Dextranase Enzyme Research Articles

Combined of ultrasound irradiation with high hydrostatic pressure (US/HHP) as a new method to improve immobilization of dextranase onto alginate gel

In this research work, dextranase was immobilized onto calcium alginate beads by the combination of ultrasonic irradiation and high hydrostatic pressure (US/HHP) treatments. Effects of US/HHP treatments on loading efficiency and immobilization yield of dextranase enzyme onto calcium alginate beads were investigated. Furthermore, the activities of immobilized enzymes prepared with and without US/HHP treatments and that prepared with ultrasonic irradiation (US) and high hydrostatic pressure (HHP), as a function of pH, temperature, recyclability and enzyme kinetic parameters, were compared with that for free enzyme. The maximum loading efficiency and the immobilization yield were observed when the immobilized dextranase was prepared with US (40W at 25kHz for 15min) combined with HHP (400MPa for 15min), under which the loading efficiency and the immobilization yield increased by 88.92% and 80.86%, respectively, compared to immobilized enzymes prepared without US/HHP treatment. On the other hand, immobilized enzyme prepared with US/HHP treatment showed Vmax, KM, catalytic and specificity constants values higher than that for the immobilized enzyme prepared with HHP treatment, indicated that, this new US/HHP method improved the catalytic kinetics activity of immobilized dextranase at all the reaction conditions studied. Compared to immobilized enzyme prepared either with US or HHP, the immobilized enzymes prepared with US/HHP method exhibited a higher: pH optimum, optimal reaction temperature, thermal stability and recyclability, and lower activation energy, which, illustrating the effectiveness of the US/HHP method. These results indicated that, the combination of US and HHP treatments could be an effective method for improving the immobilization of enzymes in polymers.

Application of statistical design for the optimization of dextranase production by a novel fungus isolated from Red Sea sponge.

Marine endophytic fungi isolated from Red Sea organisms were screened for the production of dextranase enzyme. The most potent isolate was from the Red Sea sponge Callyspongia spp. and was selected for identification. The18S rRNA amplification for phylogenetic study revealed that the isolate was highly related to Aspergillus flocculosus strain NRRL 5224 by 99 %. Medium composition and culture conditions for dextranase production were optimized by response surface methodology. A significant influence of dextran, yeast extract, K2HPO4, NaNO3, NaCl, MgSO4.7H2O and culture requirements such as incubation time, inoculum size, medium volume and inoculum age on dextranase production was evaluated by Plackett–Burman design. The most significant factors were further optimized using Box–Behnken design. The model predicted a dextranase activity of 438.15 U/ml when dextran concentration, medium volume and incubation time were 2.1 g/l, 52.47/250 ml flask and 80.48 h, respectively. Verification of the model showed that dextranase production of 440 U/ml was observed under the optimal condition confirming the validity of the model.

Influence of the probiotic Streptococcus salivarius strain M18 on indices of dental health in children: a randomized double-blind, placebo-controlled trial

The prevalence of dental caries continues to increase, and novel strategies to reverse this trend appear necessary. The probiotic Streptococcus salivarius strain M18 offers the potential to confer oral health benefits as it produces bacteriocins targeting the important cariogenic species Streptococcus mutans, as well as the enzymes dextranase and urease, which could help reduce dental plaque accumulation and acidification, respectively. In a randomized double-blind, placebo-controlled study of 100 dental caries-active children, treatment with M18 was administered for 3 months and the participants were assessed for changes to their plaque score and gingival and soft-tissue health and to their salivary levels of S. salivarius, S. mutans, lactobacilli, β-haemolytic streptococci and Candida species. At treatment end, the plaque scores were significantly (P = 0.05) lower for children in the M18-treated group, especially in subjects having high initial plaque scores. The absence of any significant adverse events supported the safety of the probiotic treatment. Cell-culture analyses of sequential saliva samples showed no differences between the probiotic and placebo groups in counts of the specifically enumerated oral micro-organisms, with the exception of the subgroup of the M18-treated children who appeared to have been colonized most effectively with M18. This subgroup exhibited reduced S. mutans counts, indicating that the anti-caries activity of M18 probiotic treatments may be enhanced if the efficiency of colonization is increased. It was concluded that S. salivarius M18 can provide oral health benefits when taken regularly.

