Focused Research Areas : Biochemical Engineering

Studies on production of bioactives, biochemicals and biologicals

 1. Studies on cell culture based protein production

 Mammalian cell culture systems are predominantly used for the production of therapeutic protein and antibodies for healthcare. Proper production and processing of antibodies require a complex cellular mechanism. Formation of disulfide bonds (inter and intra chain) along with post-translation modification primarily glycosylation are required for the proper functioning and stability of antibody. Glycosylation is a critical protein quality attribute and changes in culture conditions like pH, glucose depletion, accumulation of ammonia, lactic acid etc. lead to formation of glycoforms with probability of altered serum half life and binding affinities etc.  In our studies, cloning and expression of a biosimilar antibody in CHO-S cell line was successfully completed.  Limiting dilution technique was used for generating stable single cell clone expressing the mAb. The effects of media composition on antibody production is being studied at flask level to maximize its production. Protein A chromatography followed by size exclusion chromatography was employed for the purification of mAb and the purified mAb was partially characterized with reference to the innovator product. Further work on process scale up and optimization of mAb production and its characterization is in progress.

2. Screening and process optimization of L-Asperaginase production 

The overall aim of the project was to isolate bacterial strains capable of hyperproduction of L-asparaginase which is a potent anti-cancerous agent. L-asparaginase catalyzes the hydrolysis of L-asparagine into aspartic acid and ammonia and thus has a role in the treatment of acute leukemia lymphoblast (ALL). In this study, L-asparaginase from three new bacterial sources were identified and initial studies  showed promising results of production of this potent biotherapeutic agent. A new pH indicator dye i.e., cresol red based assay for efficient screening of L-asparaginase was also developed. Furthermore,

L-Asperaginase production from these three strains was optimized by conventional and statistical approaches. L-asparaginase from one of these strains showed free radical scavenging application by inhibiting the oxidation of 2,2-Diphenyl-1-picrylhydrazyl (DPPH) which have damaging effects on cells of organisms. Further workonuse of genetic manipulation strategies to enhance L-Asparaginase production and purification and characterization of the enzyme are in progress.


Media supplemented with Cresol Red dye

(a) Uninoculated (Control)

(b) IGS-131

(c) E. coli (T) (Positive Control)

(d) S. epidermidis BT-34 (Negative Control

(e) IGS-131 (Without Asparagine)

3. Development of Kluyveromycesmarxianus as platform for production of biomolecules 

Kluyveromycesmarxianus strains are considered as one of the best choices for industrial applications. However, some of the major limitations like its genetics are not well established. There are limited literature information available on this strain and to the best of our knowledge, though some promotors from K. marxianus such as PKmPGK, PKmTDH3 and PKmADH1are reported these are not strong native promoters. The main aim of this work is toidentifynew novel strong promoter for expression of heterologous proteins in K. marxianus using xylose as carbon source.

4. Studies on antimicrobial peptides (AMPs)

The rising emergence of antibiotic resistant bacteria has led to the exploration of alternative therapeutic agents such as ribosomally synthesized bacterial peptides known as bacteriocins. The putative gene encoding an antimicrobial peptide sonorensin, identified from the genome of B. sonorensis MT93, was successfully cloned and expressed heterologously in E.coli. Another clone producing a antimicrobial peptide into periplasm with the highest host cell viability was selected and this non-ribosomal peptide was purified to homogeneity by reverse phase chromatography. Its characterization and production using batch and fed batch processes are in progress.


Exploration of microbial diversity for useful enzymes and bioactive molecules

Microbial diversity is a valuable resource of industrially useful enzymes and bioactive molecule. Microbial enzymes are economically important due to their applications in industries such as textile, paper and pulp, biofuel and food . Enzyme based process supplants hazardous chemicals and therefore are eco-friendly.

