Purification and in vivo stability and half-life of recombinant lipid modified staphylokinase.

Staphylokinase (SAK), the thrombolytic protein holds a significant position in treating cardiovascular diseases. However, the rapid clearance of this protein from blood circulation reduces its effective usage and as a strategy to increase the half-life of SAK, initial work focussed on lipid modification of SAK (LMSAK) in E. coli GJ1158. Effective purification of the modified protein achieved using the two step method of hydrophobic interaction chromatography in succession with size exclusion chromatography, indicated a better yield. The thrombolytic activity of purified LMSAK analysed in heated plasma agar plate assay confirmed an enhanced activity. In vivo pharmacokinetic studies carried out for determining the half-life of LMSAK in blood circulation of mice presented that it has a half-life of 43.3 ± 3.4 min which is much higher than 21.6 ± 2.1 min that of the unmodified version of SAK. The studies confirmed the role of lipid modification as a crucial factor in confirming in vivo stability of LMSAK and proves to be beneficial in therapeutic usage.

Insights into phytase-containing transgenic Lemna minor (L.) as a novel feed additive.

This study assessed the effect of supplementation of novel transgenic phytase on growth performance and bone mineralization in Korean native broiler chickens. The experiment was designed using four dietary groups: those with a diet supplemented with (A) recombinant phytase, (B) transgenic phytase from the plant Lemna minor, (C) or wild-type L. minor as well as (D) a control group that was supplemented with commercially available feed. Three hundred 1-day-old Korean native broiler chicks were used and divided into these four dietary treatment groups having three replicates of 25 birds each (n = 75). The results showed increases in growth performance and bone mineralization in Groups B and C; compared with Groups A and D. Hematological analyses revealed notable contrasts in erythrocyte sedimentation rate, red blood cell count, and hemoglobin levels among the experimental groups, whereas no impacts of dietary treatment were observed on total eosinophil, lymphocyte, heterophil, monocyte, and basophil levels. The relative expression profiling of candidate genes showed that the genes involved in growth response, meat quality, and P-Ca metabolism were significantly highly expressed in the phytase-supplemented groups. Hence, it is suggested that dietary supplementation with transgenic phytase plant L. minor for enhancing growth performance is a promising new approach in the broiler feed industry. To the best of our knowledge, we report here the most comprehensive analysis using a broiler model that provides a workable platform for further research on the cost-effective production of feed with different compositions that might be beneficial in the livestock feed industry.

Statistical approach for the enhanced production of cold-active ß-galactosidase from Thalassospira frigidphilosprofundus: a novel marine psychrophile from deep waters of Bay of Bengal.

In the present investigation Thalassospira frigidphilosprofundus, a novel species from the deep waters of the Bay of Bengal, was explored for the production of cold-active ß-galactosidase by submerged fermentation using marine broth medium as the basal medium. Effects of various medium constituents, namely, carbon, nitrogen source, pH, and temperature, were investigated using a conventional one-factor-at-a-time method. It was found that lactose, yeast extract, and bactopeptones are the most influential components for ß-galactosidase production. Under optimal conditions, the production of ß-galactosidase was found to be 3,864 U/mL at 20 ± 2°C, pH 6.5 ± 0.2, after 48 hr of incubation. ß-Galactosidase production was further optimized by the Taguchi orthogonal array design of experiments and the central composite rotatable design (CCRD) of response surface methodology. Under optimal experimental conditions the cold-active ß-galactosidase enzyme production from Thalassospira frigidphilosprofundus was enhanced from 3,864 U/mL to 10,657 U/mL, which is almost three times higher than the cold-active ß-galactosidase production from the well-reported psychrophile Pseudoalteromonas haloplanktis.

Cold active ß-galactosidase from Thalassospira sp. 3SC-21 to use in milk lactose hydrolysis: a novel source from deep waters of Bay-of-Bengal.

