Yogender Pal Khasa

Optimization of cellulase production by a brown rot fungus Fomitopsis sp. RCK2010 under solid state fermentation

Culture conditions for enhanced cellulase production from a newly isolated brown rot fungus, Fomitopsis sp. RCK2010 were optimized under solid state fermentation. An initial pH of 5.5 and moisture ratio of 1:3.5 (solid:liquid) were found to be optimal for maximum enzyme production. Of the different carbon sources tested wheat bran gave the maximum production of CMCase (71.526 IU/g), FPase (3.268 IU/g), and β-glucosidase (50.696 IU/g). Among the nitrogen sources, urea caused maximum production of CMCase (81.832 IU/g), where as casein and soyabean meal gave the highest FPase (4.682 IU/g) and β-glucosidase (69.083 IU/g) production, respectively. Among amino acids tested glutamic acid gave the highest production for CMCase (84.127I U/g); however 4-hydroxy-l-proline stimulated maximum FPase production (6.762 IU/g). Saccharification of pretreated rice straw and wheat straw by crude enzyme extract from Fomitopsis sp. RCK2010 resulted in release of 157.160 and 214.044 mg/g of reducing sugar, respectively.

Bioethanol production from Lantana camara (red sage): Pretreatment, saccharification and fermentation

Lantanacamara contains 61.1% (w/w) holocellulose and can serve as a low-cost feedstock for bioethanol production. Acid hydrolysis (3.0%, v/v H(2)SO(4), 120 degrees C for 45 min) of L. camara produced 187.14 mg/g total sugars along with fermentation inhibitors such as phenolics (8.2mg/g), furfurals (5.1mg/g) and hydroxy methyl furfurals (6.7 mg/g). Sequential application of overliming (pH 10.0) and activated charcoal (1.5%, w/v) adsorption was used to remove these toxic compounds from the acid hydrolysate. The acid-pretreated biomass of L. camara was further delignified through combined pretreatment of sodium sulphite (5.0% w/v) and sodium chlorite (3.0% w/v), which resulted in about 87.2% lignin removal. The enzymatic hydrolysis of delignified cellulosic substrate showed 80.0% saccharification after 28 h incubation at 50 degrees C and pH 5.0. Fermentation of acid and enzymatic hydrolysates with Pichiastipitis and Saccharomycescerevisiae gave rise to 5.16 and 17.7 g/L of ethanol with corresponding yields of 0.32 and 0.48 g/g after 24 and 16 h, respectively.

Solid state bioconversion of wheat straw into digestible and nutritive ruminant feed by Ganoderma sp. rckk02

Solid state fermentation (SSF) of wheat straw with Ganoderma sp. rckk02 was carried out for 15 days for improving its digestibility and nutrients. Fungal growth caused a significant (P<0.05) decrease in acid detergent fiber (ADF), neutral detergent fiber (NDF), hemicellulose, lignin and cellulose content till 15th day. In vitro gas production (IVGP) test revealed that 10th day fermented feed possessed higher metabolizable energy (ME: 4.87 MJ/kg), in vitro organic matter digestibility (OMD: 334 g/kg) and short chain fatty acids (SCFAs: 1.82 mmol/g Dry Matter). The fermented feed was also evaluated in vivo in goats fed with either untreated wheat straw (T1) or fungal treated straw (T2). Dry matter intake (DMI), digestible crude protein (DCP), total digestible nutrients (TDN) and nitrogen (N) intake were found significantly (P<0.05) increased in T2 group. The study shows that fermentation of wheat straw with Ganoderma sp. rckk02 holds potential in improving its nutritive value.

