Production Of Dextranase Research Articles

Removal of bacterial dextran in sugarcane juice by Talaromyces minioluteus dextranase expressed constitutively in Pichia pastoris

A gene construct encoding the mature region of Talaromyces minioluteus dextranase (EC 3.2.1.11) fused to the Saccharomyces cerevisiae SUC2 signal sequence was expressed in Pichia pastoris under the constitutive glyceraldehyde 3–phosphate dehydrogenase promoter (pGAP). The increase of the transgene dosage from one to two and four copies enhanced proportionally the extracellular yield of the recombinant enzyme (r-TmDEX) without inhibiting cell growth. The volumetric productivity of the four-copy clone in fed batch fermentation (51 h) using molasses as carbon source was 1706 U/L/h. The secreted N-glycosylated r-TmDEX was optimally active at pH 4.5–5.5 and temperature 50–60 °C. The addition of sucrose (600 g/L) as a stabilizer retained intact the r-TmDEX activity after 1-h incubation at 50–60 °C and pH 5.5. Bacterial dextran in deteriorated sugarcane juice was completely removed by applying a crude preparation of secreted r-TmDEX. The high yield of r-TmDEX in methanol-free cultures and the low cost of the fed batch fermentation make the P. pastoris pGAP-based expression system appropriate for the large scale production of dextranase and its use for dextran removal at sugar mills.

Food-Grade Expression and Characterization of a Dextranase from Chaetomium gracile Suitable for Sugarcane Juice Clarification.

The microbial production of dextranase using cheap carbon sources is beneficial to solve the economic loss caused by the accumulation of dextran in syrup. A food-grade microbial cell factory was constructed by introducing the dextranase encoding gene DEX from Chaetomium gracile to the chromosome of Bacillus subtilis, and the antibiotic resistance marker gene was subsequently deleted via the Cre/loxP strategy. The dual-promoter system with a sequentially arranged constitutive P43 promoter resulted in an 85 % increase in DEX expression. Under the optimal fermentation conditions of 10 g/L maltose, 15 g/L casein, 1 g/L Na2 HPO4 , 1 g/L FeSO4 and 8 g/L NaCl, DEX activity was increased from 2.625 to 64.34 U/mL. Recombinant DEX was purified 5.98-fold with a recovery ratio of 26.67 % and specific activity of 3935.02 U/mg. Enzyme activity was optimal at 55 °C and pH 5.0 and remained 80.34 % and 71.36 % of the initial activity at 55 °C and pH 4.0 after 60 min, respectively. The enzyme possessed high activity in the presence of Co2+ , while Ag+ showed the strongest inhibition ability. The optimal substrate was 20 g/L dextran T-2000. The findings could facilitate the low-cost, large-scale production of food-grade DEX for use in the sugar industry.

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.

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%.

Optimal Fermentation of Saccharomyces cerevisiae Expressing a Dextranase from Chaetomium gracile

Dextranase from Chaetomium gracile is generally considered safe for use in the sugarcane industry. Herein, a truncated and codon-optimised α-dextranase gene from C. gracile was successfully cloned and expressed in Saccharomyces cerevisiae for the first time. The optimum conditions of fermentation was achieved when the maximum dextranase activity reached to 58.45 U/mL after 48 h in shake flasks. The optimal pH and temperature were 5.5 and 60 °C, respectively. The recombinant dextranase remained stable between pH 4 and 6 and temperature between 55 and 60 °C. The findings in the present study could facilitate large-scale production of food-grade recombinant dextranase for use in the sugar industry.

Production and Characteristics of Yeast Dextranase from Soil

The existence of dextran in sugar cane juice is a major problem in the sugar industry, causing substantial losses. Treatment of dextran through enzymatic hydrolysis using dextranase is highly recommended as the most suitable method at this time because this is more effective and more economical. This study investigated the production and characterization of dextranase from local isolate yeast to degrade dextran on sugar cane juice. The selected yeast was identified on the basis of molecular identification. Dextranase was produced from the culture with the best carbon and nitrogen sources then was characterized. Application of enzyme was also evaluated. As a selected isolate, F4 had the closest relationship with Pichia kudriavzevii. The highest production of dextranase was induced by the supplementation of glucose and combination of yeast extract and peptone. The enzyme had optimum working condition at pH 7, temperature at 30°C and it is more stable at 4°C of storage temperature. The cation Na+ played key role as co-factor while K+ and Ca2+ were detected as inhibitor of the enzyme. Dextranase from F4 isolate can hydrolyze dextran both in pure and in mixed dextran substrate, but with a lower hydrolysis rate.

Production and Characterization of Dextranase by Penicillium brevicompactum Isolated from Garden Soil

Production and Characterization of Dextranase by Penicillium brevicompactum Isolated from Garden Soil

Purification, characterization, and biocatalytic potential of a novel dextranase from Chaetomium globosum.

