ADSORPTION OF PEPSIN WITH POLYETHYLENEIMINE

ABSTRACT Polyethyleneimine is being increasingly applied for immobilizing enzymes employing its primary, secondary and tertiary ionisable amino groups. In this study, we attempt to immobilize pepsin on Sepabeads functionalized with Polyethyleneimine (PEI) polymers of varying molecular sizes (M w 600, 1200, 10,000 gmol −1 ). The stability and activity of pepsin bound to PEI was found to be dependent on PEI molecular weight and also on the ionic strength of medium, used for immobilization and protein hydrolysis. Immobilized pepsin exhibited good storage and operational stability which was investigated for generating F (ab') 2 fragments from hydrolysis of immunoglobulins. Digestion of IgG by soluble pepsin and immobilized pepsin showed the same electrophoresis profile (of F (ab') 2 and Fc fragments) indicating no modification in pepsin specificity after immobilization on PEI activated Sepabeads support. The above method of immobilization presents an efficient means to immobilize pepsin onto a solid support wherein the said preparation would be free from autolysis and can be reused for multiple cycles

Engineering Geobacillus thermoglucosidasius for direct utilisation of holocellulose from wheat straw

Background: A consolidated bioprocessing (CBP), where lignocellulose is converted into the desired product(s) in a single fermentative step without the addition of expensive degradative enzymes, represents the ideal solution of renewable routes to chemicals and fuels. Members of the genus Geobacillus are able to grow at elevated temperatures and are abile to utilise a wide range of oligosaccharides derived from lignocellulose. This makes them ideally suited to the development of CBP. Results: In this study, we engineered Geobacillus thermoglucosidasius NCIMB 11955 to utilise lignocellulosic biomass, in the form of nitric acid/ammonia treated wheat straw to which expensive hydrolytic enzymes had not been added. Two different strains, BZ9 and BZ10, were generated by integrating the cglT (β-1,4-glucosidase) gene from Thermoanaerobacter brockii into the genome, and localising genes encoding different cellulolytic enzymes on autonomous plasmids. The plasmid of strain BZ10 carried a synthetic cellulosomal operon comprising the celA (Endoglucanase A) gene from Clostridium thermocellum and cel6B (Exoglucanase) from Thermobifida fuscii, whereasstrain BZ9, contained a plasmid encoding the celA (multidomain cellulase) gene from Caldicellulosiruptor bescii. All of the genes were successfully expressed, and their encoded products secreted in a functionally active form, as evidenced by their detection in culture supernatants by Western blotting and enzymatic assay. In the case of the C. bescii CelA enzyme, this is one of the first times that the heterologous production of this multi-functional enzyme has been achieved in a heterologous host. Both strains (BZ9 and BZ10) exhibited improved growth on pre-treated wheat straw, achieving a higher final OD600 and producing greater numbers of viable cells. To demonstrate that cellulosic ethanol can be produced directly from lignocellulosic biomass by a single organism, we established our consortium of hydrolytic enzymes in a previously engineered ethanologenic G. thermoglucosidasius strain, LS242. We observed approximately 2-fold and 1.6-fold increase in ethanol production in the recombinant G. thermoglucosidasius equivalent to BZ9 and BZ10, respectively, compared to G. thermoglucosidasius LS242 strain at 24 hours of growth. Conclusion: We engineered G. thermoglucosidasius to utilise a real-world lignocellulosic biomass substrate and demonstrated that cellulosic ethanol can be produced directly from lignocellulosic biomass in one step. Direct conversion of biomass to desired products represents a new paradigm for CBP, offering the potential for carbon neutral, cost-effective production of sustainable chemicals and fuels

Modelling and numerical simulation of liquid–solid circulating fluidized bed system for protein purification

A novel liquid–solid circulating fluidized bed (LSCFB) was modelled for protein recovery from the feed broth. A typical LSCFB system consists of downer and riser, integrating two different operations simultaneously. A general purpose, extensible, and dynamic model was written based on the tanks-in-series framework. The model allowed adjusting the degree of backmixing in each phase for both columns. The model was validated with previously published data on extraction of bovine serum albumin (BSA) as model protein. Detailed dynamic analysis was performed on the protein recovery operation. The interaction between the riser and downer were captured. Parametric studies on protein recovery in LSCFB system were carried out using the validated model to better understand the system behaviour. Simulation results have shown that both production rate and overall recovery increased with solids circulation rate, superficial liquid velocity in the downer and riser, and feed solution concentration. The model was flexible and could use various forms of ion exchange kinetics and could simulate different hydrodynamic behaviours. It was useful to gain insight into protein recovery processes. The general nature of the model made it useful to study other protein recovery operations for plant and animal proteins. It could also be useful for further multi-objective optimization studies to optimize the LSCFB system

A rapid preparative method for the purification of vitamin E acetate by kinetic reverse phase chromatography

