Production Of Β-glucanase Research Articles

Management of Black Root Disease-Causing Fungus Fusarium solani CRP1 by Endophytic Bacillus siamensis CNE6 through Its Metabolites and Activation of Plant Defense Genes.

Black root rot disease of Cicer arietinum L. is accountable for substantial loss in chickpea production worldwide. Endophytic Bacillus siamensis CNE6 has previously shown multifaceted plant growth-promoting, broad-spectrum antifungal, and chickpea plant-colonizing potential. In the present study, the strain Bacillus siamensis CNE6 was used for controlling black root rot disease caused by Fusarium solani CRP1 in chickpea. CNE6 showed strong antagonistic potential against CRP1 both in vivo and in vitro. Scanning electron microscopic studies indicated cellular deformation of CRP1 due to production of β-glucanase, protease, and other secondary metabolites. A total of five compounds were detected from the cell-free supernatant (CFS) of the ethyl acetate (EA) fraction of CNE6 through gas chromatography-mass spectrometry analysis. A confocal microscopic study demonstrated strong inhibition of biofilm formation of the pathogen CRP1 by the EA fraction of CFS of CNE6. Molecular docking analysis revealed that one compound, (2E)-6-methoxy-2-[(4-methoxyphenyl)methylidene]-2,3-dihydro-1-benzofuran-3-one, may inhibit the activity of lanosterol 14-alpha demethylase, which is involved in ergosterol biosynthesis and beta-tubulin assembling. In vivo experiments also showed the efficacy of CNE6 for increasing chickpea growth as well as upregulation of four defense genes (CHI1, PAMP, PR2B, and TF1082) upon pathogenic challenge. Thus, our results strongly suggest a positive role for CNE6 as a prospective biocontrol agent for combating Fusarium solani in chickpea. IMPORTANCE The present work was undertaken to explore an effective biocontrol agent against the destructive black root rot disease of chickpea. We have used an efficient bacterial endophyte, CNE6, which can colonize in the chickpea root system, produce secondary metabolites and enzymes to degrade pathogenic cellular integrity, inhibit pathogenic establishment by rupturing biofilm formation, and induce host immunity upon treatment. Interaction of the bacterial metabolite was also observed with lanosterol 14-alpha demethylase, which is an important component in fungal membrane functioning. Being an endophyte, Bacillus siamensis CNE6 fulfills a suitable criterion as a biocontrol agent to control black root rot disease in chickpea and has huge prospects for use commercially.

Application of foam separation in production of β-glucanase in Pichia

Abstract β-glucanase is widely used in many fields and has great economic value and development space, but it faces the difficulties of separation and nutrient destruction in the process of industrial production. Foam separation is a simple, mild and efficient adsorption separation technique that enables efficient separation and extraction of β-glucanase. In this study, five single factors (loading volume, pH, separation gas velocity, fermentation loading concentration, surfactant concentration) of foam separation and harvest of β-glucanase produced by Pichia pastoris were studied. The best univariate condition was: 600 mL/min separation gas velocity, loading volume of 200 mL, initial enzyme concentration of 100 g/mL, surfactant concentration of 0.3 mg/mL and pH of 5. Based on the best univariate condition, the optimal separation conditions of β-glucanase were further explored, and the five-factor four-level orthogonal test was designed. From the experimental results, the best separation condition was: 600 mL/min, loading volume of 200 mL, initial enzyme concentration of 100 μg/mL, surfactant concentration of 0.5 mg/mL and pH of 5. Under this separation condition, the enrichment ratio (E) was 0.56 and the recovery rate (R) was 96.01%.

