Punit Kumar

Mycelium transformation of Streptomyces toxytricini into pellet: Role of culture conditions and kinetics

The present study envisages the role of different carbon sources, nitrogen sources, metals, pH, inoculum volume and agitation rate in pellet formation of S. toxytricini at shake-flask level. It was found that galactose, ammonium sulphate, sodium nitrate, Cu2+, Zn2+, higher inoculum volume (5% v/v) and agitation rate at 300rpm caused significant reduction in pellet size (up to the range of 30μm-0.5mm) but biomass formations was also reduced subsequently. Interestingly diffused type of morphology was obtained in Fe2+ supplemented medium with reduced biomass (1.5gL-1). Rheological study revealed that non-Newtonian behaviour of culture broth. Besides this, kinetics study was also made to understand the growth kinetics (0.39gL-1h-1), oxygen uptake rate (0.1146mgL-1h-1), and production of lipstatin (0.0072gh-1).

Current trends and future prospects of lipstatin: a lipase inhibitor and pro-drug for obesity

Obesity is an alarming state caused by an energy imbalance. It is associated with cardiovascular diseases, type-II diabetes, and cancer. Physical inactivity and overeating are the main causes of obesity but scientific reports also support the involvement of Single Nucleotide Polymorphisms (SNPs) in adiposity. Lipids are known to produce more energy than sugars and proteins, hence controlling the digestion of dietary lipids is a promising approach to treat obesity. Lipstatin is a β-lactone molecule which controls the digestive activity of pancreatic lipases and thus controls the absorption of fat in the small intestine. The three dimensional structure of pancreatic lipase revealed that its active site is masked by a lid domain which is opened by the action of bile salt micelles and colipase. Lipstatin is supposed to inhibit the catalytic activity of pancreatic lipase by acylation of the serine residue present in the active site. A binding study suggests that one molecule of lipstatin binds with one molecule of lipase. Tetra-hydro-lipstatin is an Active Pharmaceutical Ingredient (API) of lipstatin which is utilized in antiobesity medicines. Lipstatin is a natural product produced from Streptomyces toxytricini but poor productivity heightens the cost. The mechanism of lipstatin biosynthesis in bacteria is still unexplored. However, the acyl-coenzyme A carboxylase (ACCase) complex plays a key role in lipstatin biosynthesis. The present review focuses on the implications and causes of obesity, the status of antiobesity drugs, the mechanism of inhibition of pancreatic lipases, the biosynthesis of lipstatin and the present status of lipstatin production.

Modulation of fatty acid metabolism and tricarboxylic acid cycle to enhance the lipstatin production through medium engineering in Streptomyces toxytricini

This work investigated the potential of medium engineering to obtain maximum biomass, non-conventional carbon sources for lipstatin production and modulation of tricarboxylic acid (TCA) cycle to promote lipstatin synthesis. It was found that 2:3 carbon and nitrogen ratio, produced maximum biomass of 7.9g/L in growth medium and 6.6g/L in pre-seed medium. Among the studied non-conventional carbon sources i.e., soya flour 40g/L and sesame oil 30mL/L were found producing 1109.37mg/L (1.24-fold of control) and 1196.75mg/L (1.34-fold of control) lipstatin respectively. Supplementation of TCA cycle intermediates revealed that NADH and succinic acid showed lipstatin production to 1132.99mg/L and 1171.10mg/L respectively. Experimental outcome was validated in 7L bioreactor and produced 2242.63mg/L lipstatin which was ∼14% higher than shake flask.

Biotherapeutic potential and mechanisms of action of colchicine

Abstract Cancer is a clinical situation caused by uncontrolled cell division and is responsible for a large number of deaths worldwide. Colchicine is a classical antimitotic, tubulin-binding agent (TBA) which is being explored for its antitumor activities, although its tubulin-binding ability leads to some toxicity toward normal cells proliferation. Colchicine derivatives are considered as potent antitumor compounds with less toxicity compared to colchicine. Derivatives with substituted functional groups at A-ring (methoxy), B-ring (acetamide) or C-ring (methoxy) have been synthesized via chemical and microbial routes and show modified bioactivities and altered tropolonic functionality. Earlier reports, in combination with our group’s research findings, suggest that microbial biotransformation is an efficient choice for the production of bioactive colchicine derivatives. This route has gained significant interest in the mass production of regio-specific, cost-effective, safe and eco-friendly derivatives. The present review paper critically analyzes and discusses the development and application of colchicine derivatives as a potent antitumor molecule and their production through a microbial transformation process. The information provided in this review might assist in the stimulation of new ideas regarding the development of alternative therapeutic agent(s) for cancer treatment.