Impact of Dextranase on Sugar Manufacturing and its Kinetic on the Molecular Weights of Remaining Dextran

In this work, we investigated the influence of dextranase enzyme on the molecular weight parameters of remaining dextran and intrinsic viscosity after different enzymatic treatments at different steps during sugar manufacturing. A spectrophotometric method was used to determine the relative activity of dextranase and the result has been confirmed by measured reducing sugar using HPLC system. For comparison, the action patterns of concentrated and diluted enzymes were additionally included in the experiments. Addition of dextranase to juice were much more efficient and economical to reduce the Mw of remaining dextran than adding it to evaporator syrups. Addition of dextranase at juice pH 5.5 showed similar minimum Mw with the lowest intrinsic viscosity, observed at 55.0 °C, and activities decreased after 20°Brix. The highest dextran removal was observed at dextranase concentration at 100 ppm/juice which was resulted in 80.29 % removal dextran in the juice, Moreover, the higher the level of concentrated dextranase applied to the juice, the more the removal of dextran occurred. In addition, the longer the availability of the residence time in the factory, the lower dextran Mw has been observed. To reach a satisfactory level of dextran hydrolysis, it was necessary to correct the dose of dextranase enzyme according to the losses of activity caused by the high °Brix.

Hydrolytic and chromatographic studies on the PEGylation of dextranase from Penicillium sp.

Dextranases catalyze the hydrolysis of the α-l,6-glucosidic bond of the polysaccharide dextran. Dextranases have been isolated from bacteria, yeast and fungi. Purified dextranase enzyme from Penicillium sp . was PEGylated (polyethylene glycol modification) with mPEG (5000 Da) and showed an increase in the dextranase protein molecular weight as estimated by Superose 12 (23 ml) column and this increment in the molecular weight is directly proportional to mPEG (5000 Da) concentration until a complete dextranase enzyme PEGylation (disappearance of dextranase peak). The residual activity of partially PEGylated dextranase (mPEG 5000 of 5.8 mg/ml) was 33.8% and for the completely PEGylated dextranase (mPEG 5000 of 29 mg/ml) it was 25.75%. Dextranase PEGylated with mPEG (30,000 Da) showed a little PEGylation at mPEG concentration of 5.8 mg/ml but at a concentration of 29 mg/ml several PEGylated peaks were produced with a difference in dextranase activity toward dextran T500, retardation in the activity with the increasing in the molecular weight was clearly appeared with Sephadex G75 but for Sephadex G200 a little retardation than Sephadex G75 has been appeared.

Enzyme immobilisation in permselective microcapsules

The objective of this investigation was to study the permselective behaviour of calcium alginate membranes, including the modifying effects of silica additives, which were subsequently used as microcapsule shells. Diffusion experiments and HPLC were carried out to ascertain the size-exclusion property of the membranes for a mixed molecular-weight dextran solution. Hollow microcapsules containing the enzyme dextranase were prepared using double concentric nozzles and the encapsulation performance was evaluated based on an analysis of the enzyme reactivity and stability. To improve mass transport within the microcapsules, magnetic nanoparticles were introduced into the liquid core and agitated using an alternating external magnetic field. The modified membranes exhibited better size-exclusion behaviour than the unmodified membranes. The magnetic nanoparticles slightly improved mass transport inside the microcapsule. The encapsulated enzyme yielded nearly 80% of the free enzyme activity and retained about 80% of the initial catalytic activity even after being used for eight reaction cycles.

