• Journal of Periodontal and Implant Science
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• v.32 no.3
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• pp.665-683
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• 2002

A novel glucanhydrolase from a mutant of Lipomyces starkeyi KSM 22 has additional amylase activity besides mutanolytic activity and has been suggested as promising anti-plaque agent. It has been shown effective in hydrolysis of mutan, reduction of mutan formation by Streptococcus mutans and removal pre-formed sucrose-dependent adherent microbial film and has been strongly bound to hydroxyapatitie. These in vitro properties of Lipomyces starkeyi KSM 22 glucanhydrolase are desirable for its application as a dental plaque control agent. In human experimental gingivitis　model and 6 month clinical trial, mouthrinsing with Lipomyces　starkeyi KSM 22 dextranase was comparable to 0.12% chlorhexidine mouthwash in inhibition of plaque accumulation and gingival inflammation and local side effect was negligible. This study was aimed to evaluate the cytotoxic effect of Lipomyces starkeyi KSM 22 glucanhydrolase on human gingival fibroblasts. Primary culture of human gingival fibroblasts at the 4th to 6th passages were used. Glucanhydrolase solution was made from lyophilized glucanhydrolase powder from a mutant of Lipomyces stakeyi KSM 22 solved in PBS and added to DMEM medium to the final concentration of 0.5, 1, and 2 unit. Cells were exposed to glucanhydrolase solution or 0.1 % chlorhexidine and the cells cultured in DMEM with 10% FBS and 1% antibiotics as control. After exposure, the morphological change, cell attachment, and cell activity by MTT assay were evaluated in 0.5, 1.5, 3, 6, 24 hours after treatment. The cell proliferation and cell activity was also evaluated at 2 and 7 days after 1 minute exposure, twice a day. The cell morphology was similar between the Lipomyces smkeyi KSM 22 glucanhydrolase groups and control group during the incubation periods, while most fibroblasts remained as round cell regardless of incubation time in the chlorhexidine group. The numbers of the attached cells in the glucanhydrolase groups were comparable to that of control and significantly higher than the chlorhexidine group. The numbers of the proliferated cells in the glucanhydrolase groups at 7 days of incubation were comparable to the control group and higher than the chlorhexidine group. The cell activity in glucanhydrolase groups paralleled with the increased cell number by attachment and proliferation. According to these results, Lipomyces starkeyj KSM 22 glucanhydrolase has little harmful effect on attachment and proliferation of human gingival fibroblasts, in contrast to 0.1% chlorhexidine which was cytotoxic to human gingival fibroblasts. Therefore this glucanhydrolase preparation is considered as a safe and promising agent for new mouthwash formula in the near future.

• Journal of Periodontal and Implant Science
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• v.34 no.1
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• pp.195-204
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• 2004

A novel glucanhydrolase from Lipomyces starkeyi KSM 22 has been suggested as a promising anti-plaque agent because it has been shown to have additional amylase activity and mutanase activity besides dextranase activity and to strongly bind to hydroxyapatite. Mouthrinsing with Lipomyces starkeyi KSM 22 glucanhydrolase solution was comparable to 0.12% chlorhexidine mouthwash in inhibition of plaque accumulation and gingival inflammation and local side effects were less frequent and less intense in human experimental gingivitis. In this study, Lipomyces starkeyi KSM 22 glucanhydrolase mouthrinses (1 and 2 unit/ml) were compared with a control mouthrinse (commercial 0.01% benzethonium chloride mouthrinse, $Caregargle^{(R)}$, Hanmi Pharmaceuticals) in the ability to inhibit plaque formation. A 3-replicate clinical trial using 4-day plaque regrowth model was used. Fifteen volunteers were rendered plaque-free on the 1st day of each study period, ceased toothcleansing, and rinsed 2X daily with allocated mouthrinse thereafter. On day 5, plaque accumulation was scored and the washout periods was 9 days for the next trial. Lipomyces starkeyi KSM22 glucanhydrolase(1 unit and 2 unit)- containing mouthrinse resulted in Significantly lower plaque formation in plaque area and thickness, compared to the control mouthrinse. There was no significant difference in plaque inhibition between enzyme-mouthrinses at 2 different concentrations used. This glucanhydrolase- containing mouthwash resulted in significantly lower plaque area severity index score and tended to have lower plaque thickness severity index score than those of control mouthrinse. But there was no significant difference according to the enzyme concentration. From these results, Lipomyces starkeyi KSM 22 glucanhydrolase-containing benzethonium chloride mouthrinse has greater anti-plaque effect than the commercial mouthrinse alone. Therefore this glucanhydrolase preparation is a promising agent for new mouthwash formulation in the near future.

