Current efforts on sustainable green growth in the manufacturing sector to complement "make in India" for making "self-reliant India".

In an effort to become a global manufacturing giant, India has launched an ambitious "Make in India" campaign. In this article, we discussed the initiatives launched by Indian Government to create a sustainable economy, as well as opportunities for national and international start-up organisations to reinforce "Make in India" campaign. For the first time, we present an in-depth discussion of Green India policies and manufacturing sector challenges. The aim is to improve the manufacturing output of India and reduce dependence on foreign imports for which campaigns like "Atmanirbhar Bharat" which translates into "Self-Reliant-Green" India has also been started. Department of Science & Technology play a key role in establishing various programs such as infrastructure development, technological support, and green manufacturing programs all of which help to translate "discovery research" into "commercially viable technologies". It focuses specifically on "Startups", MSME's, young scientists, R&D labs and traditional manufacturing units that have little access to financial support but are an integral part of the Indian manufacturing ambit. The programs are interconnected and designed to function in a way that every support could be provided to the indigenous manufacturing, innovation and implementation of climate-resilient green growth strategies.

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.

Enzyme promiscuity in earthworm serine protease: substrate versatility and therapeutic potential.

Enzymes are the most versatile molecules in the biological world. These amazing molecules play an integral role in the regulation of various metabolic pathways and physiology subsequently. Promiscuity of an enzyme is the capacity to catalyze additional biochemical reactions besides their native one. Catalytic promiscuity has shown great impact in enzyme engineering for commercial enzyme and therapeutics with natural or engineered catalytic promiscuity. The earthworm serine protease (ESP) is a classic example of enzyme promiscuity and studied for its therapeutic potential over the last few decades. The ESP was reported for several therapeutic properties and fibrinolytic activity has been much explored. ESP, a complex enzyme exists as several isoforms of molecular weight ranging from 14 to 33 kDa. The fibrinolytic capacity of the enzyme has been studied in different species of earthworm and molecular mechanism is quite different from conventional thrombolytics. Cytotoxic and anti-tumor activities of ESP were evaluated using several cancer cell lines. Enzyme had shown tremendous scope in fighting against plant viruses and microbes. ESP is also reported for anti-inflammatory activity and anti-oxidant property. Apart from these, recently, ESP is reported for DNase activity. The daunting challenge for researchers is to understand the molecular mechanism for such diverse properties and possibility of enzyme promiscuity. This review emphasizes molecular mechanism of ESP governing various biochemical reactions. Further, the concept of enzyme promiscuity in ESP towards development of novel enzyme based drugs has been reviewed in this study.

Development and validation of an indirect competitive ELISA for quantification of recombinant staphylokinase in rabbit plasma: Application to pharmacokinetic study.

The relatively short circulatory half-life (2-3 min) of staphylokinase is a major drawback in the development of SAK- (staphylokinase) based thrombolytic drug. A rapid and sensitive method, based on indirect competitive ELISA, was developed and validated for quantitative determination of SAK in rabbit plasma. The dynamic range of the assay varied between 0.41 ± 0.16 µg/L and 9.03 ± 0.38 µg/L (R(2) = 0.98) for SAK in rabbit plasma. There were no dilution linearity issues apparent with this assay. The precision (% CV) ranged from 4.6-9.7% for the intraassay and from 17.1-19.3% for interassay. This validated method was successfully employed for evaluation of various pharmacokinetic parameters of SAK in rabbit.

Targeting therapeutics across the blood brain barrier (BBB), prerequisite towards thrombolytic therapy for cerebrovascular disorders-an overview and advancements.

