (Cellulomonas cellulans) (Ferrer et al., 1996
; Shen et al., 1991
), Thermotoga neapolitana (Zverlov et al., 1997
), Rhodothermus marinus (Spilliaert et al., 1994
), Arthrobacter sp. (Doi & Doi, 1986
) and Pyrococcus furiosus (Gueguen et al., 1997
). Although both types of enzyme catalyse the same glucanohydrolysis reaction, bacterial enzymes are classified in glycosyl hydrolase family 16 (GHF 16), while plant enzymes are grouped in GHF 17, based on differences in their amino acid sequences (Henrissat, 1991
; Henrissat & Bairoch, 1993
).
1,3-ß-Glucanase has several potential applications in biotechnology, such as utilization in the production of yeast extract (Ryan & Ward, 1985 ), and of soluble 1,3-ß-glucans that could act as immunoactivators (Mohagheghpour et al., 1995
; Rios-Hernandez et al., 1994
). It also exhibits antifungal activity for disease protection in plants (Castresana et al., 1990
; Grenier et al., 1993
; Yi & Hwang, 1997
). Recently, we have isolated a Streptomyces sioyaensis strain that exhibits antagonism against several fungal pathogens such as Pythium aphanidermatum, Colletotrichum higginsianum, Acremonium lactucum and Fusarium oxysporum (Chen et al., 2000
). To study the enzyme that may be used by this S. sioyaensis strain for the hydrolysis of fungal cell walls, we set out to clone the gene of 1,3-ß-glucanase from this bacterium and to characterize the recombinant gene product.
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METHODS |
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Bacterial strains and plasmids.
The S. sioyaensis strain was isolated from peat moss. Escherichia coli XL-1 Blue (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac[F' proAB lacIqZ M15 Tn10(Tetr)]) was used as the host cell for harbouring the DNA library, and E. coli CodonPlus (F- ompT hsdS [
] dcm+ Tetr gal endA Hte [argU proL Camr]) (Stratagene) for protein expression. Plasmid pUC18 was used as a cloning and expression vector.
Isolation of an endo-1,3-ß-glucanase gene from S. sioyaensis.
The extraction and manipulation of DNA were performed by standard protocols (Sambrook et al., 1989 ). A chromosomal DNA library of S. sioyaensis was created by inserting BamHI-cut DNA fragments (310 kb) into pUC18. The DNA library was then introduced into E. coli XL-1 Blue cells, and the cells were selected for growing on LuriaBertani (LB) agar plates containing ampicillin (100 µg ml-1), IPTG (0·25 mM) and X-Gal (0·004%, w/v). White colonies were then transferred onto agar medium containing curdlan (0·2%, w/v) and aniline blue (0·005%, w/v) for screening the 1,3-ß-glucanase-producing cells based on formation of a halo around the positive colony (Mahasneh & Stewart, 1980
).
Nucleotide sequence determination.
Both strands of the entire 3·1 kb DNA fragment containing the coding region for 1,3-ß-glucanase were sequenced on an ABI Prism 377-96 auto sequencer (Perkin-Elmer) with the BigDye terminator cycle sequencing ready reaction kit (Perkin-Elmer). The nucleotide sequence of the 3·1 kb DNA fragment and the deduced amino acid sequence of the endo-1,3-ß-glucanase are deposited in GenBank with the accession number AF217415.
Construction of 1,3-ß-glucanase expression vectors.
A putative ORF of 1437 bp was found within the cloned 3·1 kb DNA fragment. Primers 5'-GTCCGAATTCCCGAGAGGCCA-3' and 5'-CAGTTAAGCTTCTGGTGCAGCACGC-3' were designed to amplify the putative ORF in a 50 µl PCR reaction buffer containing primers (0·32 µM each), dNTP (0·2 mM each) and 2·5 units Pfu polymerase. The sequences in italic within the primers are the engineered cutting sites of EcoRI and HindIII, respectively, while the underlined sequence represents the putative ribosome-binding site. The PCR thermocycling was carried out for 35 cycles (94 °C, 1 min; 56 °C, 1 min; 72 °C, 3·5 min) followed by a 10 min extension at 72 °C. Another pair of primers, 5'-GTCCGAATTCCCGAGAGGCCA-3' and 5'-CCCGAAAGCTTGTCACCTGGACG-3', was used to amplify a 3'-deleted DNA fragment (
1·1 kb) under the same PCR conditions as described above. The amplified DNA fragments were cut with HindIII and EcoRI and then inserted into pUC18, to become expression vectors for the full-length and the C-terminally truncated enzymes, respectively.
