| Products/Services Used | Details | Operation |
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| Gene Synthesis | The codon optimized genes (for expression in E. coli) for IeOMT from Clarkia breweri(accession number of protein: O04385) and for CaOMT from Catharanthus roseus (accession number of protein: Q8W013) with C‐terminal His6‐tag were synthesized and subcloned into the E. coli expression vector pET‐21a(+) by GenScript USA Inc.. Mutagenesis to generate the triple mutant T133M/A134N/T135Q was carried out with a modified version of the FastCloning method, by amplifying the whole pET‐21a(+)‐IeOMT vector using primers with 18 bp overlapping at their 5′ ends.22 Other mutants were constructed following the QuikChange (Stratagene) protocol. Primers are as below: | Get A Quote |
An isoeugenol 4‐O‐methyltransferase (IeOMT), isolated from the plant Clarkia breweri, can be engineered to a caffeic acid 3‐O‐methyltransferase (CaOMT) by replacing three consecutive residues. Here we further investigated functions of these residues by constructing the triple mutant T133M/A134N/T135Q as well as single mutants of each residue. Phenolics with different chain lengths and different functional groups were investigated. The variant T133M improves the enzymatic activities against all tested substrates by providing beneficial interactions to residues which directly interact with the substrate. Mutant A134N significantly enhanced the regioselectivity. It is meta‐selective or even specific ag... More