High level production of active streptokinase in Pichia pastoris fed-batch culture

Streptokinase is a biological macromolecule involved in dissolution of fibrin blood clot and favourably used in various clinical applications. This protein is poorly expressed in soluble form due to its toxic effects on host physiology. The extracellular expression of recombinant streptokinase (SK) with and without 6xHis tag was obtained by cloning its gene under the α-mating factor signal sequence and alcohol inducible AOX1 promoter. Host-vector combinations were optimized to select a hyper producer. From shake flask optimization studies, a maximum expression of 582 mg/L of rSK (non-tagged) and 538 mg/L of rSK-His (His-tagged) protein was obtained when cells were induced at OD600 of 20. The high cell density fermentation increased the volumetric product concentration of rSK-His to a level of 4.25 g/L with a 7.9 folds increase from shake flask results. The specific product yield (YP/X) was 49.75 mg/g DCW along with a high volumetric productivity of 57.43 mg/L/h. The protein was predicted to have 15.43% α-helix and 26.43% β-sheet with tryptophan emission maxima of around 347 nm. The highest specific activity of rSK-His was 64,903 IU/mg with 1.48 folds purification whereas specific activity of rSK was 55,240 IU/mg with 1.22 folds purification.

Genetically Modified Microorganisms (GMOs) for Bioremediation

The increasing amount of pollutants in the environment is an alarming concern to the ecosystem. A number of organic pollutants, such as polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs), and pesticides, are resistant to degradation, which represent toxological threat to wildlife as well as human beings. Various physiological and biological measures have been employed globally to degrade these hydrocarbons to improve environment quality. Out of these, bioremediation is the most promising strategy where microorganisms are harnessed to degrade the organic and inorganic pollutants. There are many naturally existing microbes, which are routinely employed in bioremediation process. At instances, these consortia of microorganisms in various environmental conditions provide an insight about the interrelation of metabolic pathways involved in biodegradation process. Various metabolic techniques are employed to produce genetically engineered microorganisms (GEMs) with better bioremediation efficiency. Majorly biomolecular engineering approaches such as rational designing and directed evolution have been developed to genetically modify microorganisms and their enzymes for the degradation of persistent organic pollutants (POPs) like PAHs, PCBs, and pesticides. Recently, several developments in the field of recombinant DNA technologies such as development of “suicidal-GEMs” (S-GEMs) have also been carried out to achieve safe and efficient bioremediation of contaminated sites. In this chapter, we describe various techniques for the development of genetically modified microorganisms along with different examples of recombinant produced. Harmful impact of the engineered microorganisms on environment and economic consideration of viable processes development are critically discussed.

The evolution of recombinant thrombolytics: Current status and future directions

ABSTRACT Cardiovascular disorders are on the rise worldwide due to alcohol abuse, obesity, hypertension, raised blood lipids, diabetes and age-related risks. The use of classical antiplatelet and anticoagulant therapies combined with surgical intervention helped to clear blood clots during the inceptive years. However, the discovery of streptokinase and urokinase ushered the way of using these enzymes as thrombolytic agents to degrade the fibrin network with an issue of systemic hemorrhage. The development of second generation plasminogen activators like anistreplase and tissue plasminogen activator partially controlled this problem. The third generation molecules, majorly t-PA variants, showed desirable properties of improved stability, safety and efficacy with enhanced fibrin specificity. Plasmin variants are produced as direct fibrinolytic agents as a futuristic approach with targeted delivery of these drugs using liposome technlogy. The novel molecules from microbial, plant and animal origin present the future of direct thrombolytics due to their safety and ease of administration.

Single chain Fv fragment specific for human GM‐CSF: Selection and expression using a bacterial expression library

Single chain antibodies (scFvs) are replacing whole antibody molecules since they are easy to produce on large scale and amenable to genetic modifications. Here we report the development of an anti‐human granulocyte macrophage colony‐stimulating factor (hGM‐CSF) scFv as an immunoassay bio‐reagent, utilizing an easily scalable bacterial expression system. For this, the VH and VL gene repertoires were amplified from the immunoglobulin complementary DNA, derived from total RNA of mice splenocytes, pre‐sensitized with the antigen. The scFv library was expressed under the strong T7 promoter in BL21 (DE3) Escherichia coli cells. Preliminary screening led to the selection of four potential candidates, which were later subjected to light chain shuffling. Cross‐reactivity analysis involving the original and shuffled candidates resulted in the selection of one scFv (scFv196) with no cross‐reactivity against E. coli antigens. The binding affinity of the scFv196 for hGM‐CSF, measured by surface plasmon resonance, was found to be within the physiological range (KD =1.5 μM). The refolded scFv was also shown to recognize and bind the glycosylated antigen, a closer mimic of the physiological GM‐CSF, potentiating its use in immunoassays. Expression studies using shake flasks suggested periplasmic export of the scFv196 protein.