Expression of 1,3-ß-glucanase.
A 2-ml overnight culture of E. coli CodonPlus cells harbouring expression vector was transferred into 200 ml LB medium containing ampicillin (100 µg ml-1) and chloramphenicol (34 µg ml-1) and shaken vigorously at 37 °C. To induce the expression of recombinant proteins, IPTG was added to a final concentration of 0·25 mM at a cell density of OD600 0·5 and the cultivation was continued for 15 h at 32 °C. Cells were then harvested by centrifugation (13000 g, 10 min), suspended in hypertonic buffer (20% sucrose, 33 mM Tris/HCl pH 7·0, 0·1 mM EDTA) and then osmotically shocked by replacing the buffer with 0·5 mM MgCl2 as described by Nossal & Heppel (1966)
to obtain the periplasmic proteins.
Purification of the recombinant proteins.
The periplasmic proteins in sodium acetate buffer (pH 4·0, 10 mM) were first applied to a HiTrap SP column (Pharmacia) that had been equilibrated with the same buffer. While most of the E. coli proteins adsorbed onto the column, the proteins with 1,3-ß-glucanase activity came out directly. The sodium acetate buffer of the enzyme solution was then replaced with potassium phosphate buffer (10 mM, pH 8·0) and the solution applied to a HiTrap Q column (Pharmacia). The proteins adsorbed on the column were eluted with NaCl-containing buffer in a stepwise manner. The activity of 1,3-ß-glucanase was found in three fractions after HiTrap Q chromatography. A degraded form of the enzyme with an apparent molecular mass of 37 kDa (designated as Curd3) was found in the flow-through fraction; another degraded enzyme with an apparent molecular mass of 41 kDa (designated as Curd2) and the matured full-length enzyme (53 kDa, designated as Curd1) were eluted with buffer containing 50 mM and 200 mM NaCl, respectively. Both Curd2 and Curd3 were homogeneous after the HiTrap Q step. To further purify Curd1, the buffer of the enzyme was replaced with potassium phosphate buffer (50 mM, pH 8·0) containing 1 M ammonium sulfate, and the mixture was applied to an octyl-Sepharose (Pharmacia) column. The hydrophobic column was washed with potassium phosphate buffer (50 mM, pH 8·0), and Curd1 was subsequently eluted with phosphate buffer (50 mM, pH 8·0) containing ethylene glycol (60%, v/v). The C-terminal carbohydrate-binding module (CBM)-truncated protein (designated as CT) was purified by the same procedure as used for Curd3. The authentic molecular masses of Curd1 and CT were subsequently determined by LC/MS.
N-terminal amino acid sequence.
The N-terminal amino acid sequences of the proteins were determined with a gas-phase protein sequencer (Applied Biosystems, model 476A).
Enzyme activity assay.
Unless otherwise stated, the standard activity assay for 1,3-ß-glucanase was carried out at 65 °C for 5 min, using 0·5% (w/v) laminarin in 50 mM sodium acetate buffer (pH 5·5). The glucose equivalents released from enzyme reactions were determined colorimetrically by the dinitrosalicylic acid method (Wood & Bhat, 1988 ). One unit of activity is defined as the amount of enzyme required to release 1 µmol glucose equivalents min-1 under the reaction conditions described above. Protein concentration was determined by the Coomassie blue method (Bradford, 1976
) using bovine serum albumin as the standard. To determine the kinetic constants, the initial velocity at varying laminarin concentration was measured, and the values of Km and Vmax were obtained from a LineweaverBurk plot.
