Cloning,expression and characterization of a highly active thermostable alkaline phosphatase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> MTCC 1483 in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21 (DE3)

Alkaline phosphatase gene of the bacterium, Bacillus licheniformis MTCC 1483 was cloned and successfully expressed in Escherichia coli BL21 (DE3). Sequence analysis revealed an open reading frame of 1662 bp encoding a 553 amino acid protein with a molecular mass of 62 kDa, as determined by SDS-PAGE. The recombinant enzyme was purified using Ni-NTA affinity column and the purified enzyme showed a specific activity of 24890 U/mg protein, which is the highest value among any other bacterial recombinant alkaline phosphatases reported so far. The enzyme exhibited optimum activity at 50°C and pH 10.0 and showed high thermostability. The recombinant alkaline phosphatase from B. licheniformis MTCC 1483 exhibited a dephosphorylation efficiency of 92.9% to dephosphorylate linear DNA fragments. The recombinant enzyme with high catalytic efficiency and thermostability has the potential for applications in clinical diagnostics which require enzyme stability against thermal deactivation during preparation or labeling procedures.     

Hexavalent chromium removal from aqueous solutions by a novel powder prepared from Colocasia esculenta leaves

In this study, batch removal of hexavalent chromium from aqueous solutions by powdered Colocasia esculenta leaves was investigated. Batch experiments were conducted to study the effects of adsorption of Cr(VI) at different pH values, initial concentrations, agitation speeds, temperatures, and contact times. The biosorbent was characterized by scanning electron microscopy, energy-dispersive X-ray spectroscopy, and Fourier transform infrared spectrometer analysis. The biosorptive capacity of the adsorbent was dependent on the pH of the chromium solution in which maximum removal was observed at pH 2. The adsorption equilibrium data were evaluated for various adsorption isotherm models, kinetic models, and thermodynamics. The equilibrium data fitted well with Freundlich and Halsey models. The adsorption capacity calculated was 47.62 mg/g at pH 2. The adsorption kinetic data were best described by pseudo-second-order kinetic model. Thus, Colocasia esculenta leaves can be considered as one of the efficient and cheap biosorbents for hexavalent chromium removal from aqueous solutions.     

Extractive fermentation for enhanced gellan-hydrolysing enzyme production by Bacillus thuringiensis H14

A new extractive fermentation process using PEG and potassium phosphate aqueous two-phase system (ATPS) was developed for enhanced production of gellan-hydrolysing enzyme by Bacillus thuringiensis H14. Five different Bacillus sp. were tested for their ability to synthesize gellan-hydrolysing enzyme. Bacillus thuringiensis H14 was found to be the best organism for gellan-hydrolysing enzyme production. The enzyme showed maximum activity at pH 7.5 and 40 °C. The partition studies of gellan-hydrolysing enzyme in the system using PEG X (X = 9000, 6000, 4000) and potassium phosphate–water and PEG–sodium citrate–water system indicated at PEG (4000)– potassium phosphate–water is the best system for partitioning of gellan-hydrolysing enzyme into the PEG phase (K = 4.99). Gellan-hydrolysing enzyme production by Bacillus thuringiensis H14 was studied in ATPSs composed of PEG X (X = 9000, 6000, 4000) and potassium phosphate. The top phase is continuous and rich in PEG while the bottom phase is dispersed and is rich in phosphate, microbial cells being mainly retained in the bottom phase. The gellan-hydrolysing enzyme produced during fermentation partitioned into the upper PEG phase and total gellan-hydrolysing enzyme produced was 2.12, 2.29 and 2.40 times higher than that of homogeneous fermentation when the fermentations were carried out using PEG 9000–potassium phosphate–water, PEG 6000–potassium phosphate–water, PEG 4000–potassium phosphate–water systems respectively.     

Optimization of nutrients for gellan gum production by Sphingomonas paucimobilis ATCC-31461 in molasses based medium using response surface methodology

A molasses based medium for the production of gellan by Sphingomonas paucimobilis ATCC-31461 was developed. Placket-Burman design criterion was applied to study the effect of various nutrient supplements on gellan production using molasses. Among the 20 variables tested, molasses, tryptone, casaminoacid, disodium hydrogen orthophosphate and manganese chloride showed significant effect on gellan production. A central composite design was applied to determine the optimum concentrations of the significant variables obtained from Placket-Burman design. Most suitable medium composition for production of gellan was (g/l): molasses-112.5; tryptone-1; casaminoacid-1; disodium hydrogen orthophosphate-1; manganese chloride-0.947 and the optimum gellan production was 13.814 g/l.     

Improvement in Production and Quality of Gellan Gum by Sphingomonas paucimobilis Under High Dissolved Oxygen Tension Levels

The effect of agitation rate and dissolved oxygen tension (DOT) on growth and gellan production by Sphingomonas paucimobilis was studied. Higher cell growth of 5.4 g l⁻¹ was␣obtained at 700 rpm but maximum gellan (15 g l⁻¹) was produced at 500 rpm. DOT levels above 20% had no effect on cell growth but gellan yield was increased to 23 g l⁻¹ with increase in DOT level to 100%. Higher DOT levels improved the viscosity and molecular weight of the polymer with change in acetate and glycerate content of the polymer.     

Technological aspects of extractive fermentation using aqueous two-phase systems

World Journal of Microbiology and Biotechnology -     

Optimization of codon usage of the envelope protein E2 gene from various genotypes of hepatitis C virus to predict the expression level in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Hepatitis C virus infection (HCV) alarmingly increases worldwide; it causes chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, so there is urgent need of developing effective and sufficient quantity of vaccine. HCV envelope protein E2 is the main target for developing as a vaccine candidate. Presently recombinant proteins can successfully be used as a vaccine for many diseases. This concern, it is challenging to produce sufficient quantities of many recombinant proteins from their expression hosts. One of the main factors affecting the success of expression of foreign genes in heterologous hosts is the divergence of codon usage of the target gene from that used in the expression system. In this study, we optimized the various genotypes of HCV envelope protein E2 gene according to the codon usage of Pichia pastoris and predicted the expression level. Synonymous codon usage of E2 adapted to that used by P. pastoris was estimated using the relative synonymous codon usage value (RSCU), codon adaptation index (CAI) and effective number of codon (ENC). The CAI of optimized HCV E2 sequences was enhanced from 0.638 to 0.833 and %GC was decreased from 56.05 to 44.05; this was significantly (p < 0.01) different from the native sequences. Codon with RSCU value less than one was replaced with most preferred synonymous codons. The ENC values of optimized HCV E2 sequences varied from 47.00 to 47.50, with a mean value of 47.15 and an SD of 0.14. Our study suggested that, from the measured values of predicted expression level, the codon optimized HCV E2 protein could be produced in sufficient quantity in the expression host; knowledge of the codon usage patterns of E2 of various genotypes facilitate the production of a promising unique vaccine candidate for HCV.