Inversion of Lipase Stereospecificity for Fluorogenic Alkyldiacyl Glycerols

Abstract

We synthesized enantiomeric 1-O-alkyl-2,3-diacyl-sn-glycerol and 3-O-alkyl-1,2-diacyl-sn-glycerol containing pyrene as a fluorescent reporter and the trinitrophenylamino residue as a fluorescence quencher; both reporter groups were covalently bound to the omega end of the acyl chains at positions sn-2 and sn-3(1), respectively. The fluorescence of the intact substrate molecules was very low. Chemical or enzymic release of the fatty acyl chains lead to fluorescence dequenching. The rate of lipolysis could be measured from the time-dependent increase in fluorescence intensity. We used the respective substrates for the continuous determination of activity and stereopreference of four different microbial lipases from Chromobacterium viscosum, Candida rugosa, Pseudomonas sp., Rhizopus arrhizus, as well as cutinase from Fusarium solani and lipoprotein lipase from bovine milk. The stereopreference of the lipases depended, in general, on how the substrate was solubilized in the reaction medium. All lipases under investigation preferentially hydrolysed the sn-1 acyl ester bond, if the lipid analog was dispersed in albumin-containing Tris/HCl buffer in the absence of detergent or organic solvent. In mixtures of 1:1 (by vol.) water/ethanol, the enzymes showed higher activity toward the sn-3 acyl ester bond, except for lipoprotein lipase which preferred the sn-1 acyl isomer under all conditions tested. Different stereopreferences were observed with the different lipases if the substrate was solubilized by amphiphiles (micelles of N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate). C. rugosa lipase and F. solani cutinase showed high stereopreference for the sn-3 acyl ester, whereas Pseudomonas sp. lipase and C. viscosum lipase hydrolysed both enantiomers at similar rates. From spectroscopic studies, it can be inferred that the conformation of the fluorescent lipids is probably similar in water, mixtures of water and organic solvents, and in micelles. The possible effects of reaction conditions on substrate accessibility and enzyme conformation on stereoselectivity of the respective lipases are discussed.

Cutinase: From molecular level to bioprocess development

“…Lyophilized cutinase has also been used as a powder suspension to elucidate its mechanism of action, in stereoselectivity, specificity (Mannesse et al, 1995a,b;Rogalska et al, 1997;Zandonella et al, 1995), and kinetic (Sjursnes et al, 1998) studies, including esterification (Melo et al, 1995c;Sarazin et al, 1992Sarazin et al, , 1995 and transesterification (Lamare and Legoy, 1995;Lamare et al, 1997;Parvaresh et al, 1992) reactions. Cutinase was immobilized onto solid supports, either adsorbed or covalently coupled, and used in hydrolysis (Gonçalves et al, 1995(Gonçalves et al, , 1996a(Gonçalves et al, ,b, 1999Zoungrana, 1997), esterification (Sereti et al, 1997;Sjursnes et al, 1998), and transesterification (Fontes et al, 1998;Lamare and Legoy, 1995;Lamare et al, 1997;Parvaresh et al, 1992;Serralha et al, 1998) reactions.…”

“…Triacylglycerol analogs have been used to study the substrate specificity and selectivity of cutinase and mutants (Egmond and van Bemmel, 1997;Mannesse et al, 1995aMannesse et al, ,b, 1997Rogalska et al, 1997;Zandonella et al, 1995). Triglyceride analogs were synthesized by replacing one of the primary acyl ester functions with an alkyl group, whereas the secondary acyl ester bond was replaced by an acyl amino bond (Mannesse et al, 1995b).…”

Cutinase structure, function and biocatalytic applications

“…A lipase stereo-and regioselectivity towards tri-and diacylglycerols study, using a monolayer technique, was undertaken to complement the structural data (Rogalska et al, 1997 Triacylglycerol analogues have been used to study the substrate specificity and selectivity of cutinase and its mutants (Egmond and van Bemmel, 1997;Rogalska et al, 1997;Mannesse et al, 1995a;Mannesse et al, 1995b;Zandonella et al, 1995;Mannesse et al, 1997). Triglyceride analogues were synthesized replacing one of the primary acyl ester functions by an alkyl group, while the secondary acyl ester bond was replaced by an acyl amino bond (Mannesse et al, 1995a).…”

“…Lyophilized cutinase has been used to elucidate its mechanism of reaction, in stereoselectivity, specificity (Rogalska et al, 1997;Mannesse et al, 1995a;Mannesse et al, 1995b;Zandonella et al, 1995) and kinetic (Sjursnes et al, 1998) studies, including esterification (Sarazin et al, 1992;Sarazin et al, 1995;Melo et al, 1995a) and transesterification (Parvaresh et al, 1992;Lamare and Legoy, 1995;Lamare et al, 1997) reactions. Lyophilized cutinase was also added to an aqueous/triolein biphasic system to perform the hydrolysis of this triglyceride into oleic acid and glycerol (Flipsen et al, 1996).…”

O emprego de lipases como agentes de resolução cinética de enantiômeros em síntese orgânica: aspectos gerais sobre a influência do solvente

“…Em particular, a forma com que a enzima aproxima-se do substrato e as interações da enzima com a interface tem papel importante no processo de ativação e reconhecimento. É interessante notar que, embora tenha sido demonstrado que o solvente pode alterar a especificidade 61 64 das lipases e outras hidrolases, existem vários dados que indicam a independência do mecanismo de ação e estrutura enzimáticas do solvente 12 . Por exemplo: (a) o emprego do método de Hammet na análise da clivagem de vários fenil-acetatos para-substituídos, catalisada pela subtilisina em água e em diferentes solventes orgânicos, revelou que a estrutura do estado de transição para a acilação da enzima é independente do solvente; (b) estudos cinéticos de efeitos isotópicos indicaram que o estado de transição para a desacilação da subtilisina acilada não muda com o solvente; (c) estudos de RMN em estado sólido revelaram que a única rede de ligações de hidrogênio existente na tríade catalítica da proteinase a-lise, é mantida intacta tanto em octano como em acetona; (d) foram observadas estruturas indênticas para cristais da subtilisina obtidos em água e em acetonitrila.…”