Fed-Batch Cultivation and Adding Supplements to Increase Yield of β-1,3-1,4-Glucanase by Genetically Engineered Escherichia coli

The aim of this study was to analyze the major influence factors of culture medium on the expression level of β-1,3-1,4-glucanase, and to further develop an optimized process for the extracellular production of β-glucanase at a bioreactor scale (7 L) with a genetically engineered Escherichia coli (E. coli) JM109-pLF3. In this study, batch cultivation and fed-batch cultivation including the constant rate feeding strategy and the DO-stat (DO: Dissolved Oxygen) feeding strategy were conducted. At a 7 L bioreactor scale for batch cultivation, biomass reached 3.14 g/L and the maximum β-glucanase activity was 506.94 U/mL. Compared with batch cultivation, the addition of glycerol, complex nitrogen and complete medium during fed-batch cultivation increased the production of biomass and β-1,3-1,4-glucanase. The maximum biomass and β-glucanase activity, which were 7.67 g/L and 1680 U/mL, respectively, that is, 2.45 and 3.31 times higher than those obtained with batch cultivation, were obtained by feeding a complex nitrogen source at a constant rate of 1.11 mL/min. Therefore, these nutritional supplements and strategies can be used as a reference to enhance the production of other bioproducts from E. coli.

Production of β-glucanase and protease from Bacillus velezensis strain isolated from the manure of piglets

In this study, a strain producing β-glucanase and protease, identified as Bacillus velezensis Y1, was isolated from the manure of piglet. We attempted to produce β-glucanase and protease after optimization of various process parameters with the submerged fermentation. The effects of each factor on producing β-glucanase and protease were as follows: temperature > time > pH > loaded liquid volume. The properties of the β-glucanase showed that the most suitable reaction temperature was 65 °C and pH was 6.0. However for protease optimum reaction temperature was 50 °C, and pH was 6.0. The amplified PCR fragments of β-glucanase and protease were 1434 bp containing an open reading frame of 1413 bp encoding a protein with 444 amino acids and 1752 bp containing an open reading frame of 1521 bp encoding a protein with 506 amino acids, respectively. So, the study demonstrated a viable approach of using newly identified B. velezensis Y1 strain for the maximum yield of two industrially important enzymes.

Biological control of Sclerotinia disease by Aspergillus sp. on oilseed rape in the field

ABSTRACTSclerotinia sclerotiorum causes serious yield losses to many crops worldwide. Aspergillus sp. Asp-4, previously shown to inhibit germination of sclerotia of S. sclerotiorum in vitro and in the field, was evaluated in field trials for suppression of this pathogen on oilseed rape. Spray application of Asp-4 to the soil prior to sowing rice in a rice–oilseed rape rotation resulted in a significant reduction in incidence of Sclerotinia stem rot on oilseed rape compared with the non-treated control in two field trials. This application of Asp-4 also resulted in a significant reduction in germination of sclerotia relative to the non-treated control in these field trials, suggesting that this reduction in sclerotial germination led to disease control. Microscopic examination demonstrated that Asp-4 could effectively colonise external and internal portions of sclerotia of S. sclerotiorum in vitro. Incubation of Asp-4 with sterile sclerotial material induced production of β-glucanase and chitinase activities by this isolate; β-glucanase and chitinase being potentially capable of degrading the glucan and chitin polymeric components of sclerotia. Incubation of Asp-4 with sterile sclerotial material also resulted in a significant reduction in dry weight of this sclerotial material relative to the non-treated control in 96 h in vitro experiments. Experiments reported here indicate that Aspergillus sp. Asp-4 has promise as a biological control agent for S. sclerotiorum on oilseed rape. Experiments reported here suggest that disease control results from inhibition of germination of sclerotial resting structures due to mycoparasitic colonisation by Asp-4.

