Implication of substrate-assisted catalysis on improving lipase activity or enantioselectivity in organic solvents Shau-Wei Tsai a, ⁎ , Chun-Chi Chen b , Hung-Shien Yang b , I-Son Ng b , Teh-Liang Chen b a Institute of Biochemical and Biomedical Engineering, Chang Gung University, Kwei-Shan Tao-Yuan, 33302, Taiwan b Department of Chemical Engineering, National Cheng Kung University, Tainan 70101, Taiwan Received 17 May 2006; received in revised form 10 July 2006; accepted 11 July 2006 Available online 14 July 2006 Abstract In comparison with the biocatalyst engineering and medium engineering approaches, very few examples have been reported on using the substrate engineering approach such as substrate-assisted catalysis (SAC) for naturally occurring or engineered lipases and serine proteases to improve the enzyme activity and enantioselectivity. By employing lipase-catalyzed hydrolysis of (R,S)-naproxen esters in water-saturated isooctane as the model system, we demonstrate the proton shuttle device to the leaving alcohol of the substrate as a new means of SAC to effectively improve the lipase activity or enantioselectivity. The result cannot only provide a strong evidence for the rate-limiting proton transfer for the bond-breaking of tetrahedron intermediate of the acylation step, but also sheds light for performing the hydrolysis, transesterification or aminolysis in organic solvents for the ester substrate that originally lipases cannot catalyze, but now can after introducing the device. © 2006 Elsevier B.V. All rights reserved. Keywords: Lipases; Substrate-assisted catalysis; Proton transfer; Acylation step; Hydrolysis resolution 1. Introduction Lipases follow the same catalytic mechanism as observed with esterases and serine proteases for the hydrolysis, esterification, transesterification and aminolysis of a broad range of acids, esters and amines with distinct stereo- or region- preference in addition to their physiological function of cleaving triglycerides [1–4]. Three approaches of using biocatalyst engineering, medium engineering and substrate engineering such as substrate-assisted catalysis have been proposed to improve the enzyme activity and enantioselectivity [5–12]. To date, only a few examples on improving the enzyme activity or enantioselectivity via SAC have been achieved for naturally occurring or engineered lipases and serine proteases [6,7]. Here we describe a new means of using SAC to enhance the lipase activity in organic solvents. The catalytic machinery of lipases consists of a Ser–His– Asp/Glu triad to perform the same acylation–deacylation displacement mechanism for the ester hydrolysis as the amide hydrolysis for serine proteases [13,14]. In the acylation step, the imidazole moiety of the triad abstracts a proton from the serine to increase the nucleophilic attack at the carbonyl carbon, while the resultant imidazolium donates the proton to the leaving group oxygen from the tetrahedral intermediate and forms the acyl enzyme intermediate. In the deacylation step, hydrolysis proceeds via a second tetrahedral intermediate giving the acid product and free enzyme. Our hypothesis is that if the bond- breaking of the tetrahedral intermediate of the acylation step is the rate-limiting step [15–20], the substrate-assisted proton shuttle device, e.g. an organic base, will provide an additional hydrogen bond between the substrate and imidazolium moiety on lowering the free energy of the transition state (Fig. 1), and hence improves the enzyme activity. In this report, the hypothesis was confirmed by using lipase-catalyzed hydrolysis Biochimica et Biophysica Acta 1764 (2006) 1424 – 1428 www.elsevier.com/locate/bbapap Abbreviations: SAC, substrate-assisted catalysis; pCPL, Carica papaya lipase; pCPHL, Carica pentagona Heilborn lipase ⁎ Corresponding author. Tel.: +886 3 2118800x3415; fax: +886 3 2118668. E-mail address: tsai@mail.cgu.edu.tw (S.-W. Tsai). 1570-9639/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2006.07.001