1. Xylanase(EC3.2.1.8): We have explored microbial diversity for isolation of xylanase producing bacteria, their production and application. Two different potent strains producing xylanase have been isolated and identified as Bacillus altitudinis and a novel sp. belonging to Bacillus subtilis group. Xylanase treatment to Kraft pulp has shown 15 % decrease in the requirement of chemical bleaching compounds.

2. Laccase (EC A novel high redox potential laccase has been isolated from a fungal strain identified as Arthrographis sp. The laccase can degrade a variety of textile dyes including indigo. Process for production of laccase using surface culture reactor has been developed. The laccase has been tested for its ability to bleach denim fabric in industrial set-up.

3. Isolation of Quorum sensing Inhibitor (QSI) Using Chromobacterium violaceum based screen, a Streptomyces strain producing QSI activity has been isolated. Isolation, purification and characterization of the molecule is in progress.

Cephalosporin Modification for production of active intermediates 7-ACA

Semisynthetic Cephalosporins are important in clinical practice due to their antimicrobial spectrum and non allergenic.Cephalosporin C (CEPH-C) is produced by Cephalosporium acremonium. The native cephalsporin is required to be converted into 7-Aminocephalosporanic acid (7-ACA) for making its semisynthetic derivatives. A method for conversion of cephalosporin C to Glutaryl-7-ACA using D-Amino acid oxidase and conversion of GL-7-ACA to 7-ACA by Glutaryl-7-ACA acylase was done. A Bacillus strain producing GL-7-ACA acylase was screened. The enzyme has been isolated, purified and characterized.


Screening for pectinase


Streptomyces sp producing QSI


Dose dependent QSI inhibition by a compound isolated from Streptomyces sp. 671


Xylanase activity assay Novel Arthrographis strain


Colony morphology on YPD agar


Microscopic structure


Denim bleaching by laccase produced by Arthrographis sp

Discovery of new antibiotic, novel efflux pump inhibitors, β-lactamase inhibitor, anti-microbial peptides, cell-penetrating peptide(CPP) to ease the drug delivery

As institute’s commitment for application oriented and translational research, our main focus is on discovery of bioactive molecules for treatment of infectious diseases. Antimicrobial resistance (AMR) is one of the thirst areas of this institute and we continuously strive for new antibiotic with novel mode of action, alternate approaches to tackle the AMR with the help of adjuvant, potentiator, target-enzyme/protein inhibitor, etc. Our inspiration is a diverse Indian ecology and large sea shore with diverse microbes, medicinal plants, etc. We are continuously working on exploration, exploitation of natural diversity for novel bio-actives to fulfill the un-mate need of “Swastha Bharat” in the area of infectious diseases.

At present our concentrated effort is on discovery of new antibiotic, novel efflux pump inhibitors, β-lactamase inhibitor, anti-microbial peptides, cell-penetrating peptide(CPP) to ease the drug delivery, etc. To achieve these goals, we use all advance techniques for isolation of culturables/non-culturable microbes, improvement of fermentation conditions for high titre of bioactives, advanced purification/downstream techniques of purification/chromatography of secondary metabolites, chemical characterization, structural elucidation, biological (In vitro&In vivo) evaluation of hits for druggability, analogues design and lead optimization.

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Exploitation of microbes for production of commercially important polysaccharides

Exopolysaccharides (EPSs) are high molecular weight polymers that constitute a substantial component (40-95%) of the extracellular polymeric substances surrounding most microbial cells. Exopolysaccharide (EPS) of microbial origin have already generated great interest among scientists worldwide due to their unique and conserved properties that can be commercially exploited for use in variousindustrial  sectors. They are considered as an inexhaustible source for the production of the polymers. The importance of microbial exopolysaccharides has been highlighted presently not only due to their diverse chemical structure but also because of their ecological importance and various biotechnological applications. Many of these exopolysaccharides have been reported to function as viscosifying agents, stabilizers, emulsifiers, gelling and water-binding agents in food industry. Around 120 isolates obtained from several niches were screened for EPS production. These strains were obtained in collaboration with MTCC scientists. A suitable media containing glucose as the carbon source was used for screening. Among the screened isolates, 6 have shown the ability to produce the EPS.  At present we are creating a library of these EPSs which will be further investigated for their industrial potential.

