• Journal of Microbiology and Biotechnology
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• v.9 no.3
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• pp.260-264
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• 1999

The combined activities of dextranase and amylase(DXAMase) from Lipomyces starkeyi KSM 22 produced from starch fermentation inhibited or prevented dental plaque formation. The activities were stable in commercial mouthwash products; DXAMase activity retained over 93％ of original activity after 6 months at 23℃. We examined the effects of enzyme inhibitors and active ingredients in mouthwash on DXAMase activity. The DXAMase was stable with 0.29％(w/v) EDTA, 20％ (v/v) ethanol, 0.05％ (w/v) fluoride, and 0.05％ (w/v) SDS. Among the active ingredients of mouthwash, sodium benzoate (up to 1 ％, w/v) had no inhibitory effect on either dextranase or amylase activity. In the case of cetylpyridinium chloride, the addition of 0.05％ (w/v) inhibited 6％ of dextranase activity and 13％ of amylase activity. Propylene glycol (up to 1％, w/v) showed no inhibitory effect on either enzyme activity. DXAMase (5 IU/㎖) in mouthwash could remove pre-formed films of glucan-bound S. mutans cells. The addition of 0.1 IU/㎖ DXAMase in mouthwash prevented the formation of insoluble-glucan. These in vitro properties of L. starkeyi KSM 22 DXAMase are desirable for its application as a dental plaque control agent.

• 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.

• KSBB Journal
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• v.17 no.6
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• pp.586-589
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• 2002

Lipomyces starkeyi KSM 22 produces dextranase and amylase (DXAMase). The addition of 0.02％ (w/v) 2-deoxy-D-glucose or 0.5% (w/v) glycerol into a 1% (w/v) starch medium increased the final activity of DXAMase produced 2.5 fold or 2.4 fold, respectively, compared to that of a 1% (w/v) starch medium. This activity was similar to that produced with 1% (w/v) dextran. The stability of purified OXAMase at 40$\^{C}$ for 3 weeks was tested using the various enzyme stabilizers. With the addition of 25% (v/v) glycerol, 90.9% of initial activity was left after 3 weeks. For practical use, the addition of 1% (v/v) glycerol with 50 mM of CaCl$_2$ or KH$_2$PO$_4$was adequate and maintained 73.4% of the initial activity under the test conditions used.

• Biotechnology and Bioprocess Engineering:BBE
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• v.8 no.2
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• pp.106-111
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• 2003

A novel carbohydrolase, which is a DXAMase, containing both dextranase and amylase equivalent activities, was purified from Lipomyces starkeyi KSM22. The purified DXAMase was also found to hydrolyze cellobiose, gentiobiose, trehalose and melezitose, while disproportionation reactions were exhibited with various di- and tri-saccharides, such as maltose, isomaltose, gentiobiose, kojibiose, sophorose, panose, maltotriose, and isomaltotriose with various kinds of oligosaccharides produced as acceptor reaction products. Furthermore, the purified DXAMase hydrolyzed raw waxy rice Starch and produced maltodextrin to the extent of 50% as a glucose equivalent.

• Journal of Microbiology and Biotechnology
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• v.19 no.2
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• pp.172-177
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• 2009

A gene(lsd1) encoding dextranase from Lipomyces starkeyi KSM22 has been previously cloned, sequenced, and expressed in Saccharomyces cerevisiae. The gene consisting of 1,824 base pairs and encoding a protein of 608 amino acids was then cloned into and secretively expressed in Pichia pastoris under the control of the AOX1 promoter. The dextranase productivity of the P. pastoris transformant(pPIC9K-LSD1, 134,000 U/I) was approximately 4.2-fold higher than that of the S. cerevisiae transformant(pYLSD1, 32,000 U/I) cultured in an 8-1 fermentor. Over 0.63 g/l of active dextranase was secreted into the medium after methanol induction. The dextranase of the P. pastoris transformant, as analyzed by SDS-PAGE and Western blotting, showed only one homogeneous band. This dextranase of the P. pastoris transformant showed a broad band near 73 kDa. Rabbit monoclonal antibodies against a synthetic LSD1 peptide mix also recognized approximately 73 kDa.

• 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.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.402-406
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• 2005

Lipomyces starkeyi produces a novel glucanhydrolase containing endo-dextranase and ${\alpha}-amylase$ activities. A cDNA from L. starkeyi encoding a dextranase was isolated and characterized. The 2,052 kb cDNA fragment (lsd1) carrying dextranase gene showed one open reading frame (ORF) composed of 1,824 bp flanked by a 41 bp 5'-UTR and a 184 bp 3'-UTR including a poly(A) tail of 27 bp. The ORF encodes for a 608 amino acid with a predicted molecular mass of 67.6 kDa. There was 77% deduced amino acid sequence identity between the LSD1 dextranase and the dextranase from Penicillium minioluteum. The primary structure of the dextranase from L. starkeyi has distant similarity with enzymes belonging to glycosyl hydrolase family 49. The lsd1 protein was expressed in the Saccharmyces cerevisiae under control of GAL1 promoter and active dextranase was produced.

• Journal of Microbiology and Biotechnology
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• v.19 no.12
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• pp.1506-1513
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• 2009

$\alpha$-Dextranase, which can hydrolyze dextran, is largely used in the sugar industry. However, a thermostable $\alpha$-dextranase is needed to alleviate the viscosity of syrups and clean blocked machines. Thus, to improve the optimal temperature of Lipomyces starkeyi $\alpha$-dextranase expressed by Pichia pastoris, the rational introduction of a de novo designed disulfide bond was investigated. Based on the known structure of Penicillium minioluteum dextranase, L. starkeyi $\alpha$-dextranase was constructed using homology modeling. Four amino acids residues were then selected for site-directed mutagenesis to cysteine. When compared with the wild-type dextranase, the mutant DexM2 (D279C/S289C) showed a more than $13^{\circ}C$ improvement on its optimal temperature. DexM2 and DexM12 (T245C/N248C, D279C/S289C) also showed a better thermal stability than the wild-type dextranase. After the introduction of two disulfide bonds, the specific activity of DexM12 was evaluated and found to be two times higher than that of the wild-type. Moreover, DexM12 also showed the highest $V_{max}$.

• 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.

• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.211-214
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• 2002

The optimal condition for the production of DXAMase, containing the both characteristics of dextranase and amylase, was studied based on different levels of pH, temperature, and aeration rate. Response surface methodology was applied to find the optimatic condition showing the relationship between the fermentation response(dextranase and amylase activity of DXAMase) and the fermentation variables(pH, temperature, and agitation rate). In case of dextranase activity, the condition of pH 4.06, $28.08^{\circ}C$, and 235.14 rpm showed the highest activity, 2.26 U/ml, and for amylase activity, the condition of pH 4.01, $27.96^{\circ}C$, and 212.01 rpm showed the highest activity, 3.52 U/ml. For the production of DXAMase, dextranase and amylase, the optimum condition was pH 4.06, $28.08^{\circ}C$, and 234.80 rpm.