Alkyl formate ester synthesis by a fungal Baeyer-Villiger monooxygenase

TOLMIE C; FERRONI FM; SMIT M; OPPERMAN DJ

Champagne Valley, KwaZulu-Natal

Conferencia; The 27th annual conference of the Catalysis Society of South Africa; 2016

Catalysis Society of South Africa

Alkyl formate esters are valuable compounds for the fragrance and flavour industry, with heptyl formate and octyl formate possessing fruity flavours [1]. Alkyl formates are typically synthesised via the esterification of formic acid and primary alcohols or through reacting alkyl halides with formamide. Lipases have also been shown to be able to perform similar transesterification and specifically of ethyl formate with 1-octanol to produce the corresponding formic acid ester [2]. Yields of alkyl formate esters are, however, limited by equilibrium constants, which necessitate the use of excess reagents to improve yields. Here we report on the synthesis of alkyl formate esters by the fungal Baeyer-Villiger monooxygenase (BVMO) AFL838.BVMOs are flavin-containing enzymes that catalyse the conversion of ketones and cyclic ketones to esters and lactones, respectively, by using molecular oxygen and NAD(P)H under mild reaction conditions [3]. A library of 10 BVMOs cloned from the filamentous fungus A. flavus was screened for alkyl formate ester synthesis, using octanal as test substrate by cell-free extract (CFE) biotransformations, and using E. coli as expression host. CFE screening of the fungal BVMOs revealed a single BVMO, AFL838, able to produce heptyl formate from octanal at significant levels (> 1%). To our knowledge however, all BVMOs tested to date with aliphatic aldehydes yield the corresponding carboxylic acids and this is the first report of alkyl formate ester synthesis by a BVMO.AFL838 was purified to near-homogeneity and biotransformations were performed against a range of aldehydes (1a-6a, Fig. 1), yielding the alkyl formate esters with high regioselectivity, in each case. 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. Near complete conversion of 10 mM octanal was achieved within 8 h with a regiomeric excess of 82%. More than 80% conversion of 50 mM octanal was reached after 72 h, giving total turnover numbers exceeding 20 000. A 200 mL-scale biotransformation under unoptimized conditions gave a space-time yield (STY) of 4.3 g.L-1.d-1 (3.5 g.L-1.d-1 isolated product).[1] Burdock, GA. Fenaroli?s Handb. Flavor Ingredients, CRC Press, 2009.[2] Bevinakatti, HS and Newadkar, RV. Biotechnol. Lett., 1989, 11, 785-788.[3] Leisch, H; Morley, K and Lau, PCK. Chem. Rev., 2011, 111, 4165-4222.