Cellulases and related enzymes in biotechnology

Basic and applied research on microbial cellulases, hemicellulases and pectinases has not only generated significant scientific knowledge but has also revealed their enormous potential in biotechnology. At present, cellulases and related enzymes are used in food, brewery and wine, animal feed, textile and laundry, pulp and paper industries, as well as in agriculture and for research purposes. Indeed, the demand for these enzymes is growing more rapidly than ever before, and this demand has become the driving force for research on cellulases and related enzymes. The present article is an overview of the biotechnological state-of-the-art for cellulases and related enzymes.     

Production and characterization of extracellular α-amylases from the thermophilic fungus Thermomyces lanuginosus (wild and mutant strains)

-Amylases from the thermophilic fungus, Thermomyces lanuginosus ATCC 34626 (wild and mutant strains), were purified to homogeneity by a simple procedure including, consecutively, precipitation with ice-cold 2-propanol, anion-exchange and molecular-sieve chromatographic methods. The molecular masses of the purified -amylases (both with pI values of 3.0) were 58 kDa by SDS-PAGE. The optimal pH of -amylase activity was 5.0 for the wild enzyme and 4.5 for the mutant one. 1-Cyclohexyl-3-(2-morpholinyl-4-ethyl)-carbodiimide (40–100 mM) and N-bromosuccinimide (0.1–1 mM) inhibited the enzymes, suggesting the involvement of carboxylic groups and tryptophan residues in the catalytic process.     

A Moricandia arvensis– based cytoplasmic male sterility and fertility restoration system in Brassica juncea

 A cytoplasmic male-sterility system has been developed in mustard (Brassica juncea) following repeated backcrossings of the somatic hybrid Moricandia arvensis (2n=28, MM)+B. juncea (2n=36, AABB), carrying mitochondria and chloroplasts from M. arvensis, to Brassica juncea. Cytoplasmic male-sterile (CMS) plants are similar to normal B. juncea; however, the leaves exhibit severe chlorosis resulting in delayed flowering. Flowers are normal with slender, non-dehiscent anthers and excellent nectaries. CMS plants show regular meiosis with pollen degeneration occurring during microsporogenesis. Female fertility was normal. Genetic information for fertility restoration was introgressed following the development of a M. arvensis monosomic addition line on CMS B. juncea. The additional chromosome paired allosyndetically with one of the B. juncea bivalents and allowed introgression. The putative restorer plant also exhibited severe chlorosis similar to CMS plants but possessed 89% and 73% pollen and seed fertility, respectively, which subsequently increased to 96% and 87% in the selfed progeny. The progeny of the cross of CMS line with the restorer line MJR-15, segregated into 1 fertile : 1 sterile. The CMS (Moricandia) B. juncea, the restorer (MJR-15), and fertility restored F₁ plants possess similar cytoplasmic organellar genomes as revealed by ‘Southern’ analysis. Received: 17 September 1997 / Accepted: 18 February 1998     

Region-Specific Targets of p42/p44MAPK Signaling in Rat Brain

Abstract: In vitro studies indicate that p42/p44^MAPK phosphorylate both nuclear and cytoplasmic proteins. However, the functional targets of p42/p44^MAPK activation in vivo remain unclear. To address this question, we localized activated p42/p44^MAPK in hippocampus and cortex and determined their signaling effects after electroconvulsive shock treatment (ECT) in rats. Phosphorylated p42/p44^MAPK content increased in the cytoplasm of hippocampal neurons in response to ECT. Consistent with this cytoplasmic localization, inhibition of ECT-induced p42/p44^MAPK activation by the extracellular signal-regulated kinase kinase inhibitor PD098059 blocked phosphorylation of the cytoplasmic protein microtubule-associated protein 2c (MAP2c), but failed to inhibit the induction of the nuclear protein c-Fos in response to ECT. In contrast to hippocampal neurons, cortical neurons exhibited an increase in amount of phosphorylated p42/p44^MAPK in both the nucleus and cytoplasm after ECT. Accordingly, PD098059 blocked the induction of Fos-like immunoreactivity in the nuclei of cortical neurons as well as MAP2c phosphorylation in the cytoplasm. Our data indicate that both nuclear and cytoplasmic substrates can be activated by p42/p44^MAPK in vivo. However, the functional targets of p42/p44^MAPK signaling depend on the precise location of p42/p44^MAPK within different subcellular compartments of brain regions. These results indicate unique functional pathways of p42/p44^MAPK-mediated signal transduction within different brain regions in vivo.     

Distribution, endemism and threat status of freshwater fishes in the Western Ghats of India