Release of paclitaxel from pH sensitive and biodegradable dextran based hydrogels

Two different dextran based pH sensitive and enzyme degradable hydrogels: dextran maleic acid (Dex-MA), and glycidyl methacrylated dextran (Dex-GMA) were synthesized for oral delivery of nanoparticles. Poorly water soluble drug paclitaxel nanoparticles (PAX NPs) were prepared as the model, and encapsulation efficiency of paclitaxel in the nanoparticles was optimized to 43%. The prepared hydrogels were stable in simulated gastric fluid, but were prone to swelling and degradation in the presence or absence of enzyme dextranase in simulated intestinal fluid. Release profiles of nanoparticles could be tuned from 5.5 h to as long as 24 h under simulated human GI conditions. The release of PAX NPs (110 ± 10 nm) was completed with longer time (45 h–120 h). Two possible release mechanisms were discussed for Dex-MA and Dex-GMA-co-AA hydrogels respectively: degradation controlled, and diffusion controlled. These two biodegradable hydrogels, which can release nanoparticles depending on pH changes, may be suitable as potential colon targeting vehicles for oral delivery of drug nanoparticles.

Synthesis of a Range of Functional Oxide and Sulphide Nanoparticles by Biopolymer Stabilization and Enzymatic Treatment

Nanoparticles of the metal oxides magnetite (Fe3O4), cobalt oxide (Co3O4), manganese oxide (Mn3O4) and the metal sulfides CuS, Cu1.96S, Ag2S and CdS were prepared using the biopolymer dextran as a stablising agent. The corresponding enzyme dextranase could then used remove the biopolymer coat to give nanoparticles stabilized by a remnant oligomer. A green and aqueous general route to functional nanoparticles is described.

A THERMOPHILIC MICROBE PRODUCING DEXTRANASE FROM HEATED SUGAR CANE

A thermophilic aerobe microorganism designated NP4, was isolated from the heated sugar cane. It grew on dextran, and produced a thermoactive extracellular dextranase. Screening and isolation was done by assay of dextranase activity semi quantitatively on solid medium containing blue dextran. It provided several colonies with different morphology exhibited decolourized zones around, on culture plates containing blue dextran 2000R. The screening resulted in isolation of one microbe which efficiently assimilate dextran as carbon source. Dextranase production from the choised strain in liquid medium was conducted at room temperature for 8 hours with shaking speed of 125 rpm. The dextranase enzyme showed optimum pH of 8 and optimum temperature of 60 oC. Keywords: thermophilic aerobe, sugar cane, dextranase activity.

Metal sulfidenanoparticles synthesized via enzyme treatment of biopolymer stabilized nanosuspensions

Nanoparticles of CuS, Cu(x)S, Ag(2)S and CdS were successfully prepared using a novel general and green synthetic process to give dextran biopolymer stabilised metal sulfifde nanosuspensions. Following preparation, dextranase enzyme was used to remove the bulk of the bound dextran to give pure stable metal sulfide nanocrystals for application in for example aspects of medicine, photonics and solar cells. Particles of good homogeneity were obtained and the CuS nanoparticle size was controlled to 9-27 nm by adjusting the reaction conditions. Cu(2)S nanoparticles were 14 nm, Ag(2)S nanoparticles were 20-50 nm and CdS nanoparticles were 9 nm is size. The complexing mechanism of nanoparticle sulfides to dextrans was further studied using carboxylmethyl dextran as a complexing agent and crosslinked Sephadex (dextran) ;beads as substrate. Particles were characterized by TEM, XRD, TGA, FT-IR and zeta-potential measurement, and their UV-vis spectroscopic absorption properties were determined. Stabilization of the sulfide nanoparticles with soluble hydroxylated biopolymers such as dextran is previously unreported and is here interpreted in terms of viscosity, pH of the system and weak polar S-H or S(metal)OH(2)(+) interactions with dextran depending on the material. Notably, the complexing mechanism appears to differ significantly from that taking place in known dextran-metal oxide systems. The process shown here has good potential for scale-up as a biosynthetic route for a range of functional sulfide nanoparticles.