Detection of 1,3-ß-glucanase on SDS-PAGE by activity staining.
The protein samples were heated at 96 °C for 3 min in loading buffer before they were separated on SDS-PAGE (10%, w/v, acrylamide). The separated proteins were then renatured by soaking the gel successively once in potassium phosphate buffer (50 mM, pH 7·0) containing 2-propanol (25%, v/v) , and twice in potassium phosphate buffer (50 mM, pH 7·0). Each soaking lasted for 30 min at room temperature. The gel was then overlaid with an 8 mm-thick agarose gel (2%, w/v) containing 0·5% (w/v) pachyman. After 12 h incubation at 37 °C, the proteins on the polyacrylamide gel were visualized by Coomassie blue staining, and the agarose gel was stained with Congo red (Béguin, 1983 ). The activity of 1,3-ß-glucanase was evidenced by the formation of a clear band against the dark brownish background on the agarose gel.
Binding activity assays.
Binding activity of the 1,3-ß-glucanase to insoluble polysaccharides was assessed by mixing 0·6 µg of the purified enzymes with various insoluble polysaccharides (2 mg) in 1·0 ml 50 mM sodium acetate buffer (pH 5·5) at 4 °C for 1 h. After centrifugation (1000 g, 5 min), the residual activity for laminarin hydrolysis in the supernatant was determined. A decrease of the enzymic activity in the supernatant indicates that a fraction of the enzyme is bound to the insoluble polysaccharides (Yamamoto et al., 1998 ).
The affinity electrophoresis method using polyacrylamide gels containing soluble polysaccharide (Tomme et al., 1996 ) was applied to analyse the binding potency of protein samples to soluble polysaccharides. To prepare polysaccharide-containing gels, soluble glucan (final concentration 0·1%, w/v) was added to the separating gel mixtures (7·5% acrylamide) prior to polymerization. Lichenan and xylan were heated at 70 °C for 20 min and 100 °C for 10 min, respectively, to increase their solubility before they were added into separating gel mixtures. Native polyacrylamide gels, with and without polysaccharide, were polymerized side-by-side, separated by an internal spacer, within the same glass plates. Each protein sample (2 µg) was loaded into gels, with and without polysaccharide, and run at 4 °C, 80 V, until the tracking dye was 2 cm from the bottom of the gel. After electrophoresis, proteins were visualized by Coomassie blue staining.
Detection of hydrolytic products.
The hydrolytic products of laminarin after treatment with 1,3-ß-glucanase were determined by TLC. The purified enzyme (0·6 µg) and laminarin (0·5%, w/v) were incubated in 1 ml sodium acetate buffer (50 mM, pH 5·5) at 65 °C for various time intervals. The reaction was stopped by repeated extraction with phenol/chloroform (1:1, v/v). Each 10 µl volume of the reaction products was then spotted on a silica plate (silica gel 60, Merck), developed with ethyl acetate/acetic acid/water (2:1:1, by vol.) (Sakellaris et al., 1990 ), and visualized by p-anisaldehyde (Fried & Sherma, 1982
).
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RESULTS |
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Analysis of the deduced amino acid sequence of the ORF revealed that the N terminus of the putative 1,3-ß-glucanase contains a typical signal peptide sequence and a predicted cleavage site for processing between Ala-48 and Ser-49 according to the method developed by von Heijne (1986) . A similarity search based on the BLASTP program (Altschul et al., 1997
) showed that the sequence following the signal peptide may represent a catalytic domain with 1,3-ß-glucanase activity classified in GHF 16 (Fig. 1a
). The sequence at the C terminus is predicted to form a potential CBM similar to members of CBM family 6 found in several xylanases (Fig. 1b
). Between these two functional domains is a polyglycine sequence.
