Dextranase Production Research Articles

Constitutive High Expression Level of a Synthetic Deleted Encoding Gene of Talaromyces minioluteus Endodextranase Variant (r–TmDEX49A–ΔSP–ΔN30) in Komagataella phaffii (Pichia pastoris)

In the sugar industry, dextran generates difficulties in the manufacturing process. Using crude dextranase (EC 3.2.1.11) to eliminate dextran in sugar is an effective practice. In this study, a synthetic dextranase-encoding gene of the filamentous fungus Talaromyces minioluteus, lacking its putative native signal peptide (1–20 amino acids) and the next 30 amino acids (r–TmDEX49A–ΔSP–ΔN30), was fused to the Saccharomyces cerevisiae prepro α–factor (MFα–2) signal sequence and expressed in Komagataella phaffii under the constitutive GAP promoter. K. phaffii DEX49A–ΔSP–ΔN30, constitutively producing and secreting the truncated dextranase, was obtained. The specific activity of the truncated variant resulted in being nearly the same in relation to the full-length mature enzyme (900–1000 U·mg−1 of protein). At shaker scale (100 mL) in a YPG medium, the enzymatic activity was 273 U·mL−1. The highest production level was achieved in a fed-batch culture (30 h) at 5 L fermenter scale using the FM21–PTM1 culture medium. The enzymatic activity in the culture supernatant reached 1614 U·mL−1, and the productivity was 53,800 U·L−1·h−1 (53.8 mg·L−1·h−1), the highest reported thus far for a DEX49A variant. Dextran decreased r–TmDEX49A–ΔSP–ΔN30 mobility in affinity gel electrophoresis, providing evidence of carbohydrate–protein interactions. K. phaffii DEX49A–ΔSP–ΔN30 shows great potential as a methanol-free, commercial dextranase production system.

Potential application of dextranase produced by Penicillium aculeatum in solid-state fermentation from brewer's spent grain in sugarcane process factories

Dextran contamination is a serious problem of the sugar industry, which can be solved with the application of dextranases that may avoid significant product losses. However, the high costs of enzyme preparations make its use economically impracticable. Dextranase production from Penicillium aculeatum NRRL 2129 was carried out by solid-state fermentation (SSF) using brewer's spent grain (BSG) as an alternative substrate/support. Optimal conditions (dextran 15 g/L, ammonium sulfate 0.45 g/L, 70% initial humidity, pH 5 and 8 g of BSG) led to a maximum dextranase activity of 75.5 U/g dry matter. Kinetics parameters of produced dextranase were determined as Km 4.20 μM and Vmax 11.89 mol/min. The produced enzyme was efficiently applied on sugarcane juice, leading to a successful 72% decrease in dextran content after 10 min, showing a comparable performance with commercial dextranases, which brings promising perspectives for this process.

Purification, Characterization, and Biocatalytic and Antibiofilm Activity of a Novel Dextranase from Talaromyces sp.

Dextranase is a useful enzyme that catalyzes the degradation of dextran to low-molecular-weight fractions, which have many critical commercial and clinical applications. Endophytic fungi represent a source of both high heat-stable and pH-stable enzymes. In this study, from Delonix regia bark by plate assay, out of 12 isolated fungal strains, hyaline zones were detected in only one strain. By using the standard ITS rDNA sequencing analysis, the isolated strain was identified as Talaromyces sp. In the case of carbon source, in a medium containing 1% dextran T2000 as the sole carbon source, the maximum dextranase activity reached approximately 120 U/ml after incubation of 2 days where the optimum pH was 7.4. Peptone addition to the production medium as a sole nitrogen source was accompanied by a significant increase in the dextranase production. Similarly, some metal ions, such as Fe2+ and Zn2+, increased significantly enzyme production. However, there was no significant difference resulting from the addition of Cu2+. The crude dextranase was purified by ammonium sulfate fractionation, followed by Sephadex G100 chromatography with 28-fold purification. The produced dextranase was 45 kDa with an optimum activity at 37°C and a pH of 7. Moreover, the presence of MgSO4, FeSO4, and NH4SO4 increased the purified dextranase activity; however, SDS and EDTA decreased it. Interestingly, the produced dextranase expressed remarkable pH stability, temperature stability, and biofilm inhibition activity, reducing old-established biofilm by 86% and biofilm formation by 6%.

