Covering: up to end of June 2018Baeyer-Villiger monooxygenases (BVMOs) are flavin-dependent enzymes that catalyse the oxidation of ketones and cyclic ketones to esters and lactones, respectively, by using molecular oxygen and NAD(P)H. BVMOs also catalyse sulfoxidations and N-oxidations. BVMOs are widely studied as attractive biocatalysts, but also catalyse key reactions in metabolic pathways of the organisms from which they are sourced. BVMOs are involved in the primary metabolism of atypical carbon sources, thereby conferring an evolutionary advantage to the host organism. In addition, BVMOs catalyse a plethora of diverse Baeyer-Villiger and heteroatom oxidations in the construction of complex secondary metabolites. These natural products often have attractive biological properties, such as anti-bacterial, anti-cancer and anti-proliferative activity, and can have clinical applications. In contrast, BVMOs are also involved in the synthesis of microbial toxins. This review will discuss the inherent roles of type I, type II and type O BVMOs in the metabolism of microorganisms.

Baeyer-Villiger monooxygenases (BVMOs) are biocatalysts that convert ketones to esters. Due to their high regio-, stereo- and enantioselectivity and ability to catalyse these reactions under mild conditions, they have gained interest as alternatives to chemical Baeyer-Villiger catalysts. Despite their widespread occurrence within the fungal kingdom, most of the currently characterized BVMOs are from bacterial origin. Here we report the catalytic and structural characterization of BVMOAFL838 from Aspergillus flavus. BVMOAFL838 converts linear and aryl ketones with high regioselectivity. Steady-state kinetics revealed BVMOAFL838 to show significant substrate inhibition with phenylacetone, which was more pronounced at low pH, enzyme and buffer concentrations. Para substitutions on the phenyl group significantly improved substrate affinity and increased turnover frequencies. Steady-state kinetics revealed BVMOAFL838 to preferentially oxidize aliphatic ketones and aryl ketones when the phenyl group are separated by at least two carbons from the carbonyl group. The X-ray crystal structure, the first of a fungal BVMO, was determined at 1.9 Å and revealed the typical overall fold seen in type I bacterial BVMOs. The active site Arg and Asp are conserved, with the Arg found in the “in” position. Similar to phenylacetone monooxygenase (PAMO), a two residue insert relative to cyclohexanone monooxygenase (CHMO) forms a bulge within the active site. Approximately half of the “variable” loop is folded into a short α-helix and covers part of the active site entry channel in the non-NADPH bound structure. This study adds to the current efforts to rationalize the substrate scope of BVMOs through comparative catalytic and structural investigation of different BVMOs.

We investigated Baeyer-Villiger monooxygenase (BVMO)-mediated synthesis of alkyl formate esters, which are important flavor and fragrance products. A recombinant fungal BVMO from Aspergillus flavus was found to transform a selection of aliphatic aldehydes into alkyl formates with high regioselectivity. Near complete conversion of 10 mm octanal was achieved within 8 h with a regiomeric excess of ∼80 %. Substrate concentration was found to affect specific activity and regioselectivity of the BVMO, as well as the rate of product autohydrolysis to the primary alcohol. More than 80 % conversion of 50 mm octanal was reached after 72 h (TTN nearly 20 000). Biotransformation on a 200 mL scale under unoptimized conditions gave a space-time yield (STY) of 4.2 g L d (3.4 g L d extracted product).

A prototyping approach was used to determine the essential system requirements of a computerised patient record information system for a typical township primary health care clinic. A pilot clinic was identified and the existing manual system and business processes in this clinic was studied intensively before the first prototype was implemented. Interviews with users, incidental observations and analysis of actual data entered were used as primary techniques to refine the prototype system iteratively until a system with an acceptable data set and adequate functionalities were in place. Several non-functional and user-related requirements were also discovered during the prototyping period