Purification and biochemical characterization of a novel thermostable lichenase from Aspergillus niger US368

New β-1,3;1,4-glucanase was purified from Aspergillus niger US368. The pure glucanase has a molecular mass of about 32kDa. The N-terminal sequence of the purified enzyme (A-G-T-N-P-P-I-G-V) was determined. The optimum pH and temperature recorded for enzyme activity were 5 and 60°C, respectively. It also displayed marked thermostability with a half-life of 30min at 70°C. At 37°C, the enzyme showed 100% stability from pH 3 to 10. The Km and Vmax values exhibited by the enzyme on barley β-glucan were 0.62mgml−1 and 34.46Uml−1, respectively. The enzyme is a retaining-one and was only active toward glucan containing β-1,3;1,4-linkages. The production of β-glucanase with barley flour as the sole carbon source was optimized. This is the first report on the purification and characterization of a β-1,3;1,4-glucanase from A. niger. This lichenase could be considered as a candidate for future application particularly in the animal feed industry.

Medium optimization for the production of thermal stable β-glucanase by Bacillus subtilis ZJF-1A5 using response surface methodology

Polysaccharides, such as barley flour, dextrin and soluble starch, were better carbon sources than monosaccharides and disaccharides, such as glucose and maltose, for cell growth of Bacillus subtilis ZJF-1A5 and β-glucanase production. β-Glucanase produced by B. subtilis ZJF-1A5 was associated partially with cell growth and increased significantly when cells entered stationary phase; yeast extract was the best nitrogen source, followed by soybean flour. All inorganic nitrogen sources chosen in the experiments were not favorable for cell growth and enzyme production. A fractional factorial design (2 6-2) was applied to elucidate medium components that significantly affect β-glucanase production. The concentration of barley flour, corn flour and soybean flour in medium were significant factors. The steepest ascent method was used to locate the optimal domain and a central composite design was used to estimate the quadratic response surface from which the factor levels for maximum production of β-glucanase were determined. The composition of fermentation medium optimized with response surface methodology was (g/l): barley flour, 63.5; corn flour, 44.8; KH 2PO 4, 1.0; MgSO 4 · 7H 2O, 0.1; CaCl 2, 0.1. β-Glucanase activity was 251 U/ml at 48 h using optimized medium, 1.4 times higher than that in original medium.

β-Glucanase production from genetically modified recombinant Escherichia coli: Effect of growth substrates and development of a culture medium in shake flasks and stirred tank bioreactor

Cultivation conditions for the extracellular production of a hybrid β-1,3-glucanase (EC 3.2.1.39) from Bacillus were established using a recombinant Escherichia coli BL21 (DE3) pET-bgl-hisactophillin-sec. Maximal β-1,3-glucanase production of 510 U ml −1 after 28 h, was obtained using an optimized medium which had an initial lactose level of 7 and 24 g l −1 yeast extract and 5 g l −1 NaCl. Lactose was a potent inducer of β-1,3-glucanase. It is a cheap and easily available substrate for large-scale cultivations. In addition, the kinetics of extracellular production of β-glucanase depended on salt concentration (NaCl). Batch fermentation in a 3-l laboratory fermentor using the optimized medium allowed the production of 495 U ml −1 β-glucanase after only 15 h. Final enzyme activity in the optimized medium can be increased up to 3.5 times (510 U ml −1) compared with the concentration obtained with the starting medium (150 U ml −1).

Induction, screening and identification of Coniothyrium minitans mutants with enhanced β-glucanase activity

The production of β-glucanase in a mycoparasitic fungus, Coniothyrium minitans, was investigated using a wild type strain 2134. Through ultraviolet (UV) irradiation of strain 2134, four mutants, M11-3B2, A7-3D, A8-1 and A10-4, exhibiting enhanced β-glucanase activities were isolated. Strains A8-1 and A10-4 were constitutive mutants that expressed barley β-glucan hydrolysing activity in the absence of a supplemental inducer (β-glucan substrate). Supernatant from A8-1 and A10-4 cultures grown in potato dextrose broth (PDB), the medium without β-glucan, had maximum levels of β-glucanase activity on average 10 times greater than the wild type strain 2134. M11-3B2 had low levels of constitutive β-glucanase expression and enhanced laminarin hydrolysing activity when grown in presence of β-glucan-rich substrate.

