Expression, purification and characterization of a cold-adapted dextranase from marine bacteria and its ability to remove dental plaque.

Dental plaque is a high-incidence health concern, and it is caused by Streptococcus mutans. Dextranase can specifically hydrolyze ɑ-1,6-glycosidic linkages in dextran. It is commonly used in the sugar industry, in the production of plasma substitutes, and the treatment and prevention of dental plaque. In this research work, we successfully cloned and expressed a cold-adapted dextranase from marine bacteria Catenovulum sp. DP03 in Escherichia coli. The recombinant dextranase named Cadex2870 contained a 2511 bp intact open reading frame and encoded 836 amino acids. The expression condition of recombinant strain was 0.1 mM isopropylthio-galactoside (IPTG), and the reduced temperature was 16 °C. The purified enzyme activity was 16.2 U/mg. The optimal temperature and pH of Cadex2870 were 45 °C and pH 8, and it also had catalytic activity at 0 °C. The hydrolysates of Cadex2870 hydrolysis Dextran T70 are maltose, maltotetraose, maltopentose, maltoheptaose and higher molecular weight maltooligosaccharides. Interestingly, 0.5% sodium benzoate, 2% xylitol, 0.5% sodium fluoride, 5% propanediol, 5% glycerin and 2% sorbitol can enhance stability Cadex2870, which are additives in mouthwashes. Additionally, Cadex2870 reduced the formation of dental plaque and effectively degraded formed plaque. Therefore, Cadex2870 shows great promise in commercial applications.

Dextranase production by recombinant Pichia pastoris under operational volumetric mass transfer coefficient (kLa) and volumetric gassed power input (Pg/V) attainable at commercial large scale.

Most of the reported bioprocesses carried out by the methylotrophic yeast Pichia pastoris have been performed at laboratory scale using high power inputs and pure oxygen, such conditions are not feasible for industrial large-scale processes. In this study, volumetric mass transfer (kLa) and volumetric gassed power input (Pg/V) were evaluated within values attainable in large-scale production as scale-up criteria for recombinant dextranase production by MutS P. pastoris strain. Cultures were oxygen limited when the volumetric gassed power supply was limited to 2 kW m-3. Specific growth rate, and then dextranase production, increased as kLa and Pg/V did. Meanwhile, specific production and methanol consumption rates were constant, due to the limited methanol condition also achieved at 2 L bioprocesses. The specific dextranase production rate was two times higher than the values previously reported for a Mut+ strain. After a scale-up process, at constant kLa, the specific growth rate was kept at 30 L bioprocess, whereas dextranase production decreased, due to the effect of methanol accumulation. Results obtained at 30 L bioprocesses suggest that even under oxygen-limited conditions, methanol saturated conditions are not adequate to express dextranase with the promoter alcohol oxidase. Bioprocesses developed within feasible and scalable operational conditions are of high interest for the commercial production of recombinant proteins from Pichia pastoris.

Purification and Characterization of a Biofilm-Degradable Dextranase from a Marine Bacterium.

This study evaluated the ability of a dextranase from a marine bacterium Catenovulum sp. (Cadex) to impede formation of Streptococcus mutans biofilms, a primary pathogen of dental caries, one of the most common human infectious diseases. Cadex was purified 29.6-fold and had a specific activity of 2309 U/mg protein and molecular weight of 75 kDa. Cadex showed maximum activity at pH 8.0 and 40 °C and was stable at temperatures under 30 °C and at pH ranging from 5.0 to 11.0. A metal ion and chemical dependency study showed that Mn2+ and Sr2+ exerted positive effects on Cadex, whereas Cu2+, Fe3+, Zn2+, Cd2+, Ni2+, and Co2+ functioned as inhibitors. Several teeth rinsing product reagents, including carboxybenzene, ethanol, sodium fluoride, and xylitol were found to have no effects on Cadex activity. A substrate specificity study showed that Cadex specifically cleaved the α-1,6 glycosidic bond. Thin layer chromatogram and high-performance liquid chromatography indicated that the main hydrolysis products were isomaltoogligosaccharides. Crystal violet staining and scanning electron microscopy showed that Cadex impeded the formation of S. mutans biofilm to some extent. In conclusion, Cadex from a marine bacterium was shown to be an alkaline and cold-adapted endo-type dextranase suitable for development of a novel marine agent for the treatment of dental caries.

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

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.

Purification, characterization, and application of a thermostable dextranase from Talaromyces pinophilus.

Dextranase can hydrolyze dextran to low-molecular-weight polysaccharides, which have important medical applications. In the study, dextranase-producing strains were screened from various soil sources. The strain H6 was identified as Talaromyces pinophilus by a standard ITS rDNA analysis. Crude dextranase was purified by ammonium sulfate fractionation and Sepharose 6B chromatography, which resulted in a 6.69-fold increase in the specific activity and an 11.27% recovery. The enzyme was 58 kDa, lower than most dextranase, with an optimum temperature of 45 °C and an optimum pH of 6.0, and identified as an endodextranase. It was steady over a pH range from 3.0 to 10.0 and had reasonable thermal stability. The dextranase activity was increased by urea, which enhanced its activity to 115.35% and was conducive to clinical dextran production. Therefore, T. pinophilus H6 dextranase could show its superiority in practical applications.

Development of thermostable dextranase from Streptococcus mutans (SmdexTM) through in silico design employing B-factor and Cartesian-ΔΔG.

