Enzyme Functional Screening, Discovery and Engineering; Automation, Metagenomics and High-throughput Approaches

Concerned with the construction and design of novel biocatalysts, the enzyme engineering served to overcome the limitations of native enzymes, in order to create biocatalysts with tailored functions, to facilitate industrial applications. The enzymes, being recognized by screening and discovery workflows and further tailored by engineering platforms, are of immense potential as improved biocatalysts. Functional metagenomics is a powerful tool to identify novel enzymes followed by the construction of metagenome-based enzyme libraries. And the subsequent screening of these enzyme libraries is in turn facilitated by ultra-high-throughput-based, for example FACS or microfluidics, enzyme engineering technologies. Relies on the compartmentalization of reaction components, in order to detect and measure assay signal within the reaction compartments, the enzyme engineering platforms are designed which include cell-as-compartment platforms, droplet-based platforms andmicro-chamber-based platforms. The metagenomics approach and high-throughput screening by these three prime enzyme engineer platforms are the focus of this review.

Isolation and Identification of Novel IndigenousBacterial Strain as a Low Cost Pectinase Source

ABSTRACT The present study was concerned with the searching of novel bacterial cultures from different samples for the lab scale production of pectinase. Keeping in view the increasing demand of pectinase specially in Faisalabad, an industrial city of Pakistan, isolation of new hyper producer bacterial strains locally is an easy and cheap way of getting the desirable products at low cost. Therefore, isolation of new strains for industrial enzyme production has been and will be remained a part of research every time. This method alone can also provide raw material for further research such as enzyme engineering or molecular directed evolution. For the identification of hyper producer strain colony PCR was done for 16S rRNA analysis. Reason to use the 16S rRNA for identification purpose is that the gene is fairly short and can be amplified quickly and easily. The bacterial isolate (sources of pectinase enzyme) was identified based on PCR amplification of 16S rRNA and for this purpose the amplified product was run in agarose gel against a known species of Bacillus licheniformis. The 16S rRNA sequencing confirmed the Bacillus status of the strain.

Double mutation of Saccharomyces cerevisiae for enhanced β-D-fructofuranosidase fructohydrolase productivity and application of growth kinetics for parametric significance analysis

Abstract The kinetics of an extracellular β-D-fructofuranosidase fructohydrolase production by Saccharomyces cerevisiae in a chemically defined medium, i.e., sucrose peptone agar yeast extract at pH 6, was investigated. The wild-type was treated with a chemical mutagen, methyl methane sulfonate. Among the six mutants isolated, methyl methane sulfonate-V was found to be a better enzyme producing strain (52 ± 2.4a U/mL). The maximum production (74 ± 3.1a U/mL) was accomplished after at 48 h (68 ± 2.7a mg/mL protein). The mutants were stabilized at low levels of 5-fluoro-cytocine and the viable ones were further processed for optimization of cultural conditions and nutritional requirements. The sucrose concentration, incubation period and pH were optimized to be 30 g/L, 28 °C, and 6.5, respectively. The methyl methane sulfonate-V exhibited an improvement of over 10 folds in enzyme production when 5 g/L ammonium sulfate was used as a nitrogen source. Thin layer chromatography and high-performance liquid chromatography analysis illustrated the optimal enzyme activity supported by the higher rate of hydrolysis of sucrose into monosaccharides, particularly α-D-glucose and β-D-fructose. The values for Qp (2 ± 0.12c U/mL/h) and Yp/s (4 ± 1.24b U/g) of the mutant were considerably increased in comparison with other yeast strains (both isolates and viable mutants). The mutant could be exploited for enzyme production over a wider temperature range (26–34 °C), with significantly high enzyme activity (LSD 0.048, HS) at the optimal temperature.

Comprehensive Roles of TP53 in Cell Signaling, Apoptosis and Carcinogenesis – A Review.

TP53 gene is a tumor suppressor gene and it inhibits the emergence of cancerous growth. The signaling of TP53 takes part in the co-ordination of cellular response to various kinds of stress like hypoxia and DNA damage. The downstream signals start to multiple processes such as MTOR (mechanistic target of rapamycin), apoptosis, and the arrest of cell cycle. TP53 becomes inactivated when encounters tumor growth. According to estimation, more than half of all cancers imply the inactivating mutations of TP53, which leads to the expression of mutant p53 protein. An extensive range of cancers carry the mutations of TP53 or certain other defects which deregulate p53 and its cofactors, making this gene a significant and highly studied tumor suppressor gene. Most of the mutations that are found in human cancers are not inherited but are acquired. As p53 protein binds DNA, it triggers another gene to synthesize a protein named p21 inside the cell, which interferes with a cell division-stimulating kinase (cdk2). When p21 forms a complex with cdk2 the cell cannot pass onto the next phase of cell division. Therefore, Mutant p53 can no longer get itself attached to DNA effectively, and as a result, the p21 protein is not made available to function as the 'stop signal' for the division of cell.

Mutation of Aspergillus oryzae for improved production of 3, 4-dihydroxy phenyl-L-alanine ( L-DOPA ) from L-tyrosine

A produção de L-DOPA a partir de tirosina pela cepa mutante de Aspergillus orizae UV-7 foi melhorada através de mutação química. Diferentes cepas foram testadas quanto a produção de L-DOPA por fermentação submersa, observando-se que a cepa denominada SI-12 foi a melhor produtora (300 mg de L-DOPA por g de células). A produção de L-DOPA pela cepa mutante a partir de diferentes fontes de carbono foi testada em diferentes fontes de nitrogênio, pH inicial e temperatura. Em pH ótimo (5,0) e temperatura ótima (30ºC), todos os açúcares foram utilizados para formação de biomassa, com um rendimento de L-DOPA de 150 mg.g-1, e produtividade volumétrica máxima e especifica de 125 mg.l.h-1 e 150 mg.g-1.h-1, respectivamente. A velocidade de formação do produto aumentou 3 vezes, sendo esse aumento o maior já relatado na literatura. Para explicar o mecanismo cinético da formação de L-DOPA e a inativação térmica da tirosinase, os parâmetros termodinâmicos foram determinados aplicando-se o modelo de Arrhenius: no caso da cepa mutante, a entalpia de ativação e entropia foram 40kj/mol e 0,076 kj/mol.K para produção de L-DOPA e 116 kj/mol and 0,590 kj/mol.K para inativação térmica, respectivamente. Os valores para formação do produto foram mais baixos e os para desativação do produto foram mais elevados que os valores correspondentes à cultura parental, indicando que a cepa mutante foi termodinamicamente mais resistente à denaturação térmica.

Myocardial ischaemia and reperfusion injury: reactive oxygen species and the role of neutrophil

A growing body of evidence suggests that oxygen radicals can mediate myocardial tissue injury during ischaemia and, in particular, during reperfusion. This review focuses on the role of neutrophil as a mediator of myocardial damage. Upon reperfusion, neutrophils accumulate and produce an inflammatory response in the myocardium that is responsible, in part, for the extension of tissue injury associated with reperfusion. It has shown that the inhibition of neutrophil accumulation and adhesion is associated with decreased infarct size. This strongly suggests that myocardial cells at risk region undergo irreversible changes upon reperfusion and accumulation of neutrophils. Several pharmacological agents [ibuprofen, allopurinol, prostacyclin, and prostaglandin E analogues] protect the myocardium from reperfusion injury. In addition, the mechanisms by which these agents act and directions of research that may lead to therapeutically useful approaches are also discussed in this review