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To examine the substrate specificity of the 1,3-ß-glucanase, various polysaccharides were tested at a concentration of 5 mg ml-1. The results (Table 1) showed that 1,3-ß-glucans (including laminarin, curdlan and pachyman) were the most favourable substrates, while 1,31,4-ß-glucan (lichenan) was hydrolysed to a lesser extent. It should be noted that lichenan needs to be pretreated (70 °C, 20 min) to be hydrolysed by the S. sioyaensis enzyme. Zymosan A was a relatively poor substrate among the ß-1,3-glucans tested. The enzyme did not show detectable activity against the remaining polysaccharides.
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To determine the mode of action, the digested products of laminarin were analysed by TLC (Fig. 4). Initially, products larger than laminaribiose appeared. As the reaction proceeded, glucose and laminaribiose gradually accumulated. Curd1, Curd2, Curd3 and CT all had similar patterns (data not shown). This demonstrates that the S. sioyaensis enzyme is an endo-1,3-ß-glucanase.
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DISCUSSION |
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The N-terminal leader sequence was processed properly in E. coli, and could direct the recombinant proteins into the periplasmic space. In addition to the matured full-length protein (Curd1), two smaller protein fragments (Curd2 and Curd3) were produced. Curd2 and Curd3 are proposed to be the C-terminally truncated versions of Curd1 because all three proteins have identical N-terminal amino acid sequences and similar activity, pH and temperature profiles; moreover, Curd3 and CT had very similar apparent molecular masses, activity profiles and substrate preferences. We hypothesize that the Streptomyces 1,3-ß-glucanase was subjected to attacks by E. coli proteinases at the linker region when secreted into the periplasmic space. The expression of the 1,3-ß-glucanase of B. circulans WL-12 (Watanabe et al., 1992 ) and of T. neapolitana (Zverlov et al., 1997
) in E. coli cells also resulted in the production of multiple forms of the recombinant protein.
The glycosyl hydrolase domain shares sequence similarity with bacterial 1,3-ß-glucanases of GHF 16 (Fig. 1a) and contains a conserved GELDIME motif, whose catalytic importance has been suggested by the crystal structure of a Bacillus 1,31,4-ß-glucanase (Keitel et al., 1993
; Hahn et al., 1995
), and by mutational analyses of 1,31,4-ß-glucanase (Juncosa et al., 1994
) and laminarinase (Krah et al., 1998
). Two subfamilies, 1,31,4-ß-glucanases (licheninases) (EC 3.2.1.73) and 1,3-ß-glucanases (EC 3.2.1.39), are classified within GHF 16 based mainly on substrate specificity. Licheninase catalyses the hydrolysis of 1,4-ß-glucosidic linkages only when the glucosyl residue is itself linked at the O-3 position, whereas 1,3-ß-glucanase primarily catalyses the hydrolysis of 1,3-ß-glucosidic linkages in 1,3-ß-glucans. The S. sioyaensis glucanase efficiently catalyses the hydrolysis of 1,3-ß-glucans of various origins except zymosan A. In comparison to 1,3-ß-glucans, lichenan was hydrolysed at a relatively slower rate. Taken together with the enzymes endolytic mode of action, we conclude that the glycosyl hydrolase domain should be classified as an endo-1,3-ß-glucanase (EC 3.2.1 . 39). Similar substrate preference has been found in other bacterial 1,3-ß-glucanases such as those from B. circulans (Okada et al., 1995
; Yahata et al., 1990
), P. furiosus (Gueguen et al., 1997
), Cellvibrio mixtus (Sakellaris et al., 1990
) and T. neapolitana (Zverlov et al., 1997
).
