Modulation of the Catalytic Properties of Lipase B from Candida antarctica by Immobilization on Tailor-Made Magnetic Iron Oxide Nanoparticles: The Key Role of Nanocarrier Surface Engineering

The immobilization of biocatalysts on magnetic nanomaterial surface is a very attractive alternative to achieve enzyme nanoderivatives with highly improved properties. The combination between the careful tailoring of nanocarrier surfaces and the site-specific chemical modification of biomacromolecules is a crucial parameter to finely modulate the catalytic behavior of the biocatalyst. In this work, a useful strategy to immobilize chemically aminated lipase B from Candida antarctica on magnetic iