The biocatalytic, regioselective hydroxylation of 2-hydroxybiphenyl to the corresponding catechol was accomplished utilizing the monooxygenase 2-hydroxybiphenyl 3-monooxygenase (HbpA). The necessary natural nicotinamide adenine dinucleotide (NAD + ) co-factor for this biocatalytic process was replaced by a

Cell‐free enzymatic oxidations: Styrene monooxygenase (StyA) was used as a reagent for the gram‐scale preparation of enantiopure epoxides. The catalyst is highly stable in a biphasic system and results in conversions of more than 88 %. R1=H, Cl; R2=R3=H, CH3.

Summaryβ‐Peptides and their derivates are usually stable to proteolysis and have an increased half‐life compared with α‐peptides. Recently, β‐aminopeptidases were described as a new enzyme class that enabled the enzymatic degradation and formation of β‐peptides. As an alternative to the existing chemical synthesis routes, the aim of the present work was to develop a whole‐cell biocatalyst for the synthesis and production of β‐peptides using this enzymatic activity. For the optimization of the reaction system we chose the commercially relevant β,α‐dipeptide l‐carnosine (β‐alanine‐l‐histidine) as model product. We were able to show that different recombinant yeast and bacteria strains, which overexpress a β‐peptidase, could be used directly as whole‐cell biocatalysts for the synthesis of l‐carnosine. By optimizing relevant reaction conditions for the best‐performing recombinant Escherichia coli strain, such as pH and substrate concentrations, we obtained high l‐carnosine yields of up to 71%. Long‐time as well as biocatalyst recycling experiments indicated a high stability of the developed biocatalyst for at least five repeated batches. Application of the recombinant E. coli in a fed‐batch process enabled the accumulation of l‐carnosine to a concentration of 3.7 g l−1.

The b,a-dipeptide l-carnosine occurs in high concentrations in long-lived innervated mammalian tissues and is widely sold as a food additive. On a large scale l-carnosine is produced by chemical synthesis procedures. We have established two aqueous enzymatic reaction systems for the preparation of l-carnosine using the dissolved bacterial b-aminopeptidases DmpA from Ochrobactrum anthropi and BapA from Sphingosinicella xenopeptidilytica as catalysts and investigated the kinetics of the enzymecatalyzed peptide couplings. DmpA catalyzed the formation of l-carnosine from C-terminally activated b-alanine derivatives (acyl donor) and l-histidine (acyl acceptor) in an aqueous reaction mixture at pH 10 with high catalytic rates (V max = 19.2 mmol min À1 per mg of protein, k cat = 12.9 s À1), whereas V max in the BapA-catalyzed coupling reaction remained below 1.4 mmol min À1 per mg of protein (k cat = s À1). Although the equilibrium of this reaction lies on the side of the hydrolysis products, the reaction is under kinetic control and l-carnosine temporarily accumulated to concentrations that correspond to yields of more than 50% with respect to the employed acyl donor. However, competing nucleophiles caused unwanted hydrolysis and coupling reactions that led to decreased product yield and to formation of various peptidic by-products. The substitution of l-histidine for l-histidine methyl ester as acyl acceptor shifted the pK a of the amino functionality from 9.25 to 6.97, which caused a drastic reduction in the amount of coupling by-products in an aqueous reaction system at pH 8.

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