Cerebral tissues possess highly selective and dynamic protection known as blood brain barrier (BBB) that regulates brain homeostasis and provides protection against invading pathogens and various chemicals including drug molecules. Such natural protection strictly monitors entry of drug molecules often required for the management of several diseases and disorders including cerebral vascular and neurological disorders. However, in recent times, the ischemic cerebrovascular disease and clinical manifestation of acute arterial thrombosis are the most common causes of mortality and morbidity worldwide. The management of cerebral Ischemia requires immediate infusion of external thrombolytic into systemic circulation and must cross the blood brain barrier. The major challenge with available thrombolytic is their poor affinity towards the blood brain barrier and cerebral tissue subsequently. In the clinical practice, a high dose of thrombolytic often prescribed to deliver drugs across the blood brain barrier which results in drug dependent toxicity leading to damage of neuronal tissues. In recent times, more emphasis was given to utilize blood brain barrier transport mechanism to deliver drugs in neuronal tissue. The blood brain barrier expresses a series of receptor on membrane became an ideal target for selective drug delivery. In this review, the author has given more emphasis molecular biology of receptor on blood brain barrier and their potential as a carrier for drug molecules to cerebral tissues. Further, the use of nanoscale design and real-time monitoring for developed therapeutic to encounter drug dependent toxicity has been reviewed in this study.

Molecular insights into cold active polygalacturonase enzyme for its potential application in food processing.

Pectin is a complex structural heteropolysaccharide that require numerous pectinolytic enzymes for its complete degradation. Polygalacturonase from mesophilic or thermophilic origin are being widely used in fruit and vegetable processing in the recent decades to degrade pectic substances. Recently cold active pectinases are finding added advantages over meso and thermophilic counterparts, to use in industrial scale particularly in food processing industry. They facilitate in conservation of several properties of foods so that the end product retains its naturality and also generates economic benefits. In the present study, Pseudoalteromonas haloplanktis, a well reported marine psychrophile is taken as a model organism for cold active polygalacturonase and is evaluated in comparision to the routinely used mesophilic and thermophilic enzymes by insicio approach. Polygalacturonase sequences from industrially important microbial sources were subjected to MEME and Pfam wherein motifs and domains involved in the conservation were analyzed. Dendrogram revealed sequence level similarity and motifs showed uniform distribution of conserved regions that are involved in important functions. It was also observed through clustalW analysis that the amount of arginine content of psychrophiles is less when compared with thermophiles. Finally, all the modeled enzyme structures were subjected to docking studies using Autodock 4.2 with the substrate polygalacturonic acid and binding energies were found to be -5.73, -6.22 and -7.27 KCals/mole for meso, thermo and psychrophiles respectively which indicates the efficiency of psychrophilic enzymes when compared with its counterparts giving scope for further experimentation to find their better usage in various food industry applications.

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.

Perspectives on progressive strategies and recent trends in the production of recombinant human factor VIII.

Factor VIII (fVIII), a glycoprotein cofactor, plays a crucial role in the intrinsic blood coagulation pathway. As one of the most essential blood clotting factors known today, fVIII is the largest and most complex commercialized therapeutic protein used in the treatment of hemophilia A, an X-linked recessive disorder. Two lyophilized fVIII concentrates (viz., plasma fractionated and recombinant) are in use to treat hemorrhagic episodes in patients suffering with hemophilia A. Recombinant fVIII (rfVIII) products that are devoid of human and animal protein expressed in mammalian cells can be used as an alternative to plasma derived (pd) products fractionated from human blood. Although effective, the expression of rfVIII in heterologous mammalian expression systems at an industrial scale is complicated due to complex fVIII structure and non-human pattern of post-translational modifications, particularly glycosylation. Chinese hamster ovary (CHO) is the most commonly used host cell line for the production of various biotherapeutics. Product safety and adaptability to grow in suspension is the most desirable feature that makes CHO, a suitable host for rfVIII production. Even though the therapeutic and commercial application of rfVIII protein from CHO has increased extensively, further studies are required at cellular to bioprocess level to overcome the challenges in production, purification and processing. Efficient strategies are required to attain better products pertaining to the glycosylation path, productivity, stability, etc., to bring down the cost of expensive therapeutics like rfVIII that obviates these biotherapeutics affordable to common man. This review summarizes the various approaches and developments that have been in practice in fVIII production.

