Characterization of a thermostable protease from Bacillus subtilis BSP strain.

This study used conservative one variable-at-a-time study and statistical surface response methods to increase the yields of an extracellular thermostable protease secreted by a newly identified thermophilic Bacillus subtilis BSP strain. Using conventional optimization techniques, physical parameters in submerged fermentation were adjusted at the shake flask level to reach 184 U/mL. These physicochemical parameters were further optimized by statistical surface response methodology using Box Behnken design, and the protease yield increased to 295 U/mL. The protease was purified and characterized biochemically. Both Ca2+ and Fe2+ increased the activity of the 36 kDa protease enzyme. Based on its strong inhibition by ethylenediaminetetracetate (EDTA), the enzyme was confirmed to be a metalloprotease. The protease was also resistant to various organic solvents (benzene, ethanol, methanol), surfactants (Triton X-100), sodium dodecyl sulfate (SDS), Tween 20, Tween-80 and oxidants hydrogen per oxide (H2O2). Characteristics, such as tolerance to high SDS and H2O2 concentrations, indicate that this protease has potential applications in the pharmaceutical and detergent industries.

Expression and functional characterization in yeast of an endoglucanase from Bacillus sonorensis BD92 and its impact as feed additive in commercial broilers.

Some ingredients used in poultry feed formulation contain carbohydrate polymers which are difficult to digest and thus hinder nutritional feed value. Toward overcoming this limitation, exogenous enzymes have been added to poultry feed to improve its nutritive value. The present study was designed to provide first enzymatic characterization of endoglucanase (BsEgl) from the genome of B. sonorensis BD92 expressed in Pichia pastoris. Further, we tested its impact alone and in combination with a ß-glucosidase (Bteqßgluc) on growth in commercial broilers as feed additive. The expressed enzyme displayed features of GH5 family and had optimum activity against carboxymethyl cellulose at pH 5 and 50 °C. The BsEgl was stable at a range of pH from 4 to 8 for 60 min and at 50 °C for 180 min. Supplementing broilers diet with BsEgl alone or in combination with Bteqßgluc resulted in better feed conversion ratio among treatments during a five weeks testing period. Moreover, meat percentage was also highest for this treatment, and all treatments with recombinant enzymes increased intestinal length in birds compared to treatment control group. Blood parameters and serum biochemistry profile showed non-significant difference among groups. These results support that recombinant cellulolytic enzymes supplement high fiber diets improve their nutritional performance.

Biochemical characterization and molecular docking of cloned xylanase gene from Bacillus subtilis RTS expressed in E. coli.

This study employed mesophilic Bacillus subtilis RTS strain isolated from soil with high xylanolytic activity. A 642 bp (xyn) xylanase gene (GenBank accession number MT677937) was extracted from Bacillus subtilis RTS and cloned in Escherichia coli BL21 cells using pET21c expression system. The cloned gene belongs to glycoside hydrolase family 11 with protein size of approximately 23 KDa. The recombinant xylanase showed optimal enzyme activity at 60 °C and at pH 6.5. Thermostability of recombinant xylanase was observed between the temperature range of 30-60 °C. Xylanase also remained stable in different concentration of various organic solvents (ethanol, butanol). This might be due to the formation of protein/organic solvent interface which prevents stripping of essential water molecules from enzyme, thus enzyme conformation and activity remained stable. Finally, the molecular docking analysis through AutoDock Vina showed the involvement of Tyr 108, Arg140 and Pro144 in protein-ligand interaction, which stabilizes this complex. The observed stability of recombinant xylanase at higher temperature and in the presence of organic solvent (ethanol, butanol) suggested possible application of this enzyme in biofuel and other industrial applications.

Direct growth of m-BiVO4@carbon fibers for highly efficient and recyclable photocatalytic and antibacterial applications.

Owing to photocatalytic and antibacterial properties, bismuth based oxides has drawn much attention in recent past. However, non-recyclability of these oxides has restricted their practical applications. In present work, a novel nanostructured composite monoclinic bismuth vanadate@ activated carbon fibers (BiVO4@ACF) photocatalyst was efficaciously synthesized using a solvothermal method and characterized by X-ray diffraction (XRD), scanning electron microscope (SEM) and Bruner-Emmett-Teller (BET). The specific surface area, phase composition, microstructure, binding and photocatalytic activity of BiVO4@ACF pose great dependence on solvent nature and chelating agents utilized for synthesis. The photocatalytic and antibacterial potential of this composite was evaluated and optimized by using a model pollutant, Reactive Rhodamine Blue (RhB) and pathogenic microbes (Escherichia coli and Staphylococcus aureus). The composite possesses enhanced photocatalytic and antibacterial activity and was reutilized for three rounds of respective reaction without any loss of activity and structure as evident from SEM and XRD results. The photocatalytic mechanism of photodegradation of dye and bactericidal properties of samples under visible light irradiation was determined by scavenger and photoluminescence (PL) spectroscopy. The enhanced photocatalytic and antibacterial activity, chemical stability and most importantly good recyclability of BiVO4@ACFs highlight the potential application of this composite in water purification and other biological applications.

An update on carbohydrases: growth performance and intestinal health of poultry.

Poultry is an imperative domesticated livestock species that provides high quality protein and micronutrients as meat and eggs. In poultry production, feed is the single major input constituting 70-75% of total production cost. Feed mainly consists of cereal grains, those provide energy to the birds. However, these grains contain different levels of anti-nutritional factors such as non-starch polysaccharides (NSP). These NSP are indigestible by poultry birds due to the lack of vital endogenous enzymes (carbohydrases) thus increase intestinal viscosity which slower the migration and absorption of nutrients. Consequently, these NSP may also increase the chances for infection by inducing competition within gut microbiota for digestible nutrients. This affects bird's health and increases the production cost. Therefore, there is a need to find efficient and effective solutions for these problems. Carbohydrases supplementation have an important role in poultry diets with high NSP contents. Feed enzymes are being used from years to enhance growth performance and digestibility but have limited activity for selective ingredients. New generation carbohydrases with a board range of activity and stability help to degrade the complex substrates and improve growth performance of poultry. Present review summarizes the updated literature on the use of carbohydrases to improve bird's performance and intestinal health.

Statistical based experimental optimization for co-production of endo-glucanase and xylanase from Bacillus sonorensis BD92 with their application in biomass saccharification.

Endo-glucanase (cellulase) and xylanase have high industrial demand due to their vast application in industrial processes. This study reports statistical based experimental optimization for co-production of endo-glucanase and xylanase from Bacillus sonorensis BD92. Response surface methodology (RSM) involving central composite design (CCD) with full factorial experiments (23) was applied to elucidate the components that significantly affect co-production of endo-glucanase and xylanase. The optimum co-production conditions for endo-glucanase and xylanase were as follows: carboxymethyl cellulose (CMC) 20 g/L, yeast extract 15 g/L, and time 72 h. The maximum endo-glucanase and xylanase production obtained was 1.46 and 5.69 U/mL, respectively, while the minimum endo-glucanase and xylanase production obtained was 0.66 and 0.25 U/mL, respectively. This statistical model was efficient because only 20 experimental runs were necessary to assess the highest production conditions, and the model accuracy was very satisfactory as coefficient of determination (R2) was 0.95 and 0.89 for endo-glucanase and xylanase, respectively. Further, potential application of these enzymes for saccharification of lignocellulosic biomass (wheat bran, wheat straw, rice straw, and cotton stalk) was also investigated. The results revealed that the biomass was susceptible to enzymatic saccharification and the amount of reducing sugars (glucose and xylose) increased with increase in incubation time. In conclusion, Bacillus sonorensis BD92 reveals a promise as a source of potential endo-glucanase and xylanase producer that could be useful for degrading plant biomass into value-added products of economic importance using precise statistically optimized conditions.

Lignocellulosic Biomass: A Sustainable Bioenergy Source for the Future.

