A simplified and miniaturized glucometer-based assay for the detection of β-glucosidase activity / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

β-Glucosidase activity assays constitute an important indicator for the early diagnosis of neonatal necrotizing enterocolitis and qualitative changes in medicinal plants. The drawbacks of the existing methods are high consumption of both time and reagents, complexity in operation, and requirement of expensive instruments and highly trained personnel. The present study provides a simplified, highly selective, and miniaturized glucometer-based strategy for the detection of β-glucosidase activity. Single-factor experiments showed that optimum β-glucosidase activity was exhibited at 50 °C and pH 5.0 in a citric acid-sodium citrate buffer when reacting with 0.03 g/mL salicin for 30 min. The procedure for detection was simplified without the need of a chromogenic reaction. Validation of the analytical method demonstrated that the accuracy, precision, repeatability, stability, and durability were good. The linear ranges of β-glucosidase in a buffer solution and rat serum were 0.0873-1.5498 U/mL and 0.4076-2.9019 U/mL, respectively. The proposed method was free from interference from β-dextranase, snailase, β-galactosidase, hemicellulase, and glucuronic acid released by baicalin. This demonstrated that the proposed assay had a higher selectivity than the conventional dinitrosalicylic acid (DNS) assay because of the specificity for salicin and unique recognition of glucose by a personal glucose meter. Miniaturization of the method resulted in a microassay for β-glucosidase activity. The easy-to-operate method was successfully used to detect a series of β-glucosidases extracted from bitter almonds and cultured by Aspergillus niger. In addition, the simplified and miniaturized glucometer-based assay has potential application in the point-of-care testing of β-glucosidase in many fields, including medical diagnostics, food safety, and environmental monitoring.

Statistical tools application on dextranase production from Pochonia chlamydosporia (VC4) and its application on dextran removal from sugarcane juice

ABSTRACT The aim of this study was to optimize the dextranase production by fungus Pochonia chlamydosporia (VC4) and evaluate its activity in dextran reduction in sugarcane juice. The effects, over the P. chlamydosporia dextranase production, of different components from the culture medium were analyzed by Plackett-Burman design and central composite design. The response surface was utilized to determine the levels that, among the variables that influence dextranase production, provide higher production of these enzymes. The enzymatic effect on the removal of dextran present in sugarcane juice was also evaluated. It was observed that only NaNO3 and pH showed significant effect (p<0.05) over dextranase production and was determined that the levels which provided higher enzyme production were, respectively, 5 g/L and 5.5. The dextranases produced by fungus P. chlamydosporia reduced by 75% the dextran content of the sugarcane juice once treated for 12 hours, when compared to the control treatment.

Research progress in dextranase / 生物工程学报

Dextranase can degrade dextran polymer into low molecular weight polysaccharide. Dextranase and its hydrolysates are widely used in food, medicine and chemical industries. Studies on dextranase progresses rapidly in recent years. We reviewed literature reports combined with our study about the progress of dextranase and its potential applications in industry. In addition, we addressed hot topics and emphasized on the current research about dextranase, existing problems in domesticstudies and the future research needs needs.

Production of a novel halophilic dextranase from a honey isolate, bacillus subtilis NRC-B233 under solid-state fermentation

A GRAM-POSITIVE, sporulating halophilic bacteria, designated NRC-B233, was isolated from the honey produced in Saudi Arabia. It was identified by the 16-23S intergenic region as Bacillus subtilis NRC-B233. Screening of the wastes and agro-products for dextranase production under solid state fermentation showed that corn flour was the best substrate [61.323 U/g]. The optimum conditions for dextranase productions were 37°C, pH 9, 32 hr incubation period, and 200% moisture content. The most favorable nitrogen and carbon sources for enzyme production were 2% peptone and 5% starch [1076.768, 1553.364 U/g]. respectively. A unique character of this isolate is its ability to continuously produce dextranase in the absence and presence of NaCl 5-20 g/l. The addition of 0.175 Mm CrCl[3] increased the dextranase production about 4.5 fold. The enzyme has been partially purified about 112-fold from crude extract by only two purification steps involving ultra-filtration. The purified dextranase showed its maximum activity at pH 9.2 and 70°C. It retained fill activity [100%] at 75°C for one hour. Dextranase activity increased about four fold in the presence of 10% NaCl. On the other hand, CaCl[2] [0.050M], EDTA [0.100M], and KCI [0.100M] had great influence in enzyme activity. The enzyme showed variable degradation effects on different types of dextran and its derivatives. These results suggest that the dextranase secreted by Bacillus subtilis NRC-B233 is industrially important from the perspectives of its activity at across pH range [5.0-100], its thermo-activity in addition to its halophilic character and its ability to degrade different types of alpha-1,4 and alpha-1,6 glycosidic linkages

Frequency of Species and Biotypes of Mutans Streptococci Isolated from Dental Plaque in the Adolescents and Adults

The purpose of this study is to survey the frequency of mutans streptococci species and biotypes isolated from dental plaque in Korean adolescents and adults and the relationship between species of mutans streptococci and DMFT index. The dental plaque samples were collected from the anterior and molar teeth of both of jaws in the 47 human subjects (aged 14 to 29.3 years, average age was 20.2 years). A dental examination was performed for DMFT with the WHO caries diagnostic criteria. The mutans streptococci were cultured selectively on mitis-salivarius bacitracin (MSB) agar plates. The bacterial colonies growing on the MSB plates were then identified with biochemical tests (for biotyping) as well as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) targeting dextranase gene (dex) (for the determination of species). The data showed that the prevalence of Streptococcus mutans, S. sobrinus, and S. downei were 93.6%, 12.8%, and 8.5%, respectively. The biotype I (87.4%) and biotype IV (19.1%) were the most frequently detected. The DMFT scores of adolescents and adults harboring both S. mutans and S. sobrinus were significantly higher than those for S. mutans alone (p < 0.05). Above results revealed that S. mutans and biotype I were frequently detected in Korean adolescents and adults. In addition, the results suggest that subjects having plaques compared of both S. mutans and S. sobrinus may be more susceptible to caries than those having S. mutans alone.