The thermal stability of enzymes dramatically limits their application in the industrial field. Based on the crystal structure, we conducted a semi-rational design according to the B-factor and free energy values to improve the stability of dextranase from Streptococcus mutans (SmdexTM). The B-factor values of Asn102, Asn503, Asp501 and Asp500 were the highest predicted by B-FITTER. Then Rosetta was used to simulate the saturation mutations of Asn102, Asn503, Asp501 and Asp500. The mutated amino acid was designed according to the change of acG. The results showed that the thermal stability of N102P, N102C, D500G, and D500T was improved, and the half-lives of N102P/D500G and N102P/D500T at 45 °C were increased to 3.14 times and 2.44 times, respectively. Analyzing the interaction of amino acids by using Discovery Studio 4.5, it was observed that the thermal stability of dextranase was improved due to the increase in hydrophobicity and the number of hydrogen bonds of the mutant enzyme. The catalytic efficiency of N102P/D500T was increased. Compared with the hydrolyzed products of SmdexTM, the mutant enzymes do not change the specificity of hydrolysates.

A Review of the Role of Probiotic Supplementation in Dental Caries.

Dental diseases are among the common health issues experienced around the world. Dental caries is one of the most predominant oral diseases worldwide. Major factors associated with caries development include poor oral hygiene, the content of specific carbohydrates in the diet, dental biofilm formation, the cariogenic microbial load, reduction in salivary flow, insufficient fluoride exposure, gingival recession, genetic factors, and lack of personal attention to one's dental health. Several preventive measures have been implemented to reduce the risk of the development of caries. Probiotics are live microbes that when administered in suitable amounts confer health benefits on the host; they are recognized as potential adjunct therapeutic agents for several diseases. The present manuscript summarizes recent findings on the role of probiotics in dental caries prevention and the possible mechanisms of probiotic effects. Review of the literature indicates the regular consumption of probiotic products significantly reduced the risk of caries by inhibiting cariogenic bacteria and enriching commensal microbes in the oral cavity. Buffering the salivary pH, production of bacteriocin and enzymes (dextranase, mutanase, and urease), the capacity of competing for the adhesion and colonization on tooth surfaces are the possible mechanisms behind the beneficial effect of probiotics. Further studies are necessary to address the efficacy of long-term probiotic supplementation on the control of dental diseases and the influence of childhood probiotic supplementation on the risk of caries development.

Rational introduction of disulfide bond to enhance optimal temperature of Lipomyces starkeyi alpha-dextranase expressed in Pichia pastoris.

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 wildtype dextranase, the mutant DexM2 (D279C/S289C) showed a more than 13oC 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 Vmax.

[Conditions for dextranase formation by Paecilomyces lilacinus].

Induced formation conditions of dextranase by Paecilomyces lilacinus were investigated. Effect of various carbohydrates on dextranase formation was examined, dextran was the best C-source and as an inducer. The effect of dextran with different molecular weight (from 17.2 to 1000 kD) on dextranase formation was compared, productivity of dextranase increased with increase of dextran molecular weight. When dextran of 1000 kD was used as C-source. The enzyme formation was 40% higher than that 17.2 kD dextran. When other sugars were separately added to the medium with dextran, the enzyme formation was repressed. Besides C-source, the other optimum conditions of dextranase formation were as follows: N-source, beef peptone; medium initial pH, 6.0-7.0; culture temperature, 28 degrees C; inoculum amount about 10%, and the organism was cultivated for 6 days on 200 r/min shaker in 250 ml flasker with 50 ml medium.

[Conditions of dextranase formation by Penicillium funiculosum 15]. / Usloviia obrazovaniia dekstranazy Penicillium funiculosum 15

The influence of the following factors on the synthesis of extracellular dextranase by Pen. funiculosum 15 has been studied: the quantity and age of the inoculum, pH of the cultivation medium, stimulants of the microbial growth, cultivation temperature and time. The optimal amount of dextranase has been found to form under the following conditions: inoculum--3 day mycelium constituting 4%, cultivation time--4 to 7 days, temperature--28 to 29 degrees C, initial pH of the medium--6.0.

[Formation of dextranases by mycelial fungi and actinomycetes]. / Obrazovanie dekstranaz mitselial'nymi gribami i aktinomitsetami

The dextranase activity of cultures of mycelial fungi of different genera and actinomycetes from the Chromogenes species of the Actinomyces genus was studied. About one third of the mycelial fungi and 8% of actinomycetes showed dextranase activity. The resulting extracellular dextranases demonstrated on endotypic pattern of action on the substrate. Actinomycete dextranases were several times more active than fungal dextranases and exhibited a significant activity in the neutral and weakly alkaline medium. Highly productive strains that are promising as dextranase producers were isolated.

Dextranase: hyper production of dextran degrading enzyme from newly isolated strain of Bacillus licheniformis.

Dextranase, 6-alpha-D-glucan 6-glucanohydrolase catalyzes the degradation of dextran (polymer of D-glucose) in to low molecular weight fractions. Dextranolytic bacterial strains were isolated from various natural sources and plate assay methods were developed for screening of highest extracellular dextranase producing isolate. Bacillus licheniformis, identified on the basis of taxonomic characterization was subjected to UV radiation and highest enzyme producing mutant obtained led to 7 times more dextranase production than wild. Optimization of major physico-chemical parameters affecting enzyme production; including medium composition, pH, cultivation time and temperature revealed that maximum enzyme production was obtained in a self designed medium (pH 6.0) containing 1% Dextran 5000 Da, after 24 h culture incubation at 37 °C. Dextranase reported in this study is of great commercial importance as it is strictly inducible in nature and B. licheniformis being non-pathogenic removes the safety concerns associated with production of dextran fractions for clinical and pharmaceutical usage.

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.