Studies on methylmalonyl-CoA mutase from Escherichia coli

Methylmalonyl-CoA mutase (MCM, E.C. 5.4.99.2), a coenzyme B12-dependent enzyme, catalyses the inter conversion of succinyl-CoA and methylmalonyl- CoA. The gene (sbm) encoding this enzyme is found in Escherichia coli (E. coli) at 62.3min on the E. coli chromosome. However, the metabolic role of this enzyme in the organism is not known. This project involves an investigation into this metabolic obscurity. The sbm gene is part of a four gene operon which also includes argK (or ygfD) that codes for a protein kinase catalysing the phosphorylation of two periplasmic binding proteins involved in cationic amino acid transport, ygfG that codes for methylmalonyl-CoA decarboxylase and ygfH that codes for propionyl-CoA: succinyl-CoA transferase. From existing literature we suspect that this operon, including the sbm gene, could be involved in the utilisation of unusual carbon sources such as succinate and propionate. An insertion mutant of the sbm gene created by transposon mediated mutagenesis was used for investigating the role of this gene. The wild type E. coli K12 strain, E. coli TR6524 and the mutant E. coli K12 (sbm::MudJ) were used in this study. Growth of the two strains (E. coli TR6524 and FA1P1) in minimal media with three different concentrations (0.05, 0.5, 5.0μg/mL) of vitamin B12 and in the presence succinate, propionate or glucose as the sole source of carbon, was studied. Growth was typical in media with glucose with no major differences in the growth pattern of the wild type and mutant strain. However, the two strains exhibited a differential growth pattern in media containing succinate, with the wild type growing faster than the mutant, indicating the role of the sbm gene in the utilisation of this carbon source. Growth in media containing propionate as the sole carbon source indicated only marginal differences in the growth pattern of the wild type and mutant strain. This result possibly suggests that the other pathways for propionate utilisation in E. coli compensate for the lack of a functional Sbm protein in the mutant strain. Promoter analysis indicated the presence of a promoter induced by σS, a transcription factor involved in the expression of proteins under stress or stationary phase growth conditions. Reverse transcription polymerase chain reaction (RT-PCR) studies of the genes of the sbm operon (sbm-argK-ygfGygfH) under the same growth conditions were carried out. Densitometric analysis of the PCR products suggested that the transcription level of sbm was higher in E. coli grown in succinate as compared to when grown in glucose and not as much when grown in propionate indicating a transcriptional level control of the sbm gene expression during the utilisation of succinate. RT-PCR studies also indicated a higher level of transcription of the gene in the stationary phase of the culture during the utilisation of succinate. Real time reverse transcription PCR (QPCR) analysis was used for the absolute quantification of the transcription of the genes of the sbm operon. An increase in the mRNA levels corresponding to the sbm, argK and ygfG genes was observed as E. coli TR6524 growth reached stationary phase, in the presence of succinate or propionate as the sole source of carbon as compared to glucose, In contrast, the highest mRNA levels corresponding to the ygfH gene were observed in the early log-phase of growth. This indicated a differential transcriptional level control of the genes within the operon. This study further established the possible role of this operon in the utilisation of succinate and propionate. The MCM enzyme activity measurement in the whole cell extracts of the wild type E. coli K12, grown under the above mentioned conditions, led to the first ever measurement of MCM activity in wild type E. coli. These measurements also revealed a four fold increase of the MCM specific activity in the case of growth in succinate (4.76x10-3U/mg) and a two fold increase for growth in propionate (2.79x10-3U/mg) compared to that observed with growth in glucose (1.37x10-3U/mg), indicating a significant level of involvement of the enzyme in succinate utilisation, and to a lesser extent in propionate utilisation. The proteomic analysis to understand the gene expression pattern of E. coli TR6524 was carried out using cells harvested at the stationary phase. The results showed that growth conditions induced the expression of transport related (HisJ, DppA) and energy generating proteins (PckA, AceF) required by E. coli to cope with the stressful growth conditions. However, Sbm was not identified among the limited protein spots that were analysed. Finally, E. coli K12 sbm gene was successfully cloned into B. cereus SPV leading to the development of a metabolically engineered polyhydroxyalkanoate producing strain of B. cereus. The intention was to provide the bacteria with a natural intracellular source of propionyl-CoA, leading to the production of the P(3HB-co-3HV) copolymer from structurally non related carbon sources like glucose. Hence, this work has initiated investigation into the metabolic role of the sbm gene product in E. coli. In addition, it has also led to the use of this gene product in metabolic engineering applications.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

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Selection of Machining Parameters of Face Milling operation for Aluminium with HSS cutter using Response Surface Methodology and Genetic Algorithm