Exploring Prospects of Monooxygenase-Based Biocatalysts in Xenobiotics

Enzyme technology has progressed in recent years and the hunt for novel enzymes for industrial uses is a big challenge for many microbiologists. These enzymes can be used in a variety of applications provided they are efficient and are able to operate under industrial preference. This task is challenging and demanding and has not been fully covered by conventional microbiologists. The latest update on microbial monooxygenase-based biocatalysts in xenobiotics replicates its importance in hydrocarbon degradation but the work is limited with very few options of enzymes and its regioselectivity and enantioselectivity is reliant on overall recitation of such enzymes. Present chapter describes the key aspects of the overall research scenario regarding microbial monooxygenases which are currently used as a biocatalyst in xenobiotic applications.

Evaluation of Packed-Bed Reactor and Continuous Stirred Tank Reactor for the Production of Colchicine Derivatives

Bioconversion of colchicine into its pharmacologically active derivative 3-demethylated colchicine (3-DMC) mediated by P450BM3 enzyme is an economic and promising strategy for the production of this inexpensive and potent anticancer drug. Continuous stirred tank reactor (CSTR) and packed-bed reactor (PBR) of 3 L and 2 L total volumes were compared for the production of 3-demethylated colchicine (3-DMC) a colchicine derivative using Bacillus megaterium MTCC*420 under aerobic conditions. Statistical optimization technique was utilized with the most significant variables, that is, dissolved oxygen (DO), colchicine concentration, and process time for optimization. The validation of the model was performed by experiments on the predicted values in an individual run, and the optimum parameters were DO (~50%), colchicine concentration (7.5 g/L), and process time (39 h) resulted in a maximum bioconversion of 3-DMC 3.36 g/L. The PBR reactor achieved much higher productivity (6.58 g/L/h) as reported by earlier researchers. This is the first report on the use of PBR for bioconversion of colchicine.

Development of aqueous two-phase systems comprising poly ethylene glycol and dextran for purification of pullulan: Phase diagrams and fiscal analysis

Pullulan is a commercially important Exopolysaccharide (EPS) with wide‐spread applications which is produced by Aureobasidium pullulans. The alternative α (1 4) & α (1 6) configuration in pullulan provides it the specific structural and conformational properties. Pullulan is currently being exploited in food, health care, pharmacy, lithography, cosmetics. The fermented broth is processed by organic solvent precipitation for isolation and purification of pullulan. In this study, we have tried to analyze the potential of aqueous two phase system as an alternate technique to extract pullulan from fermented broth. Including this viability of ATPS was also compared with conventional organic solvent precipitation system in terms of cost and time. It was found that ATPS process produced a higher yield of pullulan (80.56%) than organic solvent precipitation method (71.6%). ATPS was also found more economical and less time consuming method.

Vaccine and antibody production in plants: developments and computational tools

Plants as bioreactors have been widely used to express efficient vaccine antigens against viral, bacterial and protozoan infections. To date, many different plant-based expression systems have been analyzed, with a growing preference for transient expression systems. Antibody expression in diverse plant species for therapeutic applications is well known, and this review provides an overview of various aspects of plant-based biopharmaceutical production. Here, we highlight conventional and gene expression technologies in plants along with some illustrative examples. In addition, the portfolio of products that are being produced and how they relate to the success of this field are discussed. Stable and transient gene expression in plants, agrofiltration and virus infection vectors are also reviewed. Further, the present report draws attention to antibody epitope prediction using computational tools, one of the crucial steps of vaccine design. Finally, regulatory issues, biosafety and public perception of this technology are also discussed.

Improving Production of Tacrolimus In Streptomyces Tacrolimicus (ATCC 55098) Through Development of Novel Mutant by Dual Mutagenesis

Tacrolimus is a polyketide macrolide produced by Streptomyces species which is widely used as anti-fibrotic agent and potent immunosuppressant. In this article dual mutagenesis approach using mutagens (NTG+EMS+UV) was used to develop a mutant strain of Streptomyces tacrolimicus (ATCC 55098) for higher tacrolimus production and this strain showed higher tacrolimus production at 82.5 mg/l. Interestingly; addition of L-Lysine (0.2 g/l) into the production medium further enhanced the tacrolimus production to ~102 mg/l at 7-L fed-batch bioreactor. To the best of our knowledge this is the first report mentioning efficient strain development for higher production of tacrolimus using dual mutagenesis. The obtained data presents an impressive model for higher production of tacrolimus and enhanced our understanding regarding improvement in production capacity of tacrolimus in Streptomyces tacrolimicus.