Identification of Mutans Streptococci isolated from dental plaque between the bracket and tooth surface in orthodontic patients / 대한치과교정학회지

The aim of this study was to compare the species and biotypes of mutans streptococci isolated from dental plaques sampled from the interfaces between the bracket and tooth surface and smooth tooth surfaces in orthodontic patients. Dental plaque was collected from the interfaces between brackets and teeth (test group), and from smooth tooth surfaces distant from brackets by more than 2 mm (control group). The dental plaque collected by a sterilized curette was transferred into a vial of 1 X PBS. The sample in the vial was vigorously vortexed for 1 min and plated on mitis-salivarius bacitracin (MSB) agar plate using cotton tips. The agar plates were incubated at 37 degrees C in a candle jar for 2 days, and again incubated for 1 more day at anambient temperature. Individual colonies were cultured in TH broth at 37 degrees C CO2 incubator. The PCR-RFLP based on dextranase gene was performed for the identification of mutans streptococci at the species-level. For biotyping of mutans streptococci, biochemical tests were performed. There was no significant difference of the species of mutans streptococci isolated from both test and control groups. However, the biotypes of the mutans streptococci isolated from test and control groups were different. These results may offer the basic data to verify the relationship between the mutans streptococci biotype and enamel decalcification or dental caries in orthodontic patients with fixed appliances.

Direct detection of cariogenic streptococci in metal brackets in vivo using polymerase chain reaction / 대한치과교정학회지

Streptococcus mutans and Streptococcus sobrinus are major etiological agents in enamel demineralization around orthodontic appliances. This study was designed to examine the prevalence of these streptococci on orthodontic brackets in vivo using polymerase chain reaction. Four incisor brackets in the upper and lower arches were removed and collected from 80 patients at the time of debonding. The genomic DNA of adhered bacteria was extracted and each dextranase gene of S. mutans and S. sobrinus was amplified using the specific oligonucleotide primers. The results showed that the maxillary incisor brackets were colonized by both cariogenic streptococci to a somewhat higher degree than that taken from the mandible. The prevalence of S. mutans was 50.0% on the maxillary incisor brackets and 33.8% on the mandibular incisor brackets, and that of S. sobrinus was 17.5% and 15.0%, respectively. Both species were detected on the maxillary incisor brackets of 7 patients (8.8%) and the mandibular incisor brackets of 5 patients (6.3%). These results suggest that cariogenic streptococci can adhere to the incisor brackets and may be resident species on the incisor brackets.

Effect of mouthrinse containing Lipomyces starkeyi KSM 22 glucanhydrolase on plaque formation during a 4-day period / 대한치주과학회지

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 KSM 22 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.

Effects of Lipomyces starkeyi KSM 22 Glucanhydrolase on human gingival fibroblasts / 대한치주과학회지

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 sucrosedependent 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 starkeyi 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 starkeyi 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 starkeyi KSM 22 glucanhydrolase has little harmful effect on attach-ment 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.

A Clinical Trial of Dextranase-Containing Mouthwash on the Inhibition of Plaque Formation and Gingivitis / 대한치주과학회지

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 toothbrushing. 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(Loe 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 gingi-val 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.

The Effect of Dextranase-Containing Mouthwash in Human Experimental Gingivitis / 대한치주과학회지

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 Loe plaque index and plaque severity index), gingivitis(Loe 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 Loe 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.

Purificación y caracterización parcial de una enzima dextranasa a partir de una cepa del hongo del género Penicillium / Purification and partial characterization of a destranase enzyme from a fungal strain of the genero Penicillium

Una fracción dextranasa (EC 3.2.1.11) excretada por un hongo del género Penicillium, fue purificada después de cinco días de inducción de la enzima en cultivo sumergido en agitación a 28-C. El crudo enzimático fue precipitado con 80 % de sulfato de amonio, resuspendido en tampóm acetato y aplicado en cromatografía sucesivas de filtración por el gel e intercambio iónico. La fracción homogénea de dextrabasa está formada por dos bandas de proteinas superpuestas con un peso molecular aproximado de 67 000 Da, un nivel de glicosidación entre 15-18 % y un punto isoeléctrico a pH 3,88. La actividad específica osciló entre 1 500 y 2 000 U/mg de proteína con un máximo de actividad a pH 5

Optimization of dextranase synthesis by a locally isolated fusarium moniliforme

Maximum dextranase synthesis was obtained by the locally isolated F. moniliforme when grown aerobically on a medium containing dextran, 2% as a carbon source, ammonium nitrate in 500 mg N/l as nitrogen source, adjusted to pH 5.5 and inoculated with 5% inoculum in the form of a 4 days mycelium after 8 days incubation. The fungus gave 66.45 units/ml dextranase activity with 9.53 units/mig mycelium. The optimum enzyme activity was found to be at pH 5 and temperature 55°