The CBM of the Streptomyces 1,3-ß-glucanase, dispensable for catalytic activity of the glycosyl hydrolase domain on laminarin, displays a high degree of homology with the CBMs classified in family 6 (Fig. 6). In general, members of CBM family 6 are linked to the xylanase domain and bind to xylan. For example, the CBM of Clostridium stercorarium xylanase A binds xylan and plays an important role in xylan hydrolysis (Sun et al., 1998
). To our knowledge, the CBM of the Streptomyces 1,3-ß-glucanase is the first instance of a family 6 CBM that associates with a 1,3-ß-glucanase domain. Binding assays demonstrated the binding of the C-terminal CBM to insoluble ß-glucans not only with 1,3- but also with 1,31,4- and 1,4- linkages (Table 2
). The strong binding of the C-terminal CBM to curdlan and pachyman probably accounts for the increase of the specific activities of Curd1 against these insoluble substrates, presumably by increasing the frequency of encounter events between the hydrolase domain and the substrates. A similar effect has been identified for Lam16A from T. neapolitana in that the presence of CBM4-2 enhanced the Lam16A activity towards gelatinized and insoluble or mixed-linkage 1,3-ß-glucan (Zverlov et al., 2001
). Despite not being hydrolysed by the glycosyl hydrolase domain, cellulose and insoluble lichenan could be bound by the C-terminal CBM. Polysaccharides in the natural environment could have very complicated structures containing various glycolytic linkages; therefore, with the ability to bind to various linkages, Curd1 should be able to function more efficiently in nature. With regard to soluble polysaccharide, gel-affinity electrophoresis showed a strong binding of the C-terminal CBM to laminarin. Binding of laminarin to the C-terminal CBM is non-productive from the point of view of the catalytic activity exerted by the hydrolase domain, and this may explain the slight increase of Km in Curd1. Soluble lichenan, xylan and CMC did not cause the retardation of either Curd1 or CT, suggesting that only a weak binding occurred (if it did occur) between the C-terminal CBM and these soluble polysaccharides. In summary, the C-terminal CBM is distinct from others of family 6 as regards binding preference, especially in its reluctant binding to xylan. The structural determinants for such difference deserve to be addressed in the future.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Béguin, P. (1983). Detection of cellulose activity in polyacrylamide gels using Congo red-stained agar replica. Anal Biochem 131, 333-336.[Medline]
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-254.[Medline]
Bucciaglia, P. A. & Smith, A. G. (1994). Cloning and characterization of Tag 1, a tobacco anther ß-1,3-glucanase expressed during tetrad dissolution. Plant Mol Biol 24, 903-914.[Medline]
Castresana, C., de Carvalho, F., Gheysen, G., Habets, M., Inze, D. & van Montagu, M. (1990). Tissue-specific and pathogen-induced regulation of a Nicotiana plumbaginifolia ß-1,3-glucanase gene. Plant Cell 2, 1131-1143.
Chang, M. M., Culley, D. E. & Hadwiger, L. A. (1993). Nucleotide sequence of a pea (Pisum sativum L.) ß-1,3-glucanase gene. Plant Physiol 101, 1121-1122.
Chen, C. H., Huang, J. W. & Tzeng, D. S. (2000). Development of PMS 502-Streptomyces biopesticide and evaluation of its efficacy on the control of crop fungal diseases. Plant Pathol Bull 9, 193 (in Chinese).
Chye, M. L. & Cheung, K. Y. (1995). ß-1,3-Glucanase is highly expressed in laticifers of Hevea brasiliensis. Plant Mol Biol 29, 397-402.[Medline]
de la Cruz, J., Pintor-Toro, J. A., Benitez, T., Llobell, A. & Romero, L. C. (1995). A novel endo-ß-1,3-glucanase, BGN13.1, involved in the mycoparasitism of Trichoderma harzianum. J Bacteriol 177, 6937-6945.[Abstract]
de Loose, M., Alliotte, T., Gheysen, G., Genetello, C., Gielen, J., Soetaert, P., van Montagu, M. & Inze, D. (1988). Primary structure of a hormonally regulated ß-glucanase of Nicotiana plumbaginifolia. Gene 70, 13-23.[Medline]
Doi, K. & Doi, A. (1986). Cloning and expression in Escherichia coli of the gene for an Arthrobacter 1,3-ß-glucanase. J Bacteriol 168, 1272-1276.[Medline]
Ferrer, P., Halkier, T., Hedegaard, L., Savva, D., Diers, I. & Asenjo, J. A. (1996). Nucleotide sequence of a ß-1,3-glucanase isoenzyme IIA gene of Oerskovia xanthineolytica LL G109 (Cellulomonas cellulans) and initial characterization of the recombinant enzyme expressed in Bacillus subtilis. J Bacteriol 178, 4751-4757.[Abstract]
Fontes, C. M. G. A., Clarke, J. H., Hazlewood, G. P., Fernandes, T. H., Gilbert, H. J. & Ferreira, L. M. A. (1998). Identification of tandemly repeated type VI cellulose-binding domains in an endoglucanase from the aerobic soil bacterium Cellvibrio mixtus. Appl Microbiol Biotechnol 49, 552-559.
