| Products/Services Used | Details | Operation |
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| Gene Synthesis | Following amplification, the template DNA was removed by DpnI digestion. The treated PCR product was separated by agarose gel electrophoresis and extracted from the gel using a Biospin Gel Extraction Kit (Bioflux, China). Afterwards the plasmids were transformed into Escherichia coli TOP10 cells (Invitrogen, Germany). The TeSADH gene was synthesized, without codon optimization, by GenScript® and supplied in pUC57. Subsequently the gene was cloned into the second multiple cloning site of the pETDuet-1 (Novagen, USA) via NdeI and AvrII. The CHMO WT and its variants were subcloned from the pET vectors into the first multiple cloning site using XbaI and BamHI, and the AFL706 with XbaI and NotI. The expression vectors used for BVMOs containing a C-terminal His-Tag were all in pET22b(+), whereas the vectors used for the co-expression of BVMOs and TeSADH were all in pETDuet-1 (Novagen, USA) without any His-Tag in frame. For individual expression of TeSADH pET21a vector (Novagen, USA) was used without any His-Tag in frame. | Get A Quote |
Technical application of cyclohexanone monooxygenase (CHMO) from Acinetobactersp. NCIMB 9871 in particular, is hindered by limited enzyme stability. In addition, substrate and product inhibition is a well-known challenge of using CHMO. By site-directed mutagenesis two new combinatorial CHMO variants, CHMO M15 L323C-A325C (M15 DS) and CHMO M16 L323C-A325C (M16 DS), were designed to stabilize the enzyme, by incorporating a reported disulfide bridge into the already published parental CHMO variants: CHMO M15 and CHMO M16. Additionally, the newly described BVMO AFL706 from Aspergillus flavus was characterized for epsilon-caprolactone (ECL) synthesis, for which the enzyme showed significantly higher substrate and... More