We aimed to identify new high-yield dextranase strains and study the catalytic potential of dextranase from the strain in industrial applications. Dextranase-producing strains were screened from soil samples, and a potential strain was identified as Chaetomium globosum according to its phenotype, biochemical characteristics, and rDNA analysis. Crude dextranase was purified to reach 10.97-fold specific activity and 18.7% recovery. The molecular weight of the enzyme was 53kDa with an optimum temperature and pH of 60°C and 5.5, respectively. Enzyme activity was stable at pH 4.0-7.0 and displayed sufficient thermal stability at temperatures < 50°C. Mn2+ (10mM) enhanced dextranase activity by 134.44%. The enzyme was identified as an endodextranase. It displayed very high hydrolytic affinity toward high-molecular weight dextran T2000, reaching 97.9% hydrolysis within 15min at 2 U/mL. Collectively, these results suggest that Chaetomium globosum shows higher production and specificity of dextranase than that from other reported strains. These findings may offer new insights into the potential of dextranase in the sugar, medical, and food industries.

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, characterization and end product analysis of dextran degrading endodextranase from Bacillus licheniformis KIBGE-IB25

Degradation of high molecular weight dextran for obtaining low molecular weight dextran is based on the hydrolysis using chemical and enzymatic methods. Current research study focused on production, purification and characterization of dextranase from a newly isolated strain of Bacillus licheniformis KIBGE-IB25. Dextranase was purified up to 36 folds with specific activity of 1405U/mg and molecular weight of 158kDa. It was found that enzyme performs optimum cleavage of dextran (5000Da, 0.5%) at 35°C in 15min at pH 4.5 with a Km and Vmax of 0.374mg/ml and 182µmol/min, respectively. Relative amino acid composition analysis of purified enzyme suggested the presence of higher number of hydrophobic, acidic and glycosylation promoting amino acids. The N-terminal sequence of dextranase KIBGE-IB25 was AYTVTLYLQG. It exhibited distinct amino acid sequence yet shared some inherent characteristics with glycosyl hydrolases (GH) family 49 and also testified the presence of O-glycosylation at N-terminal end.

Screening, production, and characterization of dextranase from Catenovulum sp.

A psychrotolerant dextranase-producing bacterium was isolated from the Gaogong island seacoast near Jiangsu, China. The bacterium, denoted as DP03, was identified as Catenovulum sp. based on its phenotype, biochemical characteristics, and 16S rRNA gene comparison. The optimal enzyme production time, initial pH, temperature, and aeration conditions of strain DP03 were found to be 28 h, 8.0, 30 °C, and 25 % volume of liquid in 100-ml Erlenmeyer flasks, respectively. The ability of 1 % dextran T20 to induce dextranase was investigated. Dextranase from strain DP03 displayed its maximum activity at pH 8.0 and 40 °C and was found to be stable at 30 °C and over a broad range of pH values (pH 6–11). Scanning electron microscopy showed that dextranase from the isolate DP03 could at least partially prevent Streptococcus mutans from forming biofilms on glass coverslips.

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.

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.

Preparation and some properties of immobilized Penicillium funiculosum 258 dextranase

The production of dextranase was investigated in static cultures of Penicillium funiculosum 258. Maximal enzyme productivity was attained at pH 8.0, with 3.5% (w/v) dextran (MW, 260 000) as carbon source, NaNO 3 (1%, w/v) and yeast extract (0.2%, w/v) as nitrogen source, 0.4% (w/v) K 2HPO 4 and 0.06% (w/v) MgSO 4. It was possible to increase the productivity of dextranase to 41.8 units ml −1 in the modified medium. The enzyme was immobilized on different carriers by different techniques of immobilization. The enzyme prepared by covalent binding on chitosan using glutaraldehyde had the highest activity, the immobilized enzyme retaining 63% of its original specific activity. Compared with the free dextranase, the immobilized enzyme exhibited: a higher pH optimum, a higher optimal reaction temperature and energy of activation, a higher Michaelis constant, improved thermal stability and higher values of deactivation rate constant. The immobilized enzyme retained about 80% of the initial catalytic activity even after being used for 12 cycles.

Purification and characterization of a dextranase from Sporothrix schenckii.

A dextranase (EC 3.2.1.11) was purified and characterized from the IP-29 strain of Sporothrix schenckii, a dimorphic pathogenic fungus. Growing cells secreted the enzyme into a standard culture medium (20 degrees C) that supports the mycelial phase. Soluble bacterial dextrans substituted for glucose as substrate with a small decrease in cellular yield but a tenfold increase in the production of dextranase. This enzyme is a monomeric protein with a molecular mass of 79 kDa, a pH optimum of 5.0, and an action pattern against a soluble 170-kDa bacterial dextran that leads to a final mixture of glucose (38%), isomaltose (38%), and branched oligosaccharides (24%). In the presence of 200 mM sodium acetate buffer (pH 5.0), the Km for soluble dextran was 0.067 +/- 0.003% (w/v). Salts of Hg2+, (UO2)2+, Pb2+, Cu2+, and Zn2+ inhibited by affecting both Vmax and Km. The enzyme was most stable between pH values of 4.50 and 4.75, where the half-life at 55 degrees C was 18 min and the energy of activation for heat denaturation was 99 kcal/mol. S. schenckii dextranase catalyzed the degradation of cross-linked dextran chains in Sephadex G-50 to G-200, and the latter was a good substrate for cell growth at 20 degrees C. Highly cross-linked grades (i.e., G-10 and G-25) were refractory to hydrolysis. Most strains of S. schenckii from Europe and North America tested positive for dextranase when grown at 20 degrees C. All of these isolates grew on glucose at 35 degrees C, a condition that is typically associated with the yeast phase, but they did not express dextranase and were incapable of using dextran as a carbon source at the higher temperature.

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

Dissociation and electrophoretic separation of dextranase and dextranase inhibitor from a tightly bound enzyme-inhibitor complex of Streptococcus sobrinus.

Endodextranase was separated from dextranase inhibitor in culture filtrates of Streptococcus sobrinus by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) in gel slabs containing blue dextran. Sample preparation included dissociation of the enzyme from its inhibitor by boiling for 1 min in SDS. During subsequent incubation of the gel, dextranase was located as clear bands on a blue background, and dextranase inhibitor appeared as blue zones on a clear background following incubation in dextranase solution. The enzyme and the inhibitor existed in multiple forms, and the range of molecular masses for dextranase (223-132 kDa) permitted an excellent separation from dextranase inhibitor (49-25 kDa). Although dextranase-negative mutants, and wild type strains grown at low dilution rate in the chemostat, were devoid of free dextranase activity, the enzyme was easily located by analytical SDS-PAGE. Likewise, analysis of filtrates from wild type strains, which contained no free inhibitor activity when growth occurred at high dilution rate, revealed dextranase inhibitor activity on the gels. The total production (free + combined) of dextranase and inhibitor by S. sobrinus was determined by dissociation of enzyme-inhibitor complexes in concentrated cell-free filtrates, their separation by preparative SDS-PAGE and electroelution from the gels, followed by renaturation of protein activity. From a comparison of activity tests of free dextranase and free inhibitor in untreated filtrates with the results of similar tests on renatured electroeluates, the proportion of each constituent bound into a complex under each growth condition could be deduced.

Study of some parameters for the production of dextranase by Penicillium aculeatum

The Study describes the effect of some parameters influencing the production of dextranase (E.C. 3.2.1.11) by Penicillium sp. molds. Penicillium aculeatum was found to produce the maximum amount of extracellular dextranase in Fukumota medium at pH 5.6 in 96 h of shake flasks. A 48-h-old culture suspension of cell mycelium (4% v/v) was found good for dextranase production using 1% dextran (40 × 10 6 MW) as substrate.

Production and Properties of Alkaline Dextranase from a Newly IsolatedBrevibacterium

About 500 strains of dextranase producing microorganisms were examined in detail for pH- activity and enzyme stability. A gram positive bacterium identified as belonging to the genus Brevibacterium was found to produce alkaline dextranase. Maximal dextranase synthesis was obtained when grown aerobically at 26°C for 3 days in a medium containing 1 % dextran, 2% ethanol, 1 % polypeptone and 0.05 % yeast extract together with trace amounts of inorganic salts.Brevibacterium dextranase had an optimum pH of 8.0 for activity at 37°C and an optimal temperature at 53°C at pH 7.5. The enzyme was quite stable over the range of pH 5.0 to 10.5 on 24 hr incubation at 37°C, especially on alkaline pH. The enzyme was also heat stable at 60°C for 10 min.

Production and Properties of Alkaline Dextranase from a Newly Isolated Brevibacterium

About 500 strains of dextranase producing microorganisms were examined in detail for pH- activity and enzyme stability. A gram positive bacterium identified as belonging to the genus Brevibacterium was found to produce alkaline dextranase. Maximal dextranase synthesis was obtained when grown aerobically at 26°C for 3 days in a medium containing 1 % dextran, 2% ethanol, 1 % polypeptone and 0.05 % yeast extract together with trace amounts of inorganic salts.Brevibacterium dextranase had an optimum pH of 8.0 for activity at 37°C and an optimal temperature at 53°C at pH 7.5. The enzyme was quite stable over the range of pH 5.0 to 10.5 on 24 hr incubation at 37°C, especially on alkaline pH. The enzyme was also heat stable at 60°C for 10 min.