Optimization of Fungal Dextranase Production and Its Antibiofilm Activity, Encapsulation and Stability in Toothpaste.

Dextranase catalyzes the degradation of the substrate dextran, which is a component of plaque biofilm. This enzyme is involved in antiplaque accumulation, which can prevent dental caries. The activity of crude dextranase from Penicillium roquefortii TISTR 3511 was assessed, and the maximum value (7.61 unit/g) was obtained at 37 °C and pH 6. The Plackett–Burman design was used to obtain significant factors for enhancing fungal dextranase production, and three influencing factors were found: Dextran, yeast extract concentration and inoculum age. Subsequently, the significant factors were optimized with the Box–Behnken design, and the most suitable condition for dextranase activity at 30.24 unit/g was achieved with 80 g/L dextran, 30 g/L yeast extract and five day- old inoculum. The use of 0.85% alginate beads for encapsulation exhibited maximum dextranase activity at 25.18 unit/g beads, and this activity was stable in toothpaste for three months of testing. This study explored the potential production of fungal dextranase under optimal conditions and its encapsulation using alginate for the possibility of applying encapsulated dextranase as an additive in toothpaste products for preventing dental caries.

Isomalto-Oligosaccharides Produced by Endodextranase Shewanella sp. GZ-7 From Sugarcane Plants

Oligosaccharides have important alimental and medical applications. Dextranase has been used to produce isomalto-oligosaccharides (IMOs). In this study, we isolated dextranase-producing bacteria from sugarcane-cultivated soil. Identification of the isolate based on its phenotypical, physiological, and biochemical characteristics, as well as 16S ribosomal deoxyribonucleic acid gene sequencing yielded Shewanella sp. strain GZ-7. The molecular weight of the dextranase produced by this strain was 100-135 kDa. The optimum temperature and pH for dextranase production were 40 °C and 7.5, respectively. The enzyme was found to be stable at the pH range of 6.0-8.0 and the temperature range of 20 °C-40 °C. Thin-layer chromatography and high-performance liquid chromatography of the enzymolysis products of the substrate confirmed the enzyme to be endodextranase. Under the optimal conditions, the ratio of IMOs could reach 91.8% of the hydrolyzate. The final products were found to efficiently scavenge the 2,2-diphenyl-1-picrylhydrazyl, hydroxyl, and superoxide anion radicals. In general, dextranase and hydrolyzates have high potential prospects for application in the future.

Production of dextranase by aspergillus fumigatus NRC-F103 and its application in cane juice treatment and enhancing ethanol production from sugarcane molasses