Fried, B. & Sherma, J. (1982). Thin-layer Chromatography: Techniques and Applications. New York: Marcel Dekker.
Grenier, J., Potvin, C. & Asselin, A. (1993). Barley pathogenesis-related proteins with fungal cell wall lytic activity inhibit the growth of yeasts. Plant Physiol 103, 1277-1283.
Gueguen, Y., Voorhorst, W. G. B., van der Oost, J. & de Vos, W. M. (1997). Molecular and biochemical characterization of an endo-ß-1,3-glucanase of the hyperthermophilic archaeon Pyrococcus furiosus. J Biol Chem 272, 31258-31264.
Hahn, M., Keitel, T. & Heinemann, U. (1995). Crystal and molecular structure at 0·16-nm resolution of the hybrid Bacillus endo-1,31,4-ß-D-glucan 4-glucanohydrolase H (A16-M). Eur J Biochem 232, 849-858.[Abstract]
Henrissat, B. (1991). A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 280, 309-316.[Medline]
Henrissat, B. & Bairoch, A. (1993). New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 293, 781-788.[Medline]
Hudspeth, R. L., Hobbs, S. L., Anderson, D. M. & Grula, J. W. (1996). Characterization and expression of chitinase and 1,3-ß-glucanase genes in cotton. Plant Mol Biol 31, 911-916.[Medline]
Juncosa, M., Pons, J., Dot, T., Querol, E. & Planas, A. (1994). Identification of active site carboxylic residues in Bacillus licheniformis 1,31,4-ß-D-glucan 4-glucanohydrolase by site-directed mutagenesis. J Biol Chem 269, 14530-14535.
Keitel, T., Simon, O., Borriss, R. & Heinemann, U. (1993). Molecular and active-site structure of a Bacillus 1,31,4-ß-glucanase. Proc Natl Acad Sci USA 90, 5287-5291.[Abstract]
Krah, M., Misselwitz, R., Politz, O., Tomsen, K. K., Welfle, H. & Borriss, R. (1998). The laminarinase from thermophilic eubacterium Rhodothermus marinus: conformation, stability, and identification of active site carboxylic residues by site directed mutagenesis. Eur J Biochem 257, 101-111.[Abstract]
Mahasneh, A. M. & Stewart, D. J. (1980). A medium for detecting ß-(1,3)-glucanase activity in bacteria. J Appl Bacteriol 48, 457-458.
Mohagheghpour, N., Dawson, M., Hobbs, P. & 7 other authors (1995). Glucans as immunological adjuvants. Adv Exp Med Biol 383, 1322.[Medline]
Nossal, N. G. & Heppel, L. A. (1966). The release of enzyme by osmotic shock from Escherichia coli in exponential phase. J Biol Chem 241, 3055-3062.
Oh, H. Y. & Yang, M. S. (1995). Nucleotide sequence of genomic DNA encoding the potato ß-1,3-glucanase. Plant Physiol 107, 1453.
Okada, T., Aisaka, M., Aida, K., Nikaidou, N., Tanaka, H. & Watanabe, T. (1995). Structure of the gene encoding ß-1,3-glucanase B of Bacillus circulans WL-12. J Ferment Bioeng 80, 229-236.
Rios-Hernandez, M., Dos-Santos, N., Silvia-Cardoso, J., Bello-Garciga, J. L. & Pedroso, M. (1994). Immunopharmacological studies of ß-1,3-glucan. Arch Med Res 25, 179-180.[Medline]
Ryan, E. M. & Ward, O. P. (1985). Study of the effect of ß-1,3-glucanase from basidiomycete QM 806 on yeast extract production. Biotechnol Lett 7, 409-412.