Aim To study (1) the large-scale distribution patterns of freshwater fishes in the Western Ghats of India; (2) the endemism and uniqueness of the fishes in various zones; and (3) the threat status of fishes by categorizing them under low risk (LR), vulnerable (VU), endangered (EN) and critically endangered (CR). Location The Western Ghats of India. Methods The scientific literature describing the freshwater fishes of the Western Ghats was reviewed. Data describing the lists of the species were extracted and complied. The species accumulation curve was plotted using Michaelis–Menten-like equation. The Western Ghats was divided into six zones and similarity of the species was calculated using Jacquard's index. Results Literature to date records 288 species belonging to 12 orders, 41 families and 109 genera, of which 118 species are endemic and 51 are unique. However, the species accumulation curve shows that there might be 345 species in this region, indicating that 16% species have not been recorded to date. An analysis of the distribution pattern of fishes in the Western Ghats suggests that the southern region is more diverse than the northern and central regions. The southern region shows high endemism and high uniqueness while the northern region shows high endemism but less uniqueness. The similarity index between the zones indicates that as the distance between the zones increases similarity decreases. The status of 105 of 288 species was not known due to data deficiency but among the remaining 183 species, 58 species were categorized as LR, 41 as VU, 54 as EN, 24 as CR while the remaining six species were introduced. Conclusions The distribution patterns of fishes in the Western Ghats are discussed in accordance with the geography of Western Ghats, its climatic conditions and ‘Satpura Hypothesis’. The threat status of fishes found in Western Ghats suggests that at least 41% of fish fauna is threatened by either being VU, EN or CR. Implication of potent conservation measures is necessary to conserve the fish fauna of Western Ghats.     

Inhibition of Mycobacterium tuberculosis topoisomerase I by m-AMSA,a eukaryotic type II topoisomerase poison

m-AMSA, an established inhibitor of eukaryotic type II topoisomerases, exerts its cidal effect by binding to the enzyme–DNA complex thus inhibiting the DNA religation step. The molecule and its analogues have been successfully used as chemotherapeutic agents against different forms of cancer. After virtual screening using a homology model of the Mycobacterium tuberculosis topoisomerase I, we identified m-AMSA as a high scoring hit. We demonstrate that m-AMSA can inhibit the DNA relaxation activity of topoisomerase I from M. tuberculosis and Mycobacterium smegmatis. In a whole cell assay, m-AMSA inhibited the growth of both the mycobacteria.     

Molecular determinants of the binding specificity of BH3 ligands to BclXL apoptotic repressor

B‐cell lymphoma extra‐large protein (BclXL) serves as an apoptotic repressor by virtue of its ability to recognize and bind to BH3 domains found within a diverse array of proapoptotic regulators. Herein, we investigate the molecular basis of the specificity of the binding of proapoptotic BH3 ligands to BclXL. Our data reveal that while the BH3 ligands harboring the LXXX[A/S]D and [R/Q]XLXXXGD motif bind to BclXL with high affinity in the submicromolar range, those with the LXXXGD motif afford weak interactions. This suggests that the presence of a glycine at the fourth position (G+4)—relative to the N‐terminal leucine (L0) within the LXXXGD motif—mitigates binding, unless the LXXXGD motif also contains arginine/glutamine at the ?2 position. Of particular note is the observation that the residues at the +4 and ?2 positions within the LXXX[A/S]D and [R/Q]XLXXXGD motifs appear to be energetically coupled—replacement of either [A/S]+4 or [R/Q]‐2 with other residues has little bearing on the binding affinity of BH3 ligands harboring one of these motifs. Collectively, our study lends new molecular insights into understanding the binding specificity of BH3 ligands to BclXL with important consequences on the design of novel anticancer drugs. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 573–582, 2014.     

Demonstration of a visual cell-based assay for screening glucose transporter 4 translocation modulators in real time

Insulin-stimulated translocation of glucose transporter 4 (GLUT4) to cell membrane leading to glucose uptake is the rate-limiting step in diabetes. It is also a defined target of antidiabetic drug research. Existing GLUT4 translocation assays are based on time-consuming immunoassays and are hampered by assay variability and low sensitivity. We describe a real-time, visual, cell-based qualitative GLUT4 translocation assay using CHO-HIRc-myc-GLUT4eGFP cells that stably express myc- and eGFP-tagged GLUT4 in addition to human insulin receptor (HIRc). GLUT4 translocation is visualized by live cell imaging based on GFP fluorescence by employing a cooled charge-coupled device camera attached to a fluorescent microscope. This video imaging method and further quantitative analysis of GLUT4 on the cell membrane provide rapid and foolproof visual evidence that this method is suitable for screening GLUT4 translocation modulators.     

Development,characterization and utilization of genomic microsatellite markers in turmeric (Curcuma longa L.)

Development of a robust set of 18 genomic microsatellite markers from turmeric (Curcuma longa L.) and its effective utilization in estimating the genetic diversity of 20 turmeric accessions are described. A total of 103 alleles were detected with an average of 5.7 alleles per locus. These markers displayed varied levels of polymorphism as evident from its discriminating power ranging from 0.19 to 0.70. The UPGMA cluster analysis of genetic distance values resolved the 20 turmeric accessions into five main groups. Three sets of genetically identical accessions were detected within the analyzed accessions, suggesting a revisit of the germplasm collection strategy based on vernacular identity. The entire grouping pattern of the entities was loose and independent of their geographical origins. These polymorphic SSR markers would be useful for the population genetic studies and germplasm management of turmeric.     

Structure-based optimization of pyrazolo-pyrimidine and -pyridine inhibitors of PI3-kinase

Starting from HTS hit 1a, X-ray co-crystallization and molecular modeling were used to design potent and selective inhibitors of PI3-kinase. Bioavailablity in this series was improved through careful modulation of physicochemical properties. Compound 12 displayed in vivo knockdown of PI3K pharmacodynamic markers such as pAKT, pPRAS40, and pS6RP in a PC3 prostate cancer xenograft model.