Optical Control of Enzyme Enantioselectivity in Solid Phase Antoni Bautista-Barrufet, ■,†,¶ Fernando Ló pez-Gallego,* ,■,‡ Víctor Rojas-Cervellera, § Carme Rovira, §,⊥ Miquel A. Perica ̀ s, ¶ Jose ́ M. Guisa ́ n, ‡ and Pau Gorostiza* ,†,⊥,□ † Institut de bioenginyeria de Catalunya (IBEC), C/Baldiri Reixac 15-21, Barcelona 08028, Spain ‡ Instituto de Cata ́ lisis y Petroleoquímica (ICP-CSIC), C/Marie Curie n 2°, Madrid 28029, Spain § Departament de Quı ́ mica Orga ̀ nica, Universitat de Barcelona (UB), Mart ı ́ i Franque ̀ s 1, Barcelona 08028, Spain ⊥ Institució Catalana de Recerca i Estudis Avanç ats (ICREA), C/Lluís Companys 23, Barcelona 08010, Spain ¶ Institut Catala ̀ d’Investigació Química (ICIQ). Avinguda Països Catalans 16, Tarragona 43007, Spain □ Centro de Investigació n Biome ́ dica en Red sobre Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), C/Poeta Mariano Esquillor s/n, Zaragoza 50018, Spain * S Supporting Information ABSTRACT: A lipase was immobilized on transparent agarose microspheres and genetically engineered to specifically anchor photochromic molecules into its catalytic site. Several combinations of azobenzene and spiropyran groups were conjugated to cysteines introduced at different positions near the active center. Light modulated the catalytic properties of the resulting solid bioconjugates, and such modulation depended on both the nature of the photochromic compound and the anchoring position. Covalent anchoring of azobenzene derivatives to the residue 295 of the lipase 2 from Bacillus thermocathenolatus triggered lipase preference for the S isomer under UV light, whereas visible light promoted preference for the R isomer. Molecular dynamics simulations indicate that conjugating photochromic compounds into the catalytic cavity allows manipulating the steric hindrance and binding energy of the substrates, leading to an enantioselective molecular fit in certain cases. Using this approach, we report for the first time the control of enzyme properties using light in the solid phase. KEYWORDS: lipase, azo compounds, photochromism, chemical modification, immobilization ■ INTRODUCTION Isolated enzymes can catalyze organic transformations at both high yield and high enantioselectivity under mild conditions. However, in situ modulation of catalytic properties has proven a great challenge, as indicated by the lack of methods to regulate enzyme enantioselectivity during in vitro biotransfor- mations. The dynamic control of enzyme selectivity for in vitro reactions schemes is thus an unmet need, especially in the context of cascade reactions catalyzed by multienzyme systems, in which biocatalysts must be switched on and off in situ according to system requirements. Optical control 1 offers the possibility to remotely manipulate enzyme activity using spatiotemporally designated patterns of illumination. 2 More- over, the immobilization of these engineered biocatalysts would enable their reuse as well as their incorporation into nanodevices. 3 Here, we present a rational approach to conjugate photochromic compounds to an immobilized enzyme in a site-directed manner and demonstrate for the first time the regulation of its enantioselectivity with light. Lipases are serin-threonin hydrolases that naturally catalyze the hydrolysis of lipids and are widely applied in chemical processes from research laboratories to industrial plants. 4 Most lipases present a hydrophobic active site shielded by an amphiphilic domain (named as “lid”) that triggers the catalytic mechanism in the presence of hydrophobic substrates. 5 This class of enzymes is the paradigm of enantioselective biocatalysis, and their hydrolytic rate and enantiomeric excess can be enhanced by adjusting the position of the substrate into the hydrophobic cavity. 6 A plethora of methodologies to alter the lipase catalytic mechanism have been devised, including enzyme engineering, reaction media engineering, immobiliza- tion, and chemical modification. 6-8 We have recently reported the alteration of both activity and selectivity of lipase 2 from Bacillus thermocathenolatus (BTL2) using site-directed chemical modification in the solid phase. 9 Building upon this method- ology, we pursued photocontrol of BTL2 catalytic properties by tethering a photochromic group inside the enzyme active center. ■ RESULTS AND DISCUSSION Site-Directed Chemical Modification of BTL2 in Solid Phase. We chemically modified BTL2 with different photo- Received: November 25, 2013 Revised: February 1, 2014 Published: February 13, 2014 Research Article pubs.acs.org/acscatalysis © 2014 American Chemical Society 1004 dx.doi.org/10.1021/cs401115s | ACS Catal. 2014, 4, 1004-1009