Isolation and properties of levanase from Streptococcus salivarius KTA-19

Takahashi, Nobuhiro; Mizuno, Fumiko; Takamori, Kenshiro

doi:10.1128/iai.42.1.231-236.1983

Cited by 22 publications

(13 citation statements)

References 35 publications

(50 reference statements)

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“…The specific levan-degrading activity of 194 U/mg (30 mg of dissolved levan per ml at 55ЊC) found with the purified levanase protein is lower but in the same order of magnitude as reported for other microbial levanases. In comparison, levanases from S. salivarius KTA-19 (38) and Actinomyces viscosus ATCC 19246 (16) and the nonspecific exo-␤-fructosidase from S. salivarius KTA-19 (39) yielded levan-degrading activities of about 850, 851, and 433 U/mg at 37ЊC with 1 mg of dissolved levan per ml.…”

Section: Discussionmentioning

confidence: 99%

Purification and characterization of the Bacillus subtilis levanase produced in Escherichia coli

Wanker

Huber

Schwab

1995

Appl Environ Microbiol

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The enzyme levanase encoded by the sacC gene from Bacillus subtilis was overexpressed in Escherichia coli with the strong, inducible tac promoter. The enzyme was purified from crude E. coli cell lysates by salting out with ammonium sulfate and chromatography on DEAE-Sepharose CL-6B, S-Sepharose, and MonoQ-Sepharose. The purified protein had an apparent molecular mass of 75,000 Da in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which is in agreement with that expected from the nucleotide sequence. Levanase was active on levan, inulin, and sucrose with K m values of 1.2 M, 6.8 mM, and 65 mM, respectively. The pH optimum of the enzyme acting on inulin was 5.5, and the temperature optimum was 55؇C. Levanase was rapidly inactivated at 60؇C, but activity could be retained for longer times by adding fructose or glycerol. The enzyme activity was completely inactivated by Ag ؉ and Hg 2؉ ions, indicating that a sulfhydryl group is involved. A ratio of sucrase to inulinase activity of 1.2 was found for the purified enzyme with substrate concentrations of 50 mg/ml. The mechanism of enzyme action was investigated. No liberation of fructo-oligomers from inulin and levan could be observed by thin-layer chromatography and size exclusion chromatography-low-angle laser light scattering-interferometric differential refractive index techniques. This indicates that levanase is an exoenzyme acting by the single-chain mode. Enzymes involved in the hydrolysis of polyfructans are of interest both for fundamental studies and for industrial applications. Especially inulin is of growing interest as a renewable carbohydrate raw material for biotechnology. Two aspects are of main importance: (i) production of pure fructose syrups, so-called high-fructose inulin syrups, by enzymatic hydrolysis of inulin (43) and (ii) direct fermentation of inulin by employing inulinase-producing microbes in order to synthesize various products such as ethanol or aceton-butanol (20, 21). ␤-D-Fructofuranosidases are usually classified upon their ability to hydrolyze levan (levanases), inulin (inulinases), and also the disaccharide sucrose (sucrases and invertases). However, many of these enzymes are capable of hydrolyzing more than one type of these substrates. Inulinases (or inulases) which are specific for inulin have been isolated only from Jerusalem artichoke tubers (7, 12), whereas levanases which are specific for levan have been isolated from bacteria only. Examples are the levanases of Streptococcus salivarius KTA-19 (38) and Actinomyces viscosus ATCC 19246 (16). Conversely, a variety of nonspecific ␤-D-fructofuranosidases have been found in bacteria, yeasts, and fungi. For example, inulinases and levanases which are capable of hydrolyzing inulin, levan, and sucrose have been isolated from Bacillus subtilis (17), Actinomyces viscosus ATCC 15987 (24), Streptococcus mutans (5), Kluyveromyces fragilis (35), Chrysosporium pannorum (46, 47) and Penicillium sp. strain (27). Enzymes active on inulin and sucrose but not on levan have been found in f...

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Section: Discussionmentioning

confidence: 99%

Purification and characterization of the Bacillus subtilis levanase produced in Escherichia coli

Wanker

Huber

Schwab

1995

Appl Environ Microbiol

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“…Fractions (5 ml) were collected at a flow rate of 15 ml/h. Peak I comprises fractions 19 to 21 as reported previously (24), and peak II contains fractions 29 to 31. (B) Bio-Gel A1.5m rechromatography.…”

Section: Methodsmentioning

confidence: 61%

“…S. salivarius KTA-19 for enzyme purification and KT-12 for levan preparation were isolated from dental plaque in our laboratory. These organisms were cultivated as described previously (24).…”

Section: Methodsmentioning

confidence: 99%

“…Thin-layer chromatography. Samples were applied to an Avicel SF (microcrystalline cellulose-acetate film) plate (Funakoshi Pharmaceutical Co. Ltd., Tokyo) and developed with n-butanol-pyridine-water (6:4:3 [vol/vol/vol]) as described previously (24). The sugar spots were visualized by spraying alkaline silver nitrate solution on the plate.…”

Section: Methodsmentioning

confidence: 99%

“…Column resins and protein samples were initially equilibrated with 20 mM phosphate buffer (pH 7.0). The cell extract was obtained from the culture supernatant of S. salivarius KTA-19 precipitated with ammonium sulfate and was followed by acetone fractionation as reported previously (24). The extract (629 mg of protein) was dissolved in 10 ml of the buffer and submitted to chromatography on a Bio-Gel A5m column (2.5 by 100 cm; Bio-Rad Laboratories, Richmond, Calif.).…”

Section: Methodsmentioning

confidence: 99%

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Purification and preliminary characterization of exo-beta-D-fructosidase in Streptococcus salivarius KTA-19

Takahashi¹,

Mizuno²,

Takamori³

1985

Infect Immun

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Streptococcus salivarius fructosidase (0-D-fructan fructohydrolase, EC 3.2.1.80) was purified to homogeneity. The molecular weight of the fructosidase was estimated to be 83,000 to 85,000 by gel filtration and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pH optimum of the enzyme was 7.0, and the isoelectric point was pH 4.7. The purified enzyme preparation hydrolyzed levan, inulin, and several 2-4-linkage-containing oligosaccharides such as sucrose and raffinose, but not melezitose, dextran, and pseudonigeran. The fructosidase was inhibited by Fe3+, Cu2+, Hg2+, and Ag+, but not by Ca2+, Co2+, Mg2+, and Zn2+, at a concentration of 10-3 M. Mn2+ was particularly effective in stimulating activity at the same concentration. The presence of either EDTA or KCN also increased fructosidase activity by 20 to 30%. The enzyme was susceptible to sulfhydryl reagents since p-chloromercuribenzoate (10-7 M) produced 63% inhibition of the activity. However, this inhibition was overcome in the presence of cysteine. This enzyme acts as an exofructosidase since thin-layer chromatographic analysis revealed that D-fructose was formed from levan or inulin by the action of the enzyme.

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Levanase

Schomburg

Salzmann

1991

Enzyme Handbook 4

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Isolation and properties of levanase from Streptococcus salivarius KTA-19

Cited by 22 publications

References 35 publications

Purification and characterization of the Bacillus subtilis levanase produced in Escherichia coli

Purification and characterization of the Bacillus subtilis levanase produced in Escherichia coli

Purification and preliminary characterization of exo-beta-D-fructosidase in Streptococcus salivarius KTA-19

Levanase

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