• Journal of Microbiology and Biotechnology
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• v.12 no.6
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• pp.993-997
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• 2002

A glucanhydrolase (a DXAMase exhibiting both dextranolytic and amylolytic activities) from Lipomyces starkeyi KSM 22 hydrolyzed polysaccharides having ${\alpha}-(1{\rightarrow}3)-,\;{\alpha}(1{\rightarrow}4)-,\;and\;{\alpha}-(1{\rightarrow}6)$-D-glucosidic linkages. The oral hygiene benefits of DXAMase-containing mouthwash were examined in relation to human experimental gingivitis during a 3-week period without brushing. The DXAMase-treated group exhibited a lower increase in plaque accumulation and gingival index score than the chlorhexidine-treated group. The DXAMase-treated group also showed less tongue accumulation, bad taste, and tooth staining, thus indicating a positive role for DXAMase as an antiplaque agent ingredient.

• Journal of Periodontal and Implant Science
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• v.31 no.2
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• pp.401-420
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• 2001

A novel glucanhydrolase from a mutant of Lipomyces starkeyi(KSM 22)has been shown effective in hydrolysis of mutan, reduction of mutan formation by Streptococcus mutans and removal pre-formed sucrose-dependent adherent microbial film and Lipomyces starkeyi KSM 22 dextranase has been strongly bound to hydroxyapatitie. These in vitro properties of Lipomyces starkeyi KSM 22 dextranase are desirable for its application as a dental plaque control agent. This study was performed to determine oral hygiene benefits and safety of dextranase(Lipomyces starkeyi KSM 22 dextranase)-containing mouthwash in human experimental gingivitis. This 3-week clinical trial was placebo-controlled double-blind design evaluating 1U/ml dextranase mouthwash and 0.12% chlorhexidine mouthwash. A total 39 systemically healthy subjects, who had moderate levels of plaque and gingivitis were included. At baseline, 1, 2 and 3 weeks, subjects were scored for plaque(Silness and $L{\ddot{o}e$ plaque index and plaque severity index), gingivitis($L{\ddot{o}e$ and Silness gingival index), and at baseline and 3 weeks of experiment, subjects were scored for plaque(Turesky-Quingley-Hein's plaque index and plaque severity index), tooth stain(Area and severity index system by Lang et al). Additionally, oral mucosal examinations were performed and subjects questioned for adverse symptoms. Two weeks after pre-experiment examinations and a professional prophylaxis, the subjects provided with allocated mousewash and instructed to use 20-ml volumes for 30s twice dailywithout toothbrushing. All the groups showed significant increase in plaque accumulation since 1 week of experiment. During 3 weeks' period, the dextranase group showed the least increase in plaque accumulation of Silness and $L{\ddot{o}e$ plaque index, compared to the chlorhexidine and placebo groups, but chlorhexidine group showed the least increase inplaque accumulation of Turesky-Quingley-Hein's plaque index. As for gingival inflammation, all the groups showed significant increase during 3 weeks of experiment. The dextranase group also showed the least increase in gingival index score, compared to the chlorhexidine as well as the placebo groups. Whereas the tooth stain was increased significantly in the chlorhexidine group, compared to the baseline score and the placebo group since 3 weeks of mouthrinsing. It was significantly increased after 3 weeks in the dextranase group, still less severe than the chlorhexidine group. As for the oral side effect, the dextranase group showed less tongue accumulation, bad taste, compared to the chlorhexidine group. From these results, mouthrinsing with Lipomyces starkeyi KSM 22 dextranase was comparable to 0.12% chlorhexidine mouthwashin inhibition of plaque accumulation and gingival inflammation and local side effects were if anything less frequent and less intense than chlorhexidine, in human experimental gingivitis. All data had provided positive evidence for Lipomyces starkeyi KSM 22 dextranase as an antiplaque agent and suggested that further development of dextranase formulations for plaque control are warranted.

