Abstract
Norcoclaurine synthases (NCS), catalyzing a Pictet-Spengler reaction in plants as one of the first enzymes in the biosynthetic benzylisoquinoline pathway, are investigated for biocatalytic transformations. The library of NCS available is extended by two novel NCSs from Argemone mexicana (AmNCS1, AmNCS2) and one new NCS from Corydalis saxicola (CsNCS); furthermore, it is shown that the NCS from Papaver bracteatum (PbNCS) is a highly productive catalyst leading to the isoquinoline product with up to >99% e.e. Under certain conditions lyophilized whole Escherichia coli cells containing the various overexpressed NCS turned out to be suitable catalysts. The reaction using dopamine as substrate bears several challenges such as the spontaneous non-stereoselective background reaction and side reactions. The PbNCS enzyme is successfully immobilized on various carriers whereby EziG3 proved to be the best suited for biotransformations. Dopamine showed limited stability in solution resulting in the coating of the catalyst over time, which could be solved by the addition of ascorbic acid (e.g., 1 mg ml-1 ) as antioxidant.
Highlights
Norcoclaurine synthases (NCS), catalyzing a Pictet–Spengler reaction in plants sant codeine, the muscle relaxant papaveras one of the first enzymes in the biosynthetic benzylisoquinoline pathway, are investigated for biocatalytic transformations
From the eight NCSs identified by BLAST three have not been described before, namely two NCSs from Argemone mexicana (AmNCS1, AmNCS2) and one from Corydalis saxicola (CsNCS) (Table S1, Supporting Information)
Related NCSs already described in literature were used for comparison or to test their substrate scope, such as the aforementioned NCS from norcoclaurine synthase from Coptis japonica (CjNCS2)[8,14,16,17,28,29] and from norcoclaurine synthase from Thalictrum flavum (TfNCS)[4,15,16,28,30,31] as well as one from Papaver bracteatum (PbNCS)[30] and two NCSs from Papaver somniferum (PsNCS1, PsNCS2).[32]
Summary
Sodium alginate and Diaion HP-2MG were obtained from Sigma–Aldrich. Phenylacetaldehyde was distilled prior to use. ABT High Density Nickel (cross-linked 6% alginate beads, loading capacity 20–40 μmol Ni2þ mlÀ1 gel) from was obtained from Agarose Bead Technologies; YTONG from OBI Graz; Amberlite FPC3500 from AcrosOrganics and EziG1-3 from EnginZyme
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