BACKGROUND: Increasing population and industrialization are continuously oppressing the existing energy resources and depleting the global fuel reservoirs. The elevated pollutions from the continuous consumption of non-renewable fossil fuels also seriously contaminating the surrounding environment. The use of alternate energy sources can be an environment-friendly solution to cope these challenges. Among the renewable energy sources biofuels (biomass-derived fuels) can serve as a better alternative to reduce the reliance on non-renewable fossil fuels. Bioethanol is one of the most widely consumed biofuels of today's world. OBJECTIVE: The main objective of this review is to highlight the significance of lignocellulosic biomass as a potential source for the production of biofuels like bioethanol, biodiesel or biogas. METHODS: We discuss the application of various methods for the bioconversion of lignocellulosic biomass to end products i.e. biofuels. The lignocellulosic biomass must be pretreated to disintegrate lignocellulosic complexes and to expose its chemical components for downstream processes. After pretreatment, the lignocellulosic biomass is then subjected to saccharification either via acidic or enzymatic hydrolysis. Thereafter, the monomeric sugars resulted from hydrolysis step are further processed into biofuel i.e. bioethanol, biodiesel or butanol etc. through the fermentation process. The fermented impure product is then purified through the distillation process to obtain pure biofuel. CONCLUSION: Renewable energy sources represent the potential fuel alternatives to overcome the global energy crises in a sustainable and eco-friendly manner. In future, biofuels may replenish the conventional non-renewable energy resources due to their renewability and several other advantages. Lignocellulosic biomass offers the most economical biomass to generate biofuels. However, extensive research is required for the commercial production of an efficient integrated biotransformation process for the production of lignocellulose mediated biofuels.

Isolation and Identification of Novel IndigenousBacterial Strain as a Low Cost Pectinase Source

ABSTRACT The present study was concerned with the searching of novel bacterial cultures from different samples for the lab scale production of pectinase. Keeping in view the increasing demand of pectinase specially in Faisalabad, an industrial city of Pakistan, isolation of new hyper producer bacterial strains locally is an easy and cheap way of getting the desirable products at low cost. Therefore, isolation of new strains for industrial enzyme production has been and will be remained a part of research every time. This method alone can also provide raw material for further research such as enzyme engineering or molecular directed evolution. For the identification of hyper producer strain colony PCR was done for 16S rRNA analysis. Reason to use the 16S rRNA for identification purpose is that the gene is fairly short and can be amplified quickly and easily. The bacterial isolate (sources of pectinase enzyme) was identified based on PCR amplification of 16S rRNA and for this purpose the amplified product was run in agarose gel against a known species of Bacillus licheniformis. The 16S rRNA sequencing confirmed the Bacillus status of the strain.

Solid State Fermentation of a Raw Starch Digesting Alkaline Alpha-Amylase from Bacillus licheniformis RT7PE1 and Its Characteristics.

The thermodynamic and kinetic properties of solids state raw starch digesting alpha amylase from newly isolated Bacillus licheniformis RT7PE1 strain were studied. The kinetic values Q p , Y p/s , Y p/X , and q p were proved to be best with 15% wheat bran. The molecular weight of purified enzyme was 112 kDa. The apparent K m and V max values for starch were 3.4 mg mL(-1) and 19.5 IU mg(-1) protein, respectively. The optimum temperature and pH for α -amylase were 55°C, 9.8. The half-life of enzyme at 95°C was 17h. The activation and denaturation activation energies were 45.2 and 41.2 kJ mol(-1), respectively. Both enthalpies (ΔH (∗)) and entropies of activation (ΔS (∗)) for denaturation of α -amylase were lower than those reported for other thermostable α -amylases.

Expression and characterization of Coprothermobacter proteolyticus alkaline serine protease.

A putative protease gene (aprE) from the thermophilic bacterium Coprothermobacter proteolyticus was cloned and expressed in Bacillus subtilis. The enzyme was determined to be a serine protease based on inhibition by PMSF. Biochemical characterization demonstrated that the enzyme had optimal activity under alkaline conditions (pH 8-10). In addition, the enzyme had an elevated optimum temperature (60°C). The protease was also stable in the presence of many surfactants and oxidant. Thus, the C. proteolyticus protease has potential applications in industries such as the detergent market.