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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Production, characterization and (co-)immobilization of dextranase from Penicillium aculeatum.

Fermentation kinetics of Penicillium aculeatum ATCC 10409 demonstrated that fungal growth and dextranase release are decoupled. Inoculation by conidia or mycelia resulted in identical kinetics. Two new isoenzymes of the dextranase were characterized regarding their kinetic constants, pI, MW, activation energy and stabilities. The larger enzyme was 3-fold more active (turnover number: 2,230 +/- 97 s(-1)). Pre-treatment of bentonite with H(2)O(2) did not affect adsorption characteristics of dextranase. Enzyme to bentonite ratios above 0.5:1 (w/w) resulted in a high conservation of activity upon adsorption. Furthermore, dextranase could be used in co-immobilizates for the direct conversion of sucrose into isomalto-oligosaccharides (e.g. isomaltose). Yields of co-immobilizates were 2-20 times that of basic immobilizates, which consist of dextransucrase without dextranase.

Enzymic, spectroscopic and calorimetric studies of a recombinant dextranase expressed in Pichia pastoris.

Conformational stability and structural characterization of an rDex (recombinant dextranase) expressed in Pichia pastoris were studied by enzymic assays, fluorescence, CD and DSC (differential scanning calorimetry). We also identified two disulphide bridges (Cys9-Cys14, Cys484-Cys488) and two free Cys residues (Cys336, Cys415) that are not conserved between bacterial and fungal dextranases of GH-49 (glycoside hydrolase family 49) by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS. Enzymic and fluorescence studies revealed that rDex is biological and conformationally stable at acidic pH, with maximum activity at pH 4.5-5.0, while CD spectra indicated a secondary structure basically composed of beta-sheets. rDex loses biological activity at neutral pH without total disruption of its conformation. In addition, rDex preserves its conformation close to 60 degrees C, but it is thermally denatured with appreciable aggregation at temperatures above 75 degrees C. DSC studies always displayed irreversible transitions and a strong dependence on the scan rate. Our combined analysis suggested that the denaturation process of rDex is under kinetic control, which is described reasonably well by the two-state kinetic scheme.

Induced systemic resistance (ISR) against pathogens in the context of induced plant defences.

Induced systemic resistance (ISR) of plants against pathogens is a widespread phenomenon that has been intensively investigated with respect to the underlying signalling pathways as well as to its potential use in plant protection. Elicited by a local infection, plants respond with a salicylic-dependent signalling cascade that leads to the systemic expression of a broad spectrum and long-lasting disease resistance that is efficient against fungi, bacteria and viruses. Changes in cell wall composition, de novo production of pathogenesis-related-proteins such as chitinases and glucanases, and synthesis of phytoalexins are associated with resistance, although further defensive compounds are likely to exist but remain to be identified. In this Botanical Briefing we focus on interactions between ISR and induced resistance against herbivores that is mediated by jasmonic acid as a central signalling molecule. While many studies report cross-resistance, others have found trade-offs, i.e. inhibition of one resistance pathway by the other. Here we propose a framework that explains many of the thus far contradictory results. We regard elicitation separately from signalling and from production, i.e. the synthesis of defensive compounds. Interactions on all three levels can act independently from each other.

Carbon source regulation of a dextranase gene from the filamentous fungus Penicillium minioluteum.

The regulation of dextranase (dexA) gene expression in the filamentous fungus Penicillium minioluteum grown on different carbon sources was studied. Growth in the presence of dextran leads to high expression of the dextranase enzyme, but growth in starch, glucose, glycerol, lactose and sorbitol did not. Dextran induced dexA gene expression at the mRNA level. However, in cultures containing dextran plus glucose or glycerol, the transcript was detected 24 h later than in the case where dextran was the only carbon source. When the glucose or glycerol concentration in the dextran-containing medium was kept at about 1% (w/v), no dextranase-transcripts were detected. It was found that both glucose and glycerol inhibited enzyme synthesis, because 1% (w/v) addition of both carbon sources to dextran-growing cultures was able to abolish the inducing effect of dextran. Our results suggest that dextran utilization responds to both specific induction and to glucose and glycerol repression, providing evidence that P. minioluteum dexA expression is regulated by the carbon source at the transcriptional level.

Characterization of the dextranase gene (dex) of Streptococcus mutans and its recombinant product in an Escherichia coli host.

The gene (dex), which encodes the Streptococcus mutans dextranase (Dex), was cloned in Escherichia coli. The E. coli host harboring a recombinant plasmid (pSD2) containing an 8-kb BamHI insert produced a Dex protein of 133 kDa as well as smaller enzymes of 118, 104, and 88 kDa. The Dex produced by the recombinant E. coli was apparently located in the cytoplasmic fraction, not in the periplasmic nor the extracellular fractions. Subcloning and deletion analysis of pSD2 showed that the structural gene of Dex was encoded by a 4-kb BamHI-SalI fragment. The fragment also contained the dex promoter which was effective in the E. coli cell.

Biochemical properties of Streptococcus sobrinus reisolates from the gastrointestinal tract of a gnotobiotic rat.

Streptococcus sobrinus strain 6715-13-201 was inoculated into the oral cavity of a gnotobiotic rat and then reisolated from different portions of the gastrointestinal tract. Fourteen isolates, selected on the basis of their colonial morphology, were then screened for their ability to adhere to saliva-coated hydroxyapatite (SHA) in vitro, and their ability to produce extracellular polysaccharide from sucrose, and low pH in glucose broth. Certain isolates were also tested for their cariogenic potential as monoinfectants in gnotobiotic rats. All isolates differed in their abilities to adhere to SHA, with most showing an increased level of adhesion in the presence of sucrose, but this did not correlate with their ability to be aggregated by dextran. Most isolates were capable of producing glucosyltransferases (with only one exception) and dextranases (also one exception). There was more variability in the production of dextranase inhibitor. No isolate was capable of producing dextranase inhibitor in the absence of dextranase production. There were no correlations between the ability of isolates to adhere in vitro or produce/utilize polysaccharides and their ability to produce caries in vivo. Due to the differences between strains in their abilities to adhere, produce polysaccharides, utilize polysaccharides or produce a low pH and the lack of correlation between any of these parameters and cariogenicity, the results suggest that the ability of strains to colonize and produce caries depends on a number of different characteristics, no one of which is essential.

[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.

Some properties of an endodextranase inhibitor from continuous cultures of Streptococcus sobrinus.

Cell-free filtrates of Streptococcus sobrinus, cultured at low growth rate in the chemostat, contain a dextranase inhibitor that can completely inhibit the activity of S. sobrinus endodextranase. The range of conditions under which inhibition occurs, and the situations in which enzyme activity can reappear, have been examined in continuous cultures of strain 6715-13WT and the dextranase-deficient mutant 6715-13-201. A purified preparation of the inhibitor was specific for S. sobrinus dextranase, having no action on dextranases from other oral streptococci. The percentage inhibition of S. sobrinus dextranase varied with the enzyme concentration, and the complete inhibition of low amounts of enzyme indicated a very tight bond between the inhibitor and the enzyme.