Mbgl Primer

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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 was digested with NdeI and HindIII restriction enzymes and…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

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