Background and objectives One of the important applications of dextranase enzyme is preventing dextran accumulation in sugarcane juice as well as, consequently, for enhancing the resultant fermentable reducing sugars and ethanol yield in the fermentation of sugarcane molasses by yeasts. Materials and methods Different materials and methods were used for fungal strains [screening, mutation ultraviolet (UV), inoculum preparation, cultivation type ‘solid-state fermentation,’, culture substrates], dextranase (production, assay, soluble protein determination), purification, characterization, application in ethanol production by fermentation from sugarcane molasses, dextran estimation, and determination of reducing sugars. Results and conclusion Six fungal strains (namely Aspergillus oryzae FK-923, Aspergillus niger F-93, A. niger F-258, Aspergillus awamori NRC-F18, Aspergillus fumigatus NRC-F103, and Trichoderma viride NRC-F107) were screened on sorghum, sugar beet pulp, wheat bran, and orange peels using the solid-state fermentation technique to produce dextranase enzyme. The fungus A. fumigatus NRC-F103 cultivated on orange peels showed promising enzyme yield than other tested fungal strains. Then, optimization of culture conditions for dextranase production was carried out. Moisture content, initial pH value, incubation temperature, and incubation period were optimized to be 2 : 1 (v/w), 5.0, 35°C and 96 h, respectively. Five inorganic nitrogen sources were trailed at equivalent levels as sole nitrogen in the fermentation medium did not result in any increase in enzyme activity. Subjecting the fungal to UV resulted in a 75% increase in enzyme activity corresponding to the mother strain before subjecting to UV. Under the above conditions, 118 U/g original substrate was obtained. Isopropanol 1 : 1 (v/v) was applied for precipitation enzyme protein, as 32% of total protein involving 68% of total enzyme activity was obtained and specific activity was 42.94 U/mg protein compared with 16.4 U/mg protein in the culture supernatant. A study on obtained dextranase showed that it has an optimum that pH ranged from 4.5 to 5.5 as well as it gave the highest activity when incubated between 35 and 40°C. Promising results were obtained when the enzyme was applied in cane juice to prevent accumulation of dextran. Enzyme supplementation to diluted sugarcane molasses (26%, w/v) resulted in an increase in reducing sugars by 2.64%. Ethanol was increased by about 2.36% (v/v) in the fermentation medium supplemented with an enzyme compared with the unsupplemented medium and the fermentation efficiency increased from 89 to 92.5%.

Characterization and mechanism of the effects of Mg\u2013Fe layered double hydroxide nanoparticles on a marine bacterium: new insights from genomic and transcriptional analyses

BackgroundLayered double hydroxides (LDHs) have received widespread attention for their potential applications in catalysis, polymer nanocomposites, pharmaceuticals, and sensors. Here, the mechanism underlying the physiological effects of Mg–Fe layered double hydroxide nanoparticles on the marine bacterial species Arthrobacter oxidans KQ11 was investigated.ResultsIncreased yields of marine dextranase (Aodex) were obtained by exposing A. oxidans KQ11 to Mg–Fe layered double hydroxide nanoparticles (Mg–Fe-LDH NPs). Furthermore, the potential effects of Mg–Fe-LDH NPs on bacterial growth and Aodex production were preliminarily investigated. A. oxidans KQ11 growth was not affected by exposure to the Mg–Fe-LDH NPs. In contrast, a U-shaped trend of Aodex production was observed after exposure to NPs at a concentration of 10 μg/L–100 mg/L, which was due to competition between Mg–Fe-LDH NP adsorption on Aodex and the promotion of Aodex expression by the NPs. The mechanism underling the effects of Mg–Fe-LDH NPs on A. oxidans KQ11 was investigated using a combination of physiological characterization, genomics, and transcriptomics. Exposure to 100 mg/L of Mg–Fe-LDH NPs led to NP adsorption onto Aodex, increased expression of Aodex, and generation of a new Shine-Dalgarno sequence (GGGAG) and sRNAs that both influenced the expression of Aodex. Moreover, the expressions of transcripts related to ferric iron metabolic functions were significantly influenced by treatment.ConclusionsThese results provide valuable information for further investigation of the A. oxidans KQ11 response to Mg–Fe-LDH NPs and will aid in achieving improved marine dextranase production, and even improve such activities in other marine microorganisms.

Characterization and application of dextranase produced by Chaetomium globosum mutant through combined application of atmospheric and room temperature plasma and ethyl methyl sulfone

Abstract Dextranase has extensive applications in the food, pharmaceutical, and chemical industries. However, enzyme production by the wild-type strain is generally low and few studies have conducted structural analysis of dextranase. This study describes an efficient and stable mutagenesis method for producing dextranase and the relationship between the enzyme structure and enzymatic properties before and after mutagenesis. Wild-type Chaetomium globosum was mutated by combined atmospheric and room temperature plasma treatment and ethyl methyl sulfone application. Mutant strains were screened for optimal dextranase production ability and genetic stability. The maximum yield of dextranase reached 824.73 U/mL. Dextranase produced by the mutant strain displayed the same optimum pH and temperature values of 5.5 and 60℃, respectively, as dextranase produced by the wild-type strain. However, dextranase from the mutant strain showed greater heat stability at 70℃. Furthermore, the three-dimensional structure of the enzyme changed slightly after mutagenesis, with the amino acid residue at position 578 changing from His to Leu. This change in the three-dimensional structure affected the properties of dextranase. Finally, dextranase significantly inhibited the growth of Streptococcus mutans. The results indicated dextranase from Chaetomium globosum would give potential implications in the medical, prevent dental caries and food industries.