The Development of β-Glucanase and Degradation of β-Glucan in Barley Grown in Scotland and Spain

The barley mutant TR49 and the cultivar from which it was derived, Troubadour, were grown in Scotland and Spain and harvested in 1991 and 1992. The development of β-glucanase together with the breakdown of β-glucan and the reduction in milling energy were followed during malting. While similar amounts of β-glucan were found in samples from both sites, TR49 showed less response to environmental conditions. Grain from Spain showed higher levels of β-glucanase activity, differences being apparent after steeping and 1–2 days of germination. The milling energy of grain was the same, but, during malting, samples from Spain showed a greater reduction in milling energy than those from Scotland. The most extreme environment in the study was the 1991 season at the Spanish site and was the driest and hottest. But even in the cooler, wetter season of 1992, Spanish grown material showed a greater production of β-glucanase than Scottish grown samples. These findings have important implications for plant breeding as well as the growing and trading of malting barley on a European scale.

Excretion into the Culture Medium of a Bacillus ß-Glucanase after Overproduction in Escherichia coli

The beta-glucanase gene (bgl) from Bacillus amyloliquefaciens was expressed in E. coli CSH 55 under the control of the PR promoter of phage lambda that is repressed by the thermosensitive repressor C1857. Production of beta-glucanase was drastically stimulated by a temperature shift to 42 degrees C. This overexpression of the bgl gene (about 20% of the total cellular protein) led to an almost complete excretion of the otherwise periplasmic protein into the extracellular medium, beta-glucanase accounted for more than 50% of the extracellular proteins. Col E 1 related plasmid (pEG 1) are amplified in E. coli relA strains in response to an amino acid limitation leading to a 10-fold increase in the activity of plasmid encoded genes. In this work we intended to maximize the expression of the bgl gene by a concerted action of a plasmid amplification and temperature induction. Surprisingly we could not increase the beta-glucanase production above the level reached by plasmid amplification or temperature induction alone. The reasons for this unexpected result will be discussed. Under all conditions tested the expression of the bgl gene was much lower in the E. coli relA strain NF 162 than in E. coli CSH 55; the low beta-glucanase production was accompanied by a reduced excretion rate of the enzyme.

β-GLUCANASE: THE SUCCESSFUL APPLICATION OF GENETIC ENGINEERING

The now well established principles of genetic engineering are described in relation to the solution of problems associated with β-glucans in beer. The application of these techniques has enabled the isolation of a Bacillus subtilis endo-1, 3–1, 4-β-D-glucanase gene which expresses a biologically active enzyme in yeast.15,16 Although this enzyme is capable of hydrolysing beer β-glucans during fermentation, thereby enhancing beer filtration, insufficient β-glucanase is produced in yeast to enable successful commercial implementation. The requirements for the efficient production of β-glucanase in genetically manipulated brewing yeast are described.

Chitin-binding hemagglutinin associated with cell wall of Conidiobolus lamprauges.

The cell wall of Conidiobolus lamprauges is composed of D-glucosamine, D-glucose and d-mannose in the ratio of 1:1:1. The first sugar originated from chitin and a part of the second sugar came from β-glucan.Cell wall-associated, chitin-binding hemagglutinin (endo-CLA) was liberated from the cell wall with β-glucanase but not with chitinase, cellulase, protease, sodium chloride, 2-mercaptoethanol, hydrochloric acid, N-acetyl-D-glucosamine or chitooligosaccharides.Endo-CLA and extracellular CLA (exo-CLA) showed almost the same sugar specificity and immunological properties in the hemagglutination inhibition test and the double immunodiffusion test, respectively.Endo-CLA was detected in the cell wall before production of β-glucanase by this organism, whereas exo-CLA was produced in the culture filtrate simultaneously with the enzyme.These results suggest that CLA was liberated from the β-glucan matrix in the cell wall by the action of β-glucanase.