Sakellaris, H., Pemberton, J. M. & Manners, J. M. (1990). Gene from Cellvibrio mixtus encoding a ß-1,3-endoglucanase. Appl Environ Microbiol 56, 3204-3208.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Shen, S. H., Chretien, P., Bastien, L. & Slilaty, S. N. (1991). Primary sequence of the glucanase gene from Oerskovia xanthineolytica: expression and purification of the enzyme from Escherichia coli. J Biol Chem 15, 1058-1063.
Spilliaert, R., Hreggvidsson, G. O., Kristjansson, J. K., Eggertsson, G. & Palsdottir, A. (1994). Cloning and sequencing of a Rhodothermus marinus gene, bglA, coding for a thermostable ß-glucanase and its expression in Escherichia coli. Eur J Biochem 224, 923-930.[Abstract]
Sun, J. L., Sakka, K., Karita, S., Kimura, T. & Ohmiya, K. (1998). Adsorption of Clostridium stercorarium xylanase A to insoluble xylan and the important of the CBDs to xylan hydrolysis. J Ferment Bioeng 85, 63-68.
Sun, L., Gurnon, J. R., Adams, B. J., Graves, M. V. & van Etten, J. L. (2000). Characterization of a ß-1,3-glucanase encoded by chlorella virus PBCV-1. Virology 276, 27-36.[Medline]
Tomme, P., Creagh, A. L., Kilburn, D. G. & Haynes, C. A. (1996). Interaction of polysaccharides with the N-terminal cellulose-binding domain of Cellulomonas fimi CenC. I. Binding specificity and calorimetric analysis. Biochemistry 35, 13885-13894.[Medline]
von Heijne, G. (1986). A new method for predicting signal sequence cleavage sites. Nucleic Acids Res 14, 4683-4690.[Abstract]
Watanabe, T., Kasahara, N., Aida, K. & Tanaka, H. (1992). Three N-terminal domains of ß-1,3-glucanase A1 are involved in binding to insoluble ß-1,3-glucan. J Bacteriol 174, 186-190.[Abstract]
Wood, T. M. & Bhat, K. M. (1988). Methods for measuring cellulase activities. Methods Enzymol 160, 87-112.
Yahata, N., Watanabe, T., Nakamura, Y., Yamamoto, Y., Kamimiya, S. & Tanaka, H. (1990). Structure of the gene encoding ß-1,3-glucanase A1 of Bacillus circulans WL-12. Gene 86, 113-117.[Medline]
Yamamoto, M., Ezure, T., Watanabe, T., Tanaka, H. & Aono, R. (1998). C-terminal domain of ß-1,3-glucanase H in Bacillus circulans IAM1165 has a role in binding to insoluble ß-1,3-glucan. FEBS Lett 433, 41-43.[Medline]
Yi, S. Y. & Hwang, B. K. (1997). Purification and antifungal activity of a basic 34 kDa ß-1,3-glucanase from soybean hypocotyls inoculated with Phytophthora sojae f. sp. glycines. Mol Cells 7, 408-413.[Medline]
Zverlov, V. V., Volkov, I. Y., Velikodvorskaya, T. V. & Schwarz, W. H. (1997). Highly thermostable endo-1,3-ß-glucanase (laminarinase) LamA from Thermotoga neapolitana: nucleotide sequence of the gene and characterization of the recombinant gene product. Microbiology 143, 1701-1708.[Abstract]
Zverlov, V. V., Volkov, I. Y., Velikodvorskaya, G. A. & Schwarz, W. H. (2001). The binding pattern of two carbohydrate-binding modules of laminarinase Lam16A from Thermotoga neapolitana: differences in ß-glucan binding within family CBM4. Microbiology 147, 621-629.
Received 3 October 2001;
revised 5 December 2001;
accepted 10 December 2001.