Dept of Biotechnology

Optimization of alkaline protease production by Streptomyces ambofaciens NRRL 2420 in free and immobilized form was investigated using submerged fermentation technique. The optimum conditions for maximum alkaline protease production 342 unit mL −1 were 30°C at pH 8.5 and incubation time 96 h in free cell cultures using starch 20 g L −1 as carbon source and yeast extract 5 g L −1 as nitrogen source. The incubation time for the best yield of 344 unit mL −1 was reduced to 72 h under the optimized fermentation conditions by immobilized cells adsorbed on synthetic cotton fibers. Data obtained during 5 reusable cycles showed higher levels of enzyme in shorter time duration. Immobilization of Streptomyces ambofaciens NRRL 2420 on synthetic cotton fiber permit repeated reuse of the cells under the optimized fermentation conditions.

A yeast strain, Aureobasidium pullulans, which could produce the high yield of protease was isolated from sediment of saltern in Qingdao, China. Maximum production of enzyme (623.1 U/mg protein; 7.2 U/ml) was obtained in a medium containing 2.5 g soluble starch and 2.0 g NaNO 3 , 100 ml seawater, initial pH 6.0, after fermentation at 24.5°C for 30 h. The protease had the highest activity at pH 9.0 and 45°C.

Bacillus licheniformis MIR 29 has been isolated and produces extracellular proteases. It is able to grow at temperatures up to 60°C and at pH values up to 9.0. Casein was the best carbon source for production of a thermostable protease activity which, in some conditions, is 90% extracellular. The synthesis of alkaline protease is not constitutive; different levels of production were found with different carbon and nitrogen sources. Casein was thought to be an inducer of enzyme synthesis. The optimal pH and temperature of the enzyme activity were 12°C and 60°C, respectively. The enzyme was stable up to 60°C in the absence of stabilizers. The protease activity was inhibited with phenylmethylsulphonyl fluoride, indicating a serine-protease activity. The proteolytic activity was lowered by molecules present in the culture supernatant, which include amino acids and peptides, indicating end-product inhibition. Electrophoresis assay on denaturating gels showed two bands with alkaline protease activity, in the 25 to 40-kDa molecular mass range.

In order to overproduce bioinsecticides production by a sporeless Bacillus thuringiensis strain, an optimal composition of a cheap medium was defined using a response surface methodology. In a first step, a Plackett-Burman design used to evaluate the effects of eight medium components on delta-endotoxin production showed that starch, soya bean and sodium chloride exhibited significant effects on bioinsecticides production. In a second step, these parameters were selected for further optimisation by central composite design. The obtained results revealed that the optimum culture medium for delta-endotoxin production consists of 30 g L -1 starch, 30 g L -1 soya bean and 9 g L -1 sodium chloride. When compared to the basal production medium, an improvement in delta-endotoxin production up to 50% was noted. Moreover, relative toxin yield of sporeless Bacillus thuringiensis S22 was improved markedly by using optimised cheap medium (148.5 mg delta-endotoxins per g starch) when compared to the yield obtained in the basal medium (94.46 mg delta-endotoxins per g starch). Therefore, the use of optimised culture cheap medium appeared to be a good alternative for a low cost production of sporeless Bacillus thuringiensis bioinsecticides at industrial scale which is of great importance in practical point of view.

Living systems are constantly under different combinations of competing gradients of chemical, thermal, pH, and mechanical stresses allied. The present work is about competing chemical and thermal gradients imposed on E. coli in a diffusive stagnant microfluidic environment. The bacterial cells were exposed to opposing and aligned gradients of an attractant (1 mM sorbitol) or a repellant (1 mM NiSO4) and temperature. The effects of the repellant/attractant and temperature on migration behavior, migration rate, and initiation time for migration have been reported. It has been observed that under competing gradients of an attractant and temperature, the nutrient gradient (gradient generated by cells itself) initiates directed migration, which, in turn, is influenced by temperature through the metabolic rate. Exposure to competing gradients of an inhibitor and temperature leads to the imposed chemical gradient governing the directed cell migration. The cells under opposing gradients of...

Biodiesel is an alternative, carbon-neutral fuel compared to fossil-based diesel, which can reduce greenhouse gas (GHGs) emissions. Biodiesel is a product of microorganisms, crop plants, and animal-based oil and has the potential to prosper as a sustainable and renewable energy source and tackle growing energy problems. Biodiesel has a similar composition and combustion properties to fossil diesel and thus can be directly used in internal combustion engines as an energy source at the commercial level. Since biodiesel produced using edible/non-edible crops raises concerns about food vs. fuel, high production cost, monocropping crisis, and unintended environmental effects, such as land utilization patterns, it is essential to explore new approaches, feedstock and technologies to advance the production of biodiesel and maintain its sustainability. Adopting bioengineering methods to produce biodiesel from various sources such as crop plants, yeast, algae, and plant-based waste is one of...

The prefatory protein-glycan interaction and stabilizing protein-protein interaction of severe acute respiratory syndrome viruses with angiotensin-converting enzyme 2 play a significant role in complex formation thereby promoting endocytosis. The microevolution of SARS-CoV-2 over a period of time has a significant role in increasing the affinity of receptor-binding domain against angiotensin converting-enzyme 2. In the current study, we have corroborated the vitality of acquired SNPs over a period of time with increased affinity by using docking studies. The results indicate that the virus modulates the undesirable glycosylation sites by a series of substitution and deletion mutations. It uses bulky residues such as Tyr/Phe for dynamic arrest for quick stabilization of the complex, and Lys residues for stabilizing via hydrogen bond formation besides increasing the binding affinity to ease the cell entry.

During translational initiation in eukaryotes, the small ribosomal subunit forms a 48S preinitiation complex (PIC) with initiation factors. The 48S PIC binds to the 5’ end of mRNA and inspects long untranslated region (UTR) for the presence of the start codon (AUG). Accurate and high speed of scanning 5’ UTR and subsequent selection of the correct start codon are crucial for protein synthesis. However, the conformational state of 48S PIC required for inspecting every codon is not clearly understood. Whether the scanning or open conformation of 48S PIC can accurately select the cognate start codon over near/non-cognate codons, or this discrimination is carried out only in the scanning-arrested or closed conformation of 48S PIC. Here, using atomistic molecular dynamics (MD) simulations and free energy calculations, we show that the scanning conformation of 48S PIC can reject all but 4 of the 63 non-AUG codons. Among nine near-cognate codons with a single mismatch, only codons with a f...

Accurate and high-speed scanning and subsequent selection of the correct start codon are important events in protein synthesis. Eukaryotic mRNAs have long 5′ UTRs that are inspected for the presence of a start codon by the ribosomal 48S pre-initiation complex (PIC). However, the conformational state of the 48S PIC required for inspecting every codon is not clearly understood. Here, atomistic molecular dynamics (MD) simulations and energy calculations suggest that the scanning conformation of 48S PIC may reject all but 4 (GUG, CUG, UUG and ACG) of the 63 non-AUG codons, and initiation factor eIF1 is crucial for this discrimination. We provide insights into the possible role of initiation factors eIF1, eIF1A, eIF2α and eIF2β in scanning. Overall, the study highlights how the scanning conformation of ribosomal 48S PIC acts as a coarse selectivity checkpoint for start codon selection and scans long 5′ UTRs in eukaryotic mRNAs with accuracy and high speed.