Current Biotechnology - Volume 3, Issue 4, 2014

The development of cancer is associated with alterations to the physiology of the cell caused by multiple changes in its genome. A frequent genomic event in cancer is chromosome translocation, an exchange of large DNA fragments between two non-homologous chromosomes that in many cases leads to the creation of new fusion genes. Chromosome translocations are key events in the initiation or progression of many neoplastic processes. The difficulty in generating specific chromosome translocations in the laboratory has meant that researchers have lacked appropriate cellular models in which to investigate the effect of these key cancer markers. Different genome-engineering strategies used to induce defined chromosome translocations have met with varying success. Recently, the use of the RGEN technology (RNA-guided endonuclease), also known as the CRISPR/Cas9 system, has demonstrated as proof-of-principle that it is possible to engineer cells to undergo these specific chromosomal translocations with high efficiency. Using this advance, it is now possible to easily and accurately generate cell models harboring the same alterations that define tumor cells from patients, allowing researchers to experimentally recapitulate the genomic alterations needed to transform a healthy cell. In this review, we summarize the different methods used to mimic cancer-related chromosomal translocations and highlight the advantages of RGEN technology for improving the generation of models for the study of cancer. These advances will in time lead to the development of better therapeutic approaches to the treatment of cancer.

Leishmaniasis is defined as a cluster of infectious diseases caused by protozoan parasites of the genus Leishmania. It is recognized worldwide as a major health problem, showing high endemicity in developing countries. The present review details this complex group of neglected diseases, with important clinical spectrum and large epidemiological diversity, and discusses the evolution of leishmaniasis treatment and the urgent need for less toxic, more specific and effective drugs against these parasites. Finally, the value of natural products and synthetic derivatives as rich sources of antileishmanial drug candidates is demonstrated, indicating a promising approach for the development of novel therapeutic alternatives.

The aim of the study was to compare the optimum immobilization conditions of lipase obtained from thermophilic Bacillus aerius and commercial lipase ‘Lipolase’. Both bacterial and commercial lipase were immobilized by surface adsorption onto silica matrix (100-200μm mesh with pore size 2.4 nm) and subsequently exposed to 4% glutaraldehyde, showed binding efficiency of 96.25% and 75.64%, respectively. Maximum enzyme loading was attained after 2 h of incubation in each case. Comparison of various independent variables like reaction time, temperature, substrate affinity, substrate concentration, buffer pH, reusability and thermostability was done to study the effect of matrix on the activity of both bacterial and commercial lipase. Bacterial lipase showed maximum activity with para-Nitrophenyl palmitate (20 mM) at pH 9.5, incubation time of 10 min at 55°C. On the other hand, commercial enzyme showed maximum activity with para-Nitrophenyl palmitate (20 mM) at pH 8.5, incubation time of 10 min and at temperature 40°C. All the metal ions studied exert an inhibitory effect on both bacterial and commercial lipase.

An extracellular cholesterol oxidase (COX) from Bacillus sp. COX-T3 was successfully purified from the cell free culture broth by successive steps of ammonium sulphate precipitation, dialysis and Riboflavin-affinity column chromatography to ∼28.4-fold with an over all yield of 6.2%. The purified COX showed a single band of Mr 21 kDa in SDS-PAGE and ~99 kDa band in Native- PAGE thus indicating an oligomeric nature of the enzyme. The purified COX was adsorbed on to Psyllium husk followed by exposure to glutarayldehyde (2%; v/v) as a cross-linking agent. The Psyllium husk-bound enzyme treated with glutaraldehyde showed an activity of 555.0 U while Psyllium husk-bound enzyme without glutaraldehyde treatment possessed a lower activity (328 U) in comparison to the enzyme used for immobilization (365 U). Thus the Psyllium husk-bound biocatalyst showed 34.2% increase in its activity at 37°C, with pH 7.5 of the free enzyme. None of the salt ions improved the enzyme activity. Approximately 30% of the original enzyme activity was noticed in the presence of Ca2+ ions (0.344 U/ml) while Mn2+ ions (0.02 U/ml) drastically reduced the activity of Psyllium husk-bound COX. The husk-bound COX retained its activity for first 4 days and lost 76.4% of its activity on the 5th day. The purified free and Psyllium husk-immobilized COX exhibited Km and Vmax values of 14.26 mM and 5.34 U/ml/min, and 18.84 mM and 6.28 U/ml/min, respectively. The Psyllium husk-immobilized enzyme however, showed 20.74 mM and 7.75 U/ml/min of Km and Vmax value, respectively in the presence of 50% toluene. Interestingly, in the presence of toluene, the husk-bound COX not only remained highly active/ stable but also provided ∼45% higher rate of reaction (Vmax) in comparison to the free enzyme.