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.

Improved Dextranase Production by Chaetomium gracile Through Optimization of Carbon Source and Fermentation Parameters

The fungus Chaetomium gracile is used for production of dextranase and removal of dextran during sugar manufacturing process, however, the productivity of dextranase is low. In this study, we investigated the influence of carbon source on dextranase production using dextrans of different sizes. Culturing C. gracile in medium containing crude dextran and use of high molecular weight of dextrans resulted in the higher yield of dextranase production (80.5 ± 4.0 U ml−1). Cells incubated in medium containing glucose as sole carbon source exhibited a high growth rate but did not produce dextranase. Based on these findings, fed-batch and two-step fermentation strategies were employed to further increase the yield of dextranase with production of 159.5 and 187.0 U ml−1, respectively.

Purification and characterization of a novel marine Arthrobacteroxydans KQ11 dextranase

Dextranases can hydrolyze dextran deposits and have been used in the sugar industry. Microbial strains which produce dextranases for industrial use are chiefly molds, which present safety issues, and dextranase production from them is impractically long. Thus, marine bacteria to produce dextranases may overcome these problems. Crude dextranase was purified by a combination of ammonium sulfate fractionation and ion-exchange chromatography, and then the enzyme was characterized. The enzyme was 66.2kDa with an optimal temperature of 50°C and a pH of 7. The enzyme had greater than 60% activity at 60°C for 1h. Moreover, 10mM Co2+ enhanced dextranase activity (196%), whereas Ni2+ and Fe3+ negatively affected activity. 0.02% xylitol and 1% alcohol enhanced activity (132.25% and 110.37%, respectively) whereas 0.05% SDS inhibited activity (14.07%). The thickness of S. mutans and mixed-species oral biofilm decreased from 54340nm to 36670nm and from 64260nm to 43320nm, respectively.

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.

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 5000Da, after 24h 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.

Biocatalytic potential of an alkalophilic and thermophilic dextranase as a remedial measure for dextran removal during sugar manufacture

The present study is focused on dextranase from Streptomyces sp. NK458 with potential to remove dextran formed during sugar manufacture. The dextranase had molecular weight of 130kDa and hydrolyzed 15–25 and 410kDa dextran. Dextranase production was optimized using statistical designs and the enzyme was purified 1.8-fold with 55.5% recovery. It displayed maximum activity at pH 9.0 and 60°C and was stable over a wide range of pH from 5.0 to 10.0. The km and Vmax values were 3.05mM and 17.97mmol/ml/h, respectively. Ten units of dextranase could reduce dextran content by 67% in 24h and 56% in 72h from sugarcane juice of cane variety CoS 86032. The enzyme was stable up to 3days at 30°C beyond which its activity decreased and dextran removal could be retained by supplementation of 5U of dextranase. These properties make it a promising biocatalyst for sugar industry.

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.

Dextranase production from Paecilomyces lilacinus and its application for dextran removal from sugarcane juice

Dextranase was produced from fungus Paecilomyces lilacinus by submerged fermentation under different cultural conditions viz. pH, incubation temperature, inoculum size and days of incubation for maximum enzyme production. Maximum enzyme production was achieved at pH 6.0 of Mandel media and at temperature 30°C. Inoculum size of 1 × 107 spores/ml was found to be optimum for maximum enzyme production. Enzyme production increased with the increase in days of incubation from 3 to 5 days and then declined thereafter. Dextranase units (from 1 to 15 U/100 ml juice) were exogenously added to sugarcane juice with an aim to optimize dextranase application for removal of dextran from cane juice. Addition of dextranase in juice of cane variety CoS 8436, resulted in relatively less increase in dextran content as compared to control during storage. Whereas, dextran content in untreated juice increased 10 times during 24 h of storage, the increase during this period was only 2.3 times in juice treated with 15 U of dextranase. With the application of 1, 2, 5, 10 and 15 units of dextranase in 100 ml of juice, dextran content was decreased by 15.51, 30.82, 50.20, 68.20 and 73.30 % as compared to the control (with no dextranase added) after 24 h of storage. This study finds application in minimizing the dextran problem in sugar industry.