Components used in chemical equipments are produced from forging, extrusion and casting processes with classic dimension tolerances due to its producing ability. So machining processes were introduced for close tolerance assembly and improve the product working efficiencies. At present,  lot of machining processes are available for producing chemical equipments such as turning, milling, drilling and grinding etc.,. Milling operation is playing critical role on making the chemical equipment’s components with high accuracy and higher productivity. Face milling operation is one of the milling processes which is used for achieving higher flatness and surface finish of chemical equipment’s parts. Thiswork concentrates the parameters influence on Material Removal Rate (MRR) and Surface Roughness (SR) by using aluminium as work piece material. Actually, aluminium alloy has the  most significant in chemical industries because of its inherent properties such as, corrosive resistance , low weight to strength ratio. The milling parameters such as feed rate, spindle speed  and depth of cut are selected as parameters for improving the quality and productivity. This work put together the link between input and response variables for developing the face milling performances. The Response Surface Methodology (RSM) is employ for making the link between dependent and independent variables. Building the empirical model by conducting regression analysis The performance of developed regression models are verified with experimental results. Verification results show the developed models have best agreement with experimental results. The developed models are used for achieving the best input parameters by using Genetic Algorithm (GA). Finally, the optimal parameters are evaluated by GA

Application of Genetic Algorithm Technique for Machining Parameters Optimization in Drilling of Stainless Steel

Abstract This work is aimed at developing relations between the pertinent variables that affect drilling process of stainless steel using artificial neural network. The experiments were conducted on vertical CNC machining centre. The parameters used were spindle speed and feed rate. The effect of machining parameters on entry burr height, exit burr height and surface roughness was experimentally evaluated for different spindle speeds and feed rates. A model was established between the drilling parameters and experimentally obtained data using ANN. The predicted values and measured values are fairly close, which indicates that the developed model can be effectively used to predict the burr height and surface roughness in drilling of stainless steel. Genetic algorithm (GA) technique was used in this work to identify the optimized drilling parameters. Confirmation test was conducted with the optimized parameters and it was found that confirmation test results were similar to that of GA-predicted output values

Large sized oilfish Ruvettus pretiosus (Cocco, 1833) ever recorded from Gulf of Mannar, south-east coast of India

Ruvettus pretiosus belonging to the Family Gempylidae of perciform fishes is closely related to Scombridae. Gempylidae fishes are commonly called as snake mackerels or escolars and this family has 16 genus and 24 valid species. R. pretiosus is one of the large sized fishes with the maximum record of 300 cm total length (Nakamura and Parin, 1993). It is bentho-pelagic, reported to be distributed along the tropical and temperate seas of the world

Occurrence of deep sea prawns in the stomach of Yellowfin tuna

The earlier studies on yellowfin tunas (Thunnus albacares) from Indian waters have shown that the fish is an opportunistic feeder, consuming fishes, cephalopods and crustaceans. The main crustacean component is the deep sea pelagic crab, Charybdis smithii. The food and feeding studies of yellowfin tuna from Thoothukudi during the period 2011-2014 were also in conformity with the results of earlier studies

Incidents of ingestion of plastic by marine fishes

Although impacts of plastic pollution on fishthrough entanglement in discarded fishing gear anddirect ingestion have been reported, the reportsfrom India especially on ingestion of plastic bymarine fishes are rare. Two instances of plasticsobserved in the stomachs of fishes caught from Gulfof Mannar off Thoothukudi, south-east coast of Indiaare reported

Occurrence of Parascolopsis eriomma (Jordan and Richardson, 1909) and P. aspinosa (Rao & Rao 1981) from Tuticorin coast

The family Nemipteridae has five genera and 69 valid species. The dwarf monocle bream belonging to the genus Parascolopsis has 12 species. They are small, bottom-living fishes usually occurring in moderately deep water on outer shelf and continental slope waters throughout tropical and sub-tropical waters of Indo-West Pacific, ranging from Western Indian Ocean to North East Australia, South Japan and Delaga Bay on the coast of East Africa. The present report on Rosy dwarf monocle bream, Parascolopsis eriomma from Tuticorin coast is evidently a range extension from the known distribution areas

First record of the twostripe goby, Valenciennea helsdingenii (Gobiidae, Gobiiformes) from the southeast coast of India

Two specimens of Valenciennea helsdingenii (Bleeker, 1858) were collected off Punnakayal coast, from Gulf of Mannar, southeast coast of India in November 2012. The morphometric and meristic characters of the recorded specimens are described and discussed. This is the first record of the species from the Indian waters that is a range extension of its known range within the Indian Ocean

First record of the twostripe goby, Valenciennea helsdingenii (Gobiidae, Gobiiformes) from the southeast coast of India

Two specimens of Valenciennea helsdingenii (Bleeker, 1858) were collected off Punnakayal coast, from Gulf of Mannar, southeast coast of India in November 2012. The morphometric and meristic characters of the recorded specimens are described and discussed. This is the first record of the species from the Indian waters that is a range extension of its known range within the Indian Ocean