O-Methyltransferase Lab Report

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Use of microbial platform overexpressing such an ideal O-methyltransferase having flexibility in its catalytic activity over different classes of substrates is of great importance in producing variety of methylated natural products. With this concept, we used the aforementioned natural compounds as substrate for biotransformation reaction. Culture of recombinant E. coli strain was prepared and induced as explained in materials and method. In vivo reaction mixtures analysis of different substrates revealed the presence of methylated product in each reaction mixtures except naringenin and quercetin. Prominent single product peak was detected in case of 7,8-DHF (product retention time (tR) of 20.7 min, substrate peak was detected at tR 18.8 min) …show more content…
peucetius and functionally characterized. Moreover, its biotechnological application for the production of diverse classes of O-methoxy natural products was experimented and investigated. Among these substrates both in the in vitro and in vivo bioconversion experiments, 7,8-dihydroxyflavone showed significant product conversion and was further analyzed by NMR study and confirmed as 7-hydroxy 8-O-methoxyflavone. Fermentation bioconversion assay was also performed with 7,8-DHF. Moreover, while analyzing the in vivo reaction mixtures by HR-QTOF-LC-ESI/MS, the mass fragments of O-methyl 3-hydroxyflavone, O-methyl phloretin, O-methyl luteolin, three mono- di- and tri-methylated genisteins, O-methyemodin and aloe-emodin were detected. Likewise, while analyzing the in vitro reaction mixture with sterol (β-sitosterol) and anthracyclines (doxorubicin and daunorubicin), the mass spectra of methoxy β-sitosterol, mono-, and di-O-methyl daunorubicin were obtained. This suggested the versatility of SpOMT7740 to accept different classes of natural products (flavonoids, sterols, anthracyclines, and anthraquinones) for the modification and production of O-methoxy compounds. These analyses indicate that OMT is non-regiospecific and capable of methylating the phenolic hydroxyl position of different classes of natural products as explained. Because it is an O-methyltransferase, the product detected with 3-hydroxyflavone should be 3-O-methoxyflavone and with sterol should be 3-O-methoxy β-sitosterol. Similarly, in the case of daunorubicin, the compound should be 6,11-di-O-methyldaunorubicin, but O-methyl daunorubicin could have attached at the 6- or 11- hydroxyl position. Moreover, the exact methylation position of O-methoxy emodin and aloe-emodin including O-methoxy genisteins, O-methoxy phloretin, and O-methoxy luteolin are

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