Dextranase in sugar industry: A review

In sugar production, dextrans are undesirable compounds synthesized by contaminant microorganisms from sucrose, increasing the viscosity of the flow and reducing industrial recovery, bringing about significant losses. The use of the dextranase enzyme is the most efficient method for hydrolyzing the dextrans at sugar mills. Some bacterial strains, filamentous fungi and a small number of yeasts have been shown to produce dextranase. The fungal dextranases showed the highest reaction rate at low Brix, with pH and temperature close to 5.0 and 50 °C, respectively, that is, conditions existing in juice extraction. Some of these dextranases formulated in enzymatic preparations have been efficiently used for hydrolyzing dextrans in sugar mill juices. In more advanced stage of the process, where the dextrans have already caused losses, the conditions of temperature and Brix are high. However, although the volumes are smaller, the treatment with these enzymes in syrup showed the need to increase the dose, equaling dextranase consumption. Some thermo tolerant bacterial dextranases identified up to now showed a much reduced specific activity that makes their industrial use unfeasible. The fungal dextranases from Chaetomium sp. have shown the best results on dextrans treatment both in juices and syrups. Any attempt to obtain a new natural or recombinant dextranase enzyme, must be comparable with the Chaetomium enzyme.

PRODUKSI, ISOLASI DAN KARAKTERISASI ENZIM DEKSTRANASE dari Arthrobacter sp. B7

Dextranase enzyme has been purified and characterized from Arthrobacter sp. B7. This enzyme was purified from the culture supernatant of Arthrobacter sp. B7 by procedure of native PAGE. The molecular size of the enzyme was estimated 72,5 kDa by SDS-PAGE. The N-terminal amino acid sequence of this enzyme determined using Edman degradation techniques were APVTADVGNLHT. SDS-PAGE and native-PAGE analysis revealed that the enzyme molecule consisted of one sub-unit.

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.

In Situ Surface Plasmon Resonance Analysis of Dextran Monolayer Degradation by Dextranase

Surface plasmon resonance (SPR) has been employed to investigate the hydrolytic degradation of thiolated dextran monolayers on silver by the enzyme dextranase. The influence of pH upon the kinetics of this enzyme-catalyzed degradative reaction has been demonstrated, and SPR experiments reveal that dextranase does not completely remove the thiolated dextran monolayers, even at the enzyme's most active pH. X-ray photoelectron spectroscopy and atomic force microscopy results support this observation, as do SPR measurements of protein adsorption to degraded samples, which show significant protein resistance after degradation. It is suggested that incomplete degradation occurs due to an inability of the enzyme to process, or complex with, the derivatized thiol bound portions of individual dextran macromolecules close to the silver surface.

Dextranase enzyme, method for its production and DNA encoding the enzyme (HR Campana et al, CU)

Dextranase enzyme, method for its production and DNA encoding the enzyme (HR Campana et al, CU)

Effect of the Chemical Modification of Dextran on the Degradation by Rat Liver Lysosomal Enzymes

The biodegradation of dextran, reduced dextran dialdehyde and dextran monosuccinate ester was investigated using isolated rat liver lysosomes. It was demonstrated that chemical modification of the parent dex tran resulted in a reduced degradability. The action of the exo-enzyme α-glucos idase (α-D-glucoside glucohydrolase) was nearly the same for parent dextran as for the derivatives. In order to explain the degradation data, the presence of the endo-acting enzyme dextranase (1,6-α-D-glucan 6-glucanohydrolase) in the lysosomes was taken into account.

Tight genetic linkage of a glucosyltransferase and dextranase of Streptococcus mutans GS-5.

