Current Biotechnology - Volume 3, Issue 3, 2014

Immobilized enzymes offer many advantages over the free counterparts. These advantages include, but are not limited to, easy separation, reuse, induced stabilities and potential of continuous operation. However, the immobilization process at the same time causes some drawbacks including loss of catalytic activity of the immobilized enzymes. Many interpretations have been given to explain the reasons behind such drawback. Some of them are related to the enzymes themselves, such as deactivation of enzymes and miss-orientation. Others refer to diffusion limitation of substrates and/or to inhibitory effects of the products. Substrate diffusion limitation presents the most serious one. In this review some strategies for overcoming this drawback are reported. Among these strategies are the ones related to enzyme immobilization on: a) soluble-insoluble matrices, b) thermally reversible hydrogels, c) pressure-sensitive gels, d) on catalytic hydrophobic membranes operating under non-isothermal conditions, and finally, on carriers' surface, are presented and discussed.

The current drug discovery and development platform is rapidly expanding with new classes of pharmaceuticals and novel biological information. The drug discovery and development process relies on the utilization of relevant and robust tools, methods, and models that are predictive of clinical effects in terms of diagnosis, prevention, therapy and prognosis. One of the methods that have gained prominence over the years is Molecular Functional Imaging that optimizes and ensures delivery, measures efficacy and toxicity of the therapeutic agents to the target cell or site in pre-clinical setting. Currently three different imaging modalities that include Radionuclide Imaging (Positron Emission Tomography or PET and Single Photon Emitted Computed Tomography or SPECT), Magnetic Resonance Imaging (MRI) and Optical Imaging (Bioluminescence and Florescence) are being extensively used in preclinical models to assess the pharmacokinetics, functional alteration of target and other sites and efficacy of treatment. These real time monitoring modalities are promoting development of novel and sophisticated molecular technologies to identify modulators of protein-protein interactions, apoptosis, protease actions and various other physiological responses. In parallel, significant paradigm shift is occurring at drug discovery and development landscape through emergence of several drug delivery systems like hydrogels, polymers, liposomes and nanoparticles. These innovative delivery vehicles are the future of modern personalized medicine which is still in experimental phases. Molecular Imaging techniques are again the final validation tools for determining the safety, efficacy and need of further improvement of these delivery vehicles. In this review we discuss the current and evolving state of drug discovery and development aided by functional molecular imaging techniques.

Protease enzyme from estuarine sediment bacterium Bacillus flexus was purified by using two steps such as ammonium sulphate precipitation (37.25% enzyme yield and 2.75 fold purification) and sephadex G-75 gel filtration chromatography (9.38% yield and 10.62 fold purification). Molecular weight of the purified protease detected was 44.3 kDa. The protease activity was found to be maximum at pH 8.0 and retained its activity in the pH range of 8-9 after 1.5 h of incubation. Also at 40°C the protease activity was maximum and remained stable between a temperature range of 40 to 50°C after 1.5 h of incubation. This enzyme is slightly halophilic and the maximum activity was observed in 0.5 M NaCl concentration. Further, the tested surfactants were found to enhance the protease activity; also this enzyme maintained its activity in the presence of SDS (5mM). Among the metal ions tested, mercuric chloride and zinc chloride completely inhibited the protease activity and optimum activity was registered in medium added with barium chloride and magnesium chloride. The serine protease inhibitor was found to inhibit 90% activity and hence it was further confirmed as a serine protease type. This enzyme effectively hydrolyzed casein when compared to BSA and gelatin.