Lipomyces starkeyi KCTC 17343에 의한 extracellular dextranase 최적생산과 덱스트란 hydrolysates 분석

We optimized dextranase culture conditions by batch fermentation using Lipomyces starkeyi KCTC 17343. Furthermore, dextranase was purified by an ultra-membrane, and then dextran hydrolyzates were characterized. Cell growth and dextranase production varied depending on the initial culture pH and temperature. The conditions of optimal dextranase production were met in a pH range of 4-5 and temperature between 25-30℃. At optimal fermentation conditions, total enzyme activity and specific enzyme activity were about 4.85 IU/㎖ and 0.79 IU/g cells, respectively. The specific growth rate was examined to be 0.076 hr-¹. The production of dextranase in culture broth was very stably maintained after mid-log phase of growth. The enzyme hydrolyzed dextran into DP (degree of polymerization) 2 to 8 oligodextran series. Analysis of the composition of hydrolysates suggested that the enzyme produced is an endo-dextranase.

Adsorption of Streptococcus sobrinus dextranase inhibitor to water-insoluble alpha-D-glucans of oral streptococci.

A low molecular weight dextranase inhibitor from Streptococcus sobrinus has previously been identified and purified. The range of conditions under which inhibition occurs, and the situations in which dextranase activity of S. sobrinus can reappear, have been examined in the chemostat. These studies have revealed that when dextranase production exceeds that of the inhibitor, all the inhibitor is tightly bound into enzyme-inhibitor complexes, and the excess enzyme remains active. Another factor that influences the activity of dextranase inhibitor has now been identified, namely the ability of the inhibitor to bind to water-insoluble glucans. Adsorption to water-insoluble alpha-D-glucans, produced by oral streptococci that were grown in batch culture, increased with their proportion of alpha-1,3-linked sequences of glucose residues. Studies with water-insoluble dextrans of Leuconostoc mesenteroides strains showed that alpha-1,6-linked sequences were also important for binding. The inhibitor was not active when adsorbed to glucan, but active inhibitor was released by incubation with soluble dextran. The interactions of sucrose, alpha-D-glucosyltransferases, alpha-D-glucans, dextranase and dextranase inhibitor are discussed in relation to the growth rate of S. sobrinus. At low growth rate in the chemostat the predominant alpha-D-glucosyltransferase (GTF) is a GTF-S that converts sucrose into soluble dextran, and the activity of free dextranase inhibitor in the culture filtrate is high. By contrast, at high growth rate the streptococci produce GTFs capable of synthesizing water-insoluble alpha-D-glucans, and no free inhibitor is found in culture filtrate. Thus the activity of free, extracellular dextranase inhibitor is controlled by (i) the extent of binding to dextranase and (ii) the extent of adsorption to water-insoluble alpha-D-glucan.

Penicillium notatum 1 a new source of dextranase

Two hundred and fifteen fungal strains were screened for extracellular dextranase production with a diffusion plate method. The best enzymatic activity (12–19 DU ml−1) was achieved byPenicillium notatum 1, a species for which the dextranase productivity has not yet been published. Some of the parameters affecting enzyme production have been standardized. The enzyme in crude state was relatively stable, its maximal activity was at 50°C and at pH 5.0. Conidia of the selected strain were mutagenized, and isolated mutants were tested for production of dextranase in submerged culture. The most active mutant,P. notatum 1-I-77, showed over two-times higher dextranase activity than the parentP. notatum 1