• Journal of Periodontal and Implant Science
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• v.31 no.2
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• pp.371-388
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• 2001

A novel glucanhydrolase(DXAMase) from a mutant of Lipomyces starkeyi(KSM 22) has been shown effective in hydrolysis of mutan, reduction of mutan formation by Streptococcus mutans and removal pre-formed sucrose-dependentadherent microbial film and DXAMase has been strongly bound to hydroxyapatitie. These in vitro properties of Lipomyces starkeyi DXAMase are desirable for its application as a dental plaque control agent. This study was performed to determine the adjunctive oral hygiene benefits and safety of dextranase(Lipomyces starkeyi KSM 22 DXAMase)-containing mouthwash when used alongside normal tooth-brushing. This 6-month clinical trial was placebo-controlled double-blind design evaluating 1U/ml dextranase mouthwash and 0.12% chlorhexidine mouthwash. A total 39 systemically healthy subjects, who had moderate levels of plaque and gingivitis were included. At baseline, 1, 3 and 6 months, subjects were scored for plaque accumulation(Turesky modification of Quingley-Hein's plaque index), gingivitis status($L\ddot{o}e$ and Silness gingival index), and tooth stain(Area and severity index system by Lang et al). Additionally, oral mucosal examinations were performed and subjects questioned for adverse symptoms. Two weeks after pre-experiment examinations and a professional prophylaxis, the subjects provided with allocated mousewash and instructed to use 20-ml volumes for 30s twice daily after toothbrushing. All the groups showed significant increase in plaque accumulation since 1 month of experiment. During 6 months' period, the Dextranase mouthwash group showed the least increase in plaque accumulation, compared to the Chlorhexidine mouthwash and placebo groups. As for gingival inflammation, all the groups showed significant increase during 6 months of experiment. The Experimental group(Dextranase mouthwash) also showed the least increase in gingival index score, compared to the Positive control(Chlorhexidine mouthwash)as well as the Negative control(placebo)groups. Whereas the tooth stain was increased significantly in the Positive control group, compared to the baseline score and the Negative controlgroup since 3 months of mouthrinsing. It was significantly increased after 6 months in the Experimental group, still less severe than the Positive control group. As for the oral side effect, the Experimental group showed less tongue accumulation, bad taste, compared to the Positive control group. From these results, mouthrinsing with Lipomyces starkeyi KSM 22 dextranase provided adjunctive benefits to toothbrushing, comparable to 0.12% chlorhexidine mouthwash in inhibition of plaque accumulation and gingival inflammation and local side effects were if anything less frequent and less intense than chlorhexidine, with long-term use of the mouthwash. All data had provided positive evidence for Lipomyces starkeyi KSM 22 dextranase as an antiplaque agent and suggested that further development of dextranase formulations for plaque control are warranted.

• Journal of Microbiology and Biotechnology
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• v.13 no.6
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• pp.993-997
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• 2003

Response surface methodology was applied to find the optimum conditions for the production of DXAMase (containing both dextranase and amylase activities) based on the cultivation variables (pH, temperature, and agitation rate). The experimental values from the model equation conceded with predicted values in which the predicted values for dextranase and amylase activities were 2.26 and 3.52 U/ml at pH 4, $28^{\circ}C$, 235 rpm, and the corresponding experimental values were 2.41 and 3.68 U/ml, respectively.