Technology development for Gellan gum production

Gellan gum (E418) has gained importance in the food or pharmaceutical industries, gel electrophoresis, immobilization of cells and enzymes, and bioremediation. Its potential use as a replacement for gelatin and agar makes it one of the most important polysaccharides. According to ICR World Market Research the world Gellan Gum market held an opportunity worth US$315 Million in 2015. The market is expected to expand at 2.17% CAGR over the period between 2016 and 2021. In the report, titled “Gellan Gum Market: Global Industry Analysis & Opportunity Assessment, 2016-2026,” it is mentioned that a majority of gellan gum manufacturers in the global market are based in China, and are incidentally factoring a rampant growth of gellan gum market in the Asia Pacific excluding Japan (APEJ) region. To the best of our knowledge there is no manufacturer of this product in India. This project would attempt to develop a commercially feasible indigenous technology for gellan gum production in pilot scale.

Technology development for Pullulan production

Pullulan, a homo-polysaccharide of maltotriose, is an extremely versatile ingredient with capability of providing a technology platform for product innovation.Due to unique physicochemical properties, pullulan has found applications in food, pharmaceuticals and cosmetic industries.  Pullulan can be formed into capsules for use with pharmaceutical and nutraceutical products. Its non-animal origin and GRAS status ensures safety and acceptability across diverse consumer groups. In CSIR-IMTECH, we have developed a process for pullulan production using osmotolerant strain of Aureobasidium pullulans. The process is ready for demonstration at 500L fermenter scale. The yield and productivity of our process is higher as compared to published reports. Moreover, the process was developed using low-cost agri-industrial residues and would result in significant reduction in the RMC vis a vis cost of production.  Hence, our process is actually about creating wealth from waste. 

Fermentation Process Development for recombinant Protein production

Our research focuses on Recombinant proteins production in E-coli and  Pombe Yeast. Fission Yeast Schizosaccharomyces Pombe an ideal host for expressing Human proteins and viral antigens for Therapeutic and Diagnostic applications. The Batch Fermentation process using synthetic media yields 43 OD in 96 hrs. The Fed-batch fermentation process with linear feeding yielded 100 OD in 90 hrs. The dry cell weight of S.pombe Fed-batch fermentation Biomass yields 50 g/l.The purified HBsAg was identified with transmission electron microscopy in range of 22 nm Virus-like particles. In another study recombinant Bacteriorhodopsin protein was sucessfully produced in E-coli in Batch fermenter which leads to pigmented E-coli cells by the addition of all-trans retinal chromophore.  


Batch Fermentation of Recombinant S.Pombe yeast for HbsAg production

To find new biomolecules to inhibit and abrogate microbial biofilm as well as kills planktonic forms of the pathogenic microbes 

Healthcare associated infections (HAI) are one of the top 10 causes of mortality worldwide [Wang et al, 2017]. Most of the microorganisms associated with HAI are biofilm formers and have capability of accruing antimicrobial resistance. Biofilm-associated microorganisms are highly resistant to antimicrobial therapies (chemical and physical treatment) and are virtually impossible to eradicate. Their relation to medical devices, implants and injuries cause extra burden to the medical andhealth-care system. Recently, Government of India, along with other agencies has declared antimicrobial resistance (AMR) as one of the priority areas. The WHO has indicated Pseudomonas aeruginosa, (carbapenum resistant), as Critical and Staphylococcus aureus (methicillin resistant, vancomycin intermediate) as High Priority microorganism for which new drug candidates are required to be developed urgently. In hospital setup these two microorganisms along with few other fungal pathogens have been found to cause devices and wound injury related-infections that are resistant to antimicrobial therapy. Biofilm formation and a variety of other molecular mechanisms such as drug efflux have been found to be the cause of drug resistance. We have designed our research work to find new biomolecules to inhibit and abrogate microbial biofilm as well as kills planktonic forms of the pathogenic microbes (OLP 0855). with prior proof of concept of biofilm eradication of the indicated microorganism [Haque et al., 2016; and Haque et al., 2017] we are also working on the development of an unique therapy for biofilm eradication combining two or more potent molecules (with different mode of action) in a single nano/micro- assembly. Primarily the project is aimed to develop antibiofilm therapy for topical application. However, other different application of the proposed research is envisaged in future. Further, we are also working on the understanding of the molecular mechanism of action of a glycolipid (sophorolipid) against pathogenic fungal infection. A part of our research is also devoted to develop drug candidates against biofilm forming microbial pathogens and to synthesize chiral pharmaceutical intermediates and fine chemicals using biocatalysis.