A genetic library consisting of over 5000 clones with an average insert size of 6.9 kilobasepairs (kbp) of Streptococcus mutans GS-5 has been constructed in a bivalent plasmid vector pMK3, which is capable of replicating in Escherichia coli and Bacillus subtilis. The recombinant plasmid pSUCRI, containing a 6.0 kbp fragment of S. mutans GS-5 DNA, was the focus of this study. Using Southern hybridization, in vitro and in vivo gene expression techniques, and biochemical analysis, this clone was shown to encode the 55 kiloDalton (kDal) GS-5 gtfA gene product, as well as a 38 and a 66 kDal polypeptide. In addition to the gtfA gene, pSUCRI encodes a dextranase activity with specificity for alpha(1----6)-linked glucans, and with no detectable activity on mutan. The dextranase enzyme had an apparent molecular weight of 66 kDal as demonstrated by SDS-PAGE analysis of the proteins produced by a dextranase-negative deletion derivative. The pH optimum of the enzyme was approximately 6.0, and there was no detectable activity below pH 5.0. By subcloning various combinations of DNA fragments from pSUCRI, it was demonstrated that the dextranase gene (designated dexB) can be separated from the gtfA gene and still be efficiently expressed in both E. coli and B. subtilis. The dexB gene contained its own promoter and ribosome-binding site. The genetic linkage of the gtfA and dexB genes in the S. mutans GS-5 chromosome was confirmed by Southern hybridization and by the independent isolation of four distinct clones containing the gtfA gene and common flanking sequences. In addition to a glucosyltransferase and dextranase, an invertase-like activity is also encoded on pSUCRI, indicating that there is a cluster of genes on the S. mutans GS-5 chromosome which is devoted to the dissimilation of sucrose and concomitant synthesis or modification of glucans into a water-insoluble form, perhaps constituting an operon for glucan modification which can be coordinately regulated in response to environmental alterations.

Role of antigen and, antibody in the regulation of the immune response

The enzyme dextranase could degrade antigenic dextran in vivo even when given 6-15 d after the antigen. Dextranase injected after the antigen suppressed the immune response when given 24 but not 48 h after the antigen, indicating that the antigen must interact with the immune system for 48 h to initiate a response. Thereafter, the B cells are independent of further antigen stimulation. To show whether antibody-mediated suppression of the immune response was determinant specific FITC-conjugated SRC were applied as immunogen and antibodies were raised both against the carrier (SRC) and the FITC hapten. When these antibodies were injected 1-3 h after the immunogen they only suppressed the immune response to the corresponding determinant. Anti-carrier antibodies usually enhanced the response to the hapten. Therefore, antibody-mediated suppression of the immune response is determinant-specific and cannot be mediated in vivo to a detectable extent by the Fc part of the antibodies.

Modification of acid attack on enamel surfaces in vitro by aggregations of bacteria.

When enamel is exposed to dilute lactic acid <i>in vitro</i> the surface of the tissue is rapidly etched, unlike that seen in caries. Experiments have been carried out to see whether enamel surface deposits, produced by bacteria <i>in vitro</i>, could modify simple acid attack on the tissue. Results show that by growing deposits of dextran-producing strains of <i>Streptococcus sanguis</i> and <i>Streptococcus mutans</i> on the surface of teeth prior to exposure to buffered lactic acid, the pattern of acid attack can be modified. Control teeth show demineralization of the outer surface layer after the formation of the ‘plaque-like’ deposits. Removal of the microbial deposit and exposure to buffered lactic acid results in loss of surface tissue and subsequent cavitation. However, after exposure of experimental teeth, having surface deposits, to buffered lactic acid <i>in vitro</i> lesions are produced that demonstrate well-mineralized surface zones superficial to demineralized subsurface regions. Quantitative imbibition studies have demonstrated that the initially demineralized surfaces produced by ‘plaque’ formation show an increase in mineral content after subsequent lesion formation. Attempts have been made to produce similar effects with other micro-organisms and also to alter the effects produced by treating the microbial deposits with the enzyme dextranase prior to acid exposure.