Broad substrate affinity and catalytic diversity of fibrinolytic enzyme from Pheretima posthumous-Purification and molecular characterization study.

In this research, a serine protease was isolated and purified from Indian earthworm Pheretima posthumous by fractionation with ammonium sulfate followed by ion exchange and size exclusion chromatography. The molecular weight of purified protease was determined 29.5kDa by Maldi-TOF/MS. The enzyme exhibited a maximum proteolytic activity of 1.2U/ml with specific activity of 17.65U/mg at pH 8 and temperature 40°C. 2D electrophoresis study illustrated purity of enzyme, purified as a single peptide and isoelectric point (pI) 4.5. The enzyme has shown tremendous stability and proteolytic activity in the wide range of pH range (4-12) and temperatures (20-60°C). The kinetic constant Km and Vmax of purified protease were reported 0.09mg/ml and 23.25mg/ml/min. The enzyme also possesses excellent catalytic capacity with Kcat (341.9min-1) and catalytic efficiency (3798.88). The N-terminal sequence of purified protease Arg-Lys-Lys-Gly-Ala-Ser-Try-Phe-Try-Pro-Trp-Ser-Val-Lys-Lys-Arg, PMF and MS/MS studies had shown a partial homology with Lumbrokinase-P2 (2) from Lumbricus rubellus. The CD spectroscopy result provided an evidence for broad substrate affinity and stability of enzyme. The different forms of secondary structures determined in EFE result broad substrate affinity of enzyme.

Cold active pectinases: advancing the food industry to the next generation.

Pectinase has been an integral part of commercial food processing, where it is used for degradation of pectin and facilitates different processing steps such as liquefaction, clarification and juice extraction. The industry currently uses pectinases from mesophilic or thermophilic microorganisms which are well established, but recently, there has been is a new trend in the food industry to adopt low-temperature processing. This trend is due to the potential economic and environmental advantages which the industry envisages. In order to achieve this change, an alternative for the existing pectinases, which are mostly mesophilic and temperature-dependent, must be identified, which can function efficiently at low temperatures. Psychrophilic pectinases derived from cold-adapted microorganisms, are known to function at low to freezing temperatures and may be an alternative to address the problem. Psychrophilic pectinases can be obtained from the vast microflora inhabiting various cold regions on earth such as oceans, Polar Regions, snow-covered mountains, and glaciers. This article is intended to study the advantages of cold active pectinases, its sources, and the current state of the research.

In vitro characterization of a multifunctional staphylokinase variant with reduced reocclusion, produced from salt inducible E. coli GJ1158.

The thrombolytic therapy with clinically approved drugs often ensues with recurrent thrombosis caused by thrombin-induced platelet aggregation from the clot debris. In order to minimize these problems, a staphylokinase (SAK)-based bacterial friendly multifunctional recombinant protein SRH (staphylokinase (SAK) linked with tripeptide RGD and dodecapeptide Hirulog (SRH)) was constructed to have Hirulog as an antithrombin agent and RGD (Arg-Gly-Asp) as an antiplatelet agent in the present study. This multifunctional fusion protein SRH was expressed in osmotically inducible E. coli GJ1158 as soluble form and purified with a yield of 0.27 g/L and functionally characterized in vitro. SRH retained the fibrinolytic activity and plasminogen activation rate comparable to the parental counterpart SAK. The antithrombin activity of SRH was significantly higher than SAK. The platelet rich clot lysis assay indicated that SRH had enhanced platelet binding activity and T(50%) and C50 of SRH were significantly lower than that of SAK. Furthermore, SRH inhibited the ADP-induced platelet aggregation in dose-dependent manner while SAK had no significant effect on platelet aggregation. Thus, the current study suggests that the SAK variant produced from osmotically inducible GJ1158 is more potent thrombolytic agent with antithrombin and antiplatelet aggregation activities for reduction of reocclusion in thrombolytic therapy.

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.