Technology development for sophorolipid production

Sophorolipid is an important microbial glycolipid biosurfactant, containing a sugar moiety (sophorose) and a fatty acid, is available in lactonic and open chain acidic form. These molecules have versatile application in cosmetic and pharmaceutical industries along with its application as detergent and cleaning agent. Sophorolipid is also known to be a less-toxic, non-allergic biosurfactant with significant biomedical potential as anticancer, virucidal, bactericidal and antifungal agents. These molecules have been approved for external application by the USFDA and are used in the shampoo, body washes, perfume and fragrance products in the cosmetic and body care industries.  We are developing a cost effective technology for the production of sophorolipid using agri-industry by product. The technology is ready at bench scale and has scope of development at the pilot scale.

Glycolipid Based Niosomal Delivery System for Drugs and Biomolecules

Efficient and safer delivery of drugs (with low solubility and poor bioavailability) and is a challenging task in pharmaceutical and biotechnological industries. Niosomes, by virtue of its vesicular structure, can house both hydrophilic and hydrophobic molecules within its compartments. By exploring the structure-forming attributes of acidic sophorolipid (a microbial glycolipid) we have developed a carrier system for the delivery of drugs (with low solubility and poor bioavailability) and biomolecules. This versatile system can be synthesized with different charges (+Ve/-Ve), size and physical characteristics. A prototype of the system has been tested for the delivery of poorly soluble drug amphotericin B. The application of this system has also been envisaged for gene and protein delivery and for bio-imaging purposes.

Development of new antimicrobial and antibiofilm compounds

Microbial infection poses increasing threat to human community and is a matter of great concern in hospital and healthcare setups. Recently, World Health Organization (WHO) has released the list of priority microorganism causing threat to human community and exhibits resistance to one or more classes of antibiotics. These microorganisms are capable of forming biofilms that make them more resistant to antibiotic treatment (biofilm dwelling microorganisms are 10-1000 times more resistant to antibiotics compared to their planktonic counterpart). Microbial biofilms are potential sources of recurrent infection making them virtually impossible to eradicate. Therefore, there is an urgent need to improve the existing therapies to eradicate the biofilm related infections or to develop new therapies to eradicate biofilms as well as the planktonic cells. Nature provides a vast reservoir of chemicals and macromolecules that exhibit potential antimicrobial and antibiofilm activity. We are exploring the microbial resources isolated from unexplored niches of the country to identify molecules that exhibit antimicrobial and antibiofilm activity. By screening the yeast biodiversity of the high altitude Himalayas, we have been able to identify few biomolecules that inhibit and abrogate microbial biofilm as well as kills planktonic forms of the pathogenic microbes. These molecules are being purified and characterized and their mode of action is being studied in our laboratory.

Biocatalytic synthesis of chiral intermediates and fine chemicals

Biocatalysis is the greener and environment-friendly method for the synthesis a wide range of chemicals, pharmaceuticals, polymers and various other molecules. A part of our research is devoted to develop chiral alcohols that are used as pharmaceutical intermediates. Further, we are also developing molecular candidates for antibiofilm and antimicrobial applications synthesized by biocatalysis. 


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