Microbes as Biocontrol Agents

Phytopathogens pose a major threat to ecosystem stability and food production, indicating the need for developing methods to control the severe losses caused by these pathogens. To control these pathogens, the use of various chemical pesticides is majorly practiced. These pesticides are associated with environmental and health hazards and also pose a risk of resistance development in phytopathogens against them forcing the researcher towards the development of alternative and innovative methods by which sustainable management of plant diseases can be achieved. To control plant diseases and have pesticide-free food worldwide, the use of natural antagonistic microorganisms known as biocontrol agents or biological control agents (BCA) is employed. BCA can act on these pathogens through a number of mechanisms such as antibiosis, hyperparasitism, enzyme production, induction of plant resistance mechanisms and competition for essential nutrients and space and through plant growth promotion. Apart from controlling phytopathogens, these microbial agents also promote plant growth and stress tolerance. BCA can be used as bioinsecticides, bionematicides and biopesticides. They are also used for the management of post-harvest diseases. Recently, recombinant microbes have been developed with enhanced biocontrol capabilities. Several commercially available BCA are currently being used for the efficient control of plant disease with improved productivity of many crops. These majorly include GB34, Kodiak, Serenade and Companion containing Bacillus as the active ingredient, Biosave 10LP and Bio-jet containing Pseudomonas as the active ingredient and Soilguard, Trichodex and Trichojet containing Trichoderma as the active ingredient. Thus, use of microbes such as fungi, bacteria, yeast and viruses holds an enormous potential as biocontrol agents to replace conventional chemical-based pesticides and provide food security in a safe and eco-friendly manner.

Combined effect of gene dosage and process optimization strategies on high-level production of recombinant human interleukin-3 (hIL-3) in Pichia pastoris fed-batch culture

In this work, the combined effects of gene dosage and process optimization strategies were studied to achieve higher hIL-3 expression in Pichia system. The in-vitro multimerization method was used to generate various Pichia X-33 transformants having multi-copy expression cassettes. The quantitative polymerase chain reaction (qPCR) strategy was used to further confirm the genome integration of hIL-3 expression cassette. From shake flask expression studies, the recombinant hIL-3 concentration in culture supernatant increased upto 8 copies to a level of 310mg/L, thereafter a considerably lower expression was observed. The small scale optimization experiments at shake flask level resulted in an improved product concentration of 350mg/L. The batch and fed-batch fermentation runs in complex medium showed a product concentration of 1.81 and 1.49g/L, respectively. To further enhance the production level, the fermentation runs were conducted in modified minimal media where a maximum hIL-3 protein level of 2.23g/L was obtained in batch fermentation. The specific product yield (YP/X) was at a level of 25.65mg/g DCW, whereas the overall volumetric productivity of the process was 27.31mg/L/h. The biological activity of the partially purified hIL-3 protein was confirmed via the proliferation of human erythroleukemia TF-1 cells using MTT assay.

Microbial Decolorization of Colored Industrial Effluents

Rapid industrialization and urbanization results in the discharge of large amount of organic wastes not easily degradable into water bodies and thereby polluting the environment. The effluent from textile industries itself is of major concern, which contains variety of dyes. The dye wastes being toxic affect the aquatic life and eventually the humans. Therefore, the degradation and/or decolorization of these dyes has become a necessity. The physical and chemical methods used for treating industrial effluents containing dyes are costlier and require high energy. In contrast, various biological methods have been studied to minimize pollution along with the toxicity in the industrial effluents. This chapter will discuss the status about the possibilities of developing various the status about the possibilities of developing various methods or technologies for treating dyes containing waste waters.