Alpha-tocopherol or vitamin E is an essential food ingredient. Due to high purity requirement in food applications, it is majorly produced in the acetate form through chemical routes. However, the isolation of food grade vitamin E acetate (VEA) is critical due to the co-generation of close characteristic impurities in the production processes. The work undertaken here provides a new kinetic chromatography method for production of food grade VEA. In this work, suitable adsorbent for chromatography separation was selected based on shallow-bed binding–elution characteristics. Loading conditions were identified by frontal adsorption behavior. Kinetic parameters and bed height for the purification was predicted by establishing the relationship between bed height, purity and the relative separation factor (RSF). The RSF of 2.8 and corresponding bed height is predicted from the established relationship for the separation of VEA from impurities. The bed height of 170 cm with isocratic mobile phase of 2% (wt/wt) water in methanol provides 98 wt.% purity of VEA with recovery of 90%. This work has highlighted methodology based on kinetic parameters for the separation of close characteristic impurities from target molecule. This work also provided the application of simple handy tools like RSF and dual wavelength detection for the rapid development of preparative separation process for VEA and other such valuable components from unknown impurities

Depolymerization of starch and pectin using superporous matrix supported enzymes

253-258Immobilized enzyme catalyzed biotransformations involving macromolecular substrates and/or products are greatly retarded due to slow diffusion of large substrate molecules in and out of the typical enzyme supports. Slow diffusion of macromolecules into the matrix pores can be speeded up by use of macroporous supports as enzyme carriers. Depolymerization reactions of polysaccharides like starch, pectin, and dextran to their respective low molecular weight products are some of the reactions that can benefit from use of such superporous matrices. In the present work, an indigenously prepared rigid cross-linked cellulose matrix (called CELBEADS) has been used as support for immobilizing alpha amylase (1,4-α-Dglucan glucanohydrolase, EC 3.2.1 . 1.) and pectinase (endo-PG: poly( 1 ,4-α-galactouronide) glycanohydrolase, EC 3.2. 1.1 5). The immobilized enzymes were used for starch and pectin hydrolysis respectively, in batch, packed bed and expanded bed modes. The macroporosity of CELBEADS was found to permit through-flow and easy diffusion of substrates pectin and starch to enzyme sites in the porous supports and gave reaction rates comparable to the rates obtained using soluble enzymes.</span

Evaluation of antiangiogenic activity through tubulin interaction of chloroform fraction of the feather star, <i style="">Lamprometra palmata palmata</i>

28-37 Tubulin binding agents have received considerable interest as potential tumour-selective angiogenesis-targeting drugs. The present study elucidates that chloroform fraction (CC) isolated from methanol extract of the feather star Lamprometra palmata palmata has a tubulin binding property. With the quantification of chick chorioallantoic (CAM) assay, we further demonstrate that CC significantly and dose-dependently inhibits proliferation, migration of endothelial cells and exhibits antiangiogenic effect with ID50 10ng/10ml. It showed moderate cytotoxicity with IC50 192 mg/ml. In addition, CC arrested onion root tip cells at prometaphase phase. We demonstrate that these effects of CC are attributable to its property to inhibit polymerization of tubulin. These findings show that CC is a candidate antiangiogenic agent and needs further purification for the specific compound, which is responsible for all these activities. </smarttagtype

Green synthesis of isopropyl myristate in novel single phase medium Part I: Batch optimization studies

Isopropyl myristate finds many applications in food, cosmetic and pharmaceutical industries as an emollient, thickening agent, or lubricant. Using a homogeneous reaction phase, non-specific lipase derived from Candida antartica, marketed as Novozym 435, was determined to be most suitable for the enzymatic synthesis of isopropyl myristate. The high molar ratio of alcohol to acid creates novel single phase medium which overcomes mass transfer effects and facilitates downstream processing. The effect of various reaction parameters was optimized to obtain a high yield of isopropyl myristate. Effect of temperature, agitation speed, organic solvent, biocatalyst loading and batch operational stability of the enzyme was systematically studied. The conversion of 87.65% was obtained when the molar ratio of isopropyl alcohol to myristic acid (15:1) was used with 4% (w/w) catalyst loading and agitation speed of 150 rpm at 60 °C. The enzyme has also shown good batch operational stability under optimized conditions

Green synthesis of isopropyl myristate in novel single phase medium Part II: Packed bed reactor (PBR) studies

Isopropyl myristate is a useful functional molecule responding to the requirements of numerous fields of application in cosmetic, pharmaceutical and food industry. In the present work, lipase-catalyzed production of isopropyl myristate by esterification of myristic acid with isopropyl alcohol (molar ratio of 1:15) in the homogenous reaction medium was performed on a bench-scale packed bed reactors, in order to obtain suitable reaction performance data for upscaling. An immobilized lipase B from Candida antartica was used as the biocatalyst based on our previous study. The process intensification resulted in a clean and green synthesis process comprising a series of packed bed reactors of immobilized enzyme and water dehydrant. In addition, use of the single phase reaction system facilitates efficient recovery of the product with no effluent generated and recyclability of unreacted substrates. The single phase reaction system coupled with a continuous operating bioreactor ensures a stable operational life for the enzyme