Cyclodextrin glycosyltransferase (CGTase) from the thermophilic anaerobe Thermoanaerobacter sp. 5K was purified and characterized. The enzyme was purified with ammonium sulfate precipitation followed by α-CD-bound, epoxy-activated Sepharose 6B affinity chromatography. Molecular weight of the purified enzyme was 70.6 kDa. The enzyme had optimal activity at 80-90°C and retained greater than 90% activity between 75°C and 95°C. Optimal pH activity was observed at 7.0, with at least 50% activity between pH 4.0 and 9.0. It was highly stable at elevated temperature, with no loss of activity after incubation at 80°C for 4 hours or at 90°C for 30 min. Km and Vmax values were 0.222 mg/mL and 0.206 mg β-CD/mL/min, respectively, with soluble starch. Amino acid composition of the enzyme was deduced from the sequence of the cloned CGTase gene. The mature enzyme has a deduced molecular weight of 75.63 kDa and contains residues conserved in the CGTase class of amylase enzymes.

Bulk production of recombinant proteins and therapeutic vaccines largely depends on cellculture techniques. Suspension culture using microcarrier is a common manufacturing practice, despite its production limitations caused by shear force damage. Here we report a simple and scalable method to overcome this and improve the healthy cell mass. We hypothesized to curb the cell damage and increase the yield of cellular products through unique functionalization of microcarrier with an analog of peptide that is known to induce collagen and fibronectin like extracellular matrix (ECM) molecules. To test the hypothesis polystyrene beads (75-150 μm) were covalently grafted with HPLC purified peptide through spacer in a manner that produces ∼ 4.2 micromole/g substitution. After UV and FTIR characterization, the functionalized beads (BSP), were evaluated by culturing Vero (African green monkey kidney) and MDCK (Madin-Darby canine kidney) cells. Microscopic study after 24 hours of culture exhibited cells in clusters over the beads and 81% of functionalized versus 41% unmodified beads (UB) were observed to be occupied by the cells. Alamar-blue assay confirmed that functionalization facilitates microcarrier to sustain more viable cells. Even with the present density of functional moiety, BSP sustained significantly (p<0.001) more viable cells in 24-30 hours in comparison to Cytodex, the most commonly used microcarrier. Thus, present technique provides an option to improve the number of healthy cells by stimulating a native mechanism in situ. Accomplished at room temperature, similar yet customized functionalization with defined moieties can potentially produce differentiated stem cells for tissue engineering and regenerative medicine.

The biotechnological production of intracellular tyrosine phenol lyase finds important applications in pharmaceutical industry for the development of novel therapeutic molecules like L-DOPA. Cell disruption is one of the important steps in the downstream processing of intracellular enzymes and bioprocess industry demands this operation to be energy, cost and time efficient. The efficiency of the disruption process for the release of tyrosine phenol lyase from Citrobacter freundii in a bead beater with zirconium beads was studied. Under optimum conditions of cell disruption (0.1mm zirconium beads, 6mg/mL dcw cells, 1mM EDTA, 0.7mM DTT and 9 disruption cycle), the specific activity of enzyme reached to 0.038 IU/mg. The stability of cell free tyrosine phenol lyase increased by 5.8-fold by addition of EDTA and DTT. The disruption of C. freundii cells with beads seems to be effective as evidenced by transmission electron microscopy. This cell free enzyme with enhanced stability can be used subsequently in suspension or immobilized form for the biotransformtion reactions.