Aspergillus is a widely used host organism for the industrial production of homologous and heterologous proteins. Although Aspergillus niger is most commonly used, a close relative of this species, Aspergillus vadensis, has been suggested as a suitable and more favourable alternative due in part, to the low levels of extracellular proteases which it produces. Despite much progress being made in the hyper production of homologous proteins, the yields obtained for heterologous proteins have still not reached a comparable level. Genetic strategies, including the development of strong constitutional promoters have been shown to lead to an increase in the levels of recombinant protein production. In this study, six novel constitutional promoters from A. niger (pef1α, ptktA, pef1β, ptal1, pcetA and ppgkA) and a further five from A. vadensis (pef1α, prps31, pgpdA, pubi1 and poliC) were tested in A. vadensis using a gene encoding a secreted arabinofuranosidase from Fusarium oxysporum as a reporter for heterologous protein production. Remarkably, 9 of the 11 promoter constructs tested resulted in higher ABF activity than the commonly used gpdA promoter. While this could partly be assigned to the number of copies of the expression cassette in the transformants, clear differences in productivity of the promoters could be observed.

Aspergillus vadensis is a close relative of Aspergillus niger and is particularly interesting for hemicellulase production as it does not produce extracellular proteases. The main regulator driving hemicellulase production in Aspergilli and other fungi is XlnR and overexpression of this regulator leads to increased hemicellulase levels in some species. To increase hemicellulase production in A. vadensis strains were generated that contain multiple copies of two XlnR target genes, xlnD and faeA, encoding β-xylosidase and feruloyl esterase A respectively, and/or multiple copies of xlnR. The strains were compared with respect to β-xylosidase and feruloyl esterase activity, respectively, to determine the effect of overexpression of xlnR in comparison to overexpression of the target genes (faeA, xlnD). The results demonstrate that insertion of additional copies of faeA and xlnD had a much larger effect on the corresponding activities than additional copies of xlnR in A. vadensis, suggesting that the amount of XlnR present in A. vadensis is sufficient to activate additional copies of the target genes without much effect on the production of other XlnR-regulated activities.

Sixteen mesophilic fungal strains of Aspergilli (10) and Penicilli (3) and Cochliobolus lunatus isolated from deciduous green forests and wetlands were characterized at molecular level and screened for cellulases (endoglucanase, β- glucosidase (cellobiase), cellobiohydrolase (CBHI/EGI) and FPase) and hemicellulase (xylanase, α -Larabinofuranosidase, β -xylosidase, acetyl xylan esterase and feruloyl esterase) production on rice straw & wheat bran containing solidified substrate. Penicillium janthinellum (3CHP) was found to produce high levels of β-glucosidase and α- L-arabinofuranosidase activities, whereas, Aspergillus tubingensis (8 DAL) produced maximal levels of endoglucanase, Fpase, xylanase and β-xylosidase. The cultures extracts from different strains were analysed for expression of multiple isoforms of endoglucanases, β-glucosidases, xylanases, α-L-arabinofuranosidases, β-xylosidases and acetyl xylan esterase. The enzyme cocktails were used for saccharification of alkali treated rice straw at substrate loading rate of (7%). The maximal saccharification (56%) was observed in enzymatic hydrolysates from Aspergillus terreus.

The production of cold shock proteins constitutes an adaptive response in psychrotrophic and psychrophillic microorganisms to allow survival after cold shock. In the present study a small 10kDa cold resistant protein (CRP) was expressed and purified by immuno-affinty chromatography from CRPF1, a cold resistant mutant of Pseudomonas fluorescens. Sequence analysis and homology studies of cold resistant gene revealed 91% sequence homology with the Pseudomonas fluorescens Pf-5 gene encoding cold shock DNA-binding protein. Three dimensional computational model of CRP was found similar to the known structures of cold shock proteins. It was consisted of five-stranded β- barrel-fold and highly conserved ribonucleoprotein consensus sequences, RNP1 (KGFGFI) and RNP2 (VHF) motif on its surface. Functional characterization of purified protein confirmed the DNA binding property of protein. From the sequence analysis and functional characterization of protein, it was concluded that a low molecular weight, DNA -binding cold resistant protein is induced in CRPF1 that helps in the survival of mutant under low temperature stress conditions.