The cold active ß-galactosidase from psychrophilic bacteria accelerate the possibility of outperforming the current commercial ß-galactosidase production from mesophilic sources. The present study is carried out to screen and isolate a cold active ß-galactosidase producing bacterium from profound marine waters of Bay-of-Bengal and to optimize the factors for lactose hydrolysis in milk. Isolated bacterium 3SC-21 was characterized as marine psychrotolerant, halophile, gram negative, rod shaped strain producing an intracellular cold active ß-galactosidase enzyme. Further, based upon the 16S rRNA gene sequence, bacterium 3SC-21 was identified as Thalassospira sp. The isolated strain Thalassospira sp. 3SC-21 had shown the enzyme activity between 4 and 20 °C at pH of 6.5 and the enzyme was completely inactivated at 45 °C. The statistical method, central composite rotatable design of response surface methodology was employed to optimize the hydrolysis of lactose and to reveal the interactions between various factors behind this hydrolysis. It was found that maximum of 80.18 % of lactose in 8 ml of raw milk was hydrolysed at pH of 6.5 at 20 °C in comparison to 40 % of lactose hydrolysis at 40 °C, suggesting that the cold active ß-galactosidase from Thalassospira sp. 3SC-21 would be best suited for manufacturing the lactose free dairy products at low temperature.

In silico engineering of L-asparaginase to have reduced glutaminase side activity for effective treatment of acute lymphoblastic leukemia.

L-Asparaginase, an enzyme drug used for the treatment of acute lymphoblastic leukemia and its effective usage in clinical arena is complicated owing to the significant Glutaminase side activity. To develop variants of the enzyme with reduced Glutaminase activity, in silico mutagenesis was done by replacing amino acids in the vicinity of the ligand binding site. It was identified that replacement of enzyme's active site amino acid Asp96 with Alanine decreased the Glutaminase activity by 30% and also increased the Asparaginase activity by 40%. Docking studies were carried out by Autodock 4.0 and binding energy for native enzyme when docked with glutamine was found to be -8.08 Kcal/mole, whereas for mutated protein it was found to be -5.97 Kcal/mole. It was also observed that replacement of active site with amino acids other than alanine did not show considerable change in both Asparaginase and Glutaminase activities. The designed enzyme model with reduced Glutaminase side activity may help to develop a variant of enzyme drug through protein engineering by site-directed mutagenesis and thus to produce a drug with reduced side effect for treating acute lymphoblastic leukemia in children.

Optimization of polygalacturonase production from a newly isolated Thalassospira frigidphilosprofundus to use in pectin hydrolysis: statistical approach.

The present study deals with the production of cold active polygalacturonase (PGase) by submerged fermentation using Thalassospira frigidphilosprofundus, a novel species isolated from deep waters of Bay of Bengal. Nonlinear models were applied to optimize the medium components for enhanced production of PGase. Taguchi orthogonal array design was adopted to evaluate the factors influencing the yield of PGase, followed by the central composite design (CCD) of response surface methodology (RSM) to identify the optimum concentrations of the key factors responsible for PGase production. Data obtained from the above mentioned statistical experimental design was used for final optimization study by linking the artificial neural network and genetic algorithm (ANN-GA). Using ANN-GA hybrid model, the maximum PGase activity (32.54 U/mL) was achieved at the optimized concentrations of medium components. In a comparison between the optimal output of RSM and ANN-GA hybrid, the latter favored the production of PGase. In addition, the study also focused on the determination of factors responsible for pectin hydrolysis by crude pectinase extracted from T. frigidphilosprofundus through the central composite design. Results indicated 80% degradation of pectin in banana fiber at 20 °C in 120 min, suggesting the scope of cold active PGase usage in the treatment of raw banana fibers.

L-Asparaginase as potent anti-leukemic agent and its significance of having reduced glutaminase side activity for better treatment of acute lymphoblastic leukaemia.

Acute lymphoblastic leukaemia (ALL) is one of the leading types of malignant disorder seen in children. Viral infections, genetic factors and exposure to chemical carcinogens are some of the factors responsible for causing ALL. Treatment strategies followed for curing ALL include chemotherapy or radiation therapy, wherein, chemotherapy involves the use of the enzymatic drug L-Asparaginase. The enzyme can be produced from various plants, animals, bacterial and fungal sources but, among them, bacterial sources are widely used for production of this enzyme. The enzyme is non-human in origin having certain bottle necks with L-Asparaginase therapy in the form of side effects such as pancreatitis, thrombosis which are mainly due to glutaminase side activity. Hence, present-day research is mainly focussed on minimizing or completely eliminating the glutaminase activity of the enzyme L-Asparaginase. This review is focussed on the complications associated with glutaminase side activity and use of glutaminase free enzymatic drug L-Asparaginase in treating ALL and the other developments related to the modification of the drug for quality treatment.