Mbgl Lab Report

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Materials and methods Gene cloning and protein over-expression in E.coli: The gene encoding Mbgl was amplified from the genome of Methylococcuscapsulatus(bath)with the following primers: forward primer- TGGTTGGTTCATATGAGCAGATACGAGTTTCCAGAGCGATTCCTCand reverse primer-TATATATTAAAGCTTATAGTCCCGATCCAGGACGGCACCGTTGGTC (restriction site NdeI and HindIII are in italics and underlined). Prime STAR HS DNA Polymerase premix (DSS Takara, New Delhi, India) and the set of above-mentioned primers were used in the program consist of the following polymerase chain reaction (PCR) steps: (1) 95°C for 5 minutes (1 cycle), (2) 98°C for 20 seconds, (3) 50°C for 20 seconds, and (4) 72°C for 2 minutes (steps 2 to 4 was repeated for 35 cycles). The obtained PCR product…show more content…
Approximately 1µg Mbgl was used with 5 mM 4-nitropheny-β-D-glucopyranoside (PNPG) in the reaction mixture of either 2 ml or 1 ml. The reaction was stopped by adding an equal volume of 0.2M Na2CO3and the released product 4-nitrophenol was quantified based on the millimolar extinction coefficient of 18.1 mM-1cm-1at 400 nm (Workman and Day 1982). The optimum pH was determined using the same assay in the 100 mM phosphate-citrate buffer in the pH range of 3.0 to 7.0 and for pH 8.0; 100 mMtris buffer was used. Similarly, the temperature optimum was determined in 100mM citrate buffer of pH-6.0. Thermal stability was determined by incubating the protein solution at 50°C and 55°Cfor different times followed by standard PNPG assay, as described earlier. The glucose sensitivity of Mbgl was studied by the PNPG assay together with the different glucose concentrations (50-1000 mM) in the same reaction mixture. Relative activities of the assays were determined by assuming a 100 % activity corresponds to the assay containing no…show more content…
PNPG was incubated with different substrate concentrations at 55°C and released 4-nitrophenol was continuously measured at 410nm for 5 minutes in UV-2550 (Shimadzu, Kyoto, Japan) spectrophotometer coupled to an accessory TCC-240A to control the temperature of the glass cuvette. Initial velocity was calculated based on the milimolar extinction coefficient of 2.8 for 4-nitrophenol at 55°C. The calculated initial velocity data were linearly fitted to the Lineweaver Burk plot equation to calculate Km and Vmax (Wilkinson 1961). For cellobiose, under similar conditions the reactions were performed for 1 hour, and the initial velocity was calculated by taking data points at different time intervals during one hour of the incubation. The reaction product glucose was measured by the GOD-POD kit (Accurex Biomedical Private Limited, Mumbai, India), as described

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