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. 2021 Aug 5:31:e00664.
doi: 10.1016/j.btre.2021.e00664. eCollection 2021 Sep.

Imine reduction by an Ornithine cyclodeaminase/μ-crystallin homolog purified from Candida parapsilosis ATCC 7330

V N M Uma Mahesh  1 , Anju Chadha  1   2
Affiliations

Imine reduction by an Ornithine cyclodeaminase/μ-crystallin homolog purified from Candida parapsilosis ATCC 7330

V N M Uma Mahesh et al. Biotechnol Rep (Amst). .

Abstract

We report a stereospecific imine reductase from Candida parapsilosis ATCC 7330 (CpIM1), a versatile biocatalyst and a rich source of highly stereospecific oxidoreductases. The recombinant gene was overexpressed in Escherichia coli and the protein CpIM1 was purified to homogeneity. This protein belongs to the Ornithine cyclodeaminase/ μ-crystallin (OCD-Mu) family of proteins which has only a few characterized members. CpIM1 catalyzed the alkylamination of α-keto acids/esters producing exclusively (S)-N-alkyl amino acids/esters e.g. N-methyl-l-alanine with > 90% conversion and > 99% enantiomeric excess (ee). The enzyme showed the highest activity for the alkylamination of pyruvate and methylamine leading to N-methyl-l-alanine with an apparent KM of 15.04 ± 2.8 mM and Vmax of 13.75 ± 1.07 μmol/min/mg. CpIM1 also catalyzed (i) the reduction of imines e.g. 2-methyl-1-pyrroline to (S)-2-methylpyrrolidine with ∼30% conversion and 75% ee and (ii) the dehydrogenation of cyclic amino acids e.g. l-Proline (as monitered by reduction of cofactor NADP+ spectrophotometrically).

Keywords: Alkylamination; Candida parapsilosis; Chiral amines; Imine reductase; N-alkyl amino acid/esters; μ-crystallin/Ornithine cyclodeaminase family.

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Conflict of interest statement

The authors whose names are listed immediately below certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.

Figures

Fig 1:
Fig. 1
Multiple sequence alignment showing the key conserved residues among the sequences of CpIM1 and other characterized OCD-Mu proteins.
Fig 2:
Fig. 2
(a) SDS page showing purified CpIM1 (∼42 kDa), protein ladder in lane A; (b) CpIM1 confirmed to be a dimer (∼ 90 kDa) by analytical gel filtration.
Fig 3:
Fig. 3
Activity-pH profile for CpIM1 (a) for methylamination of sodium pyruvate, (b) for reduction of 2-MPN.
Fig 4:
Fig. 4
Plot of specific activity vs substrate concentration for CpIM1 at various concentrations of (a) pyruvate (0.05–50 mM) with 50 mM methyl amine and 0.2 mM NADPH (b) methyl amine (0.5–50 mM) with 10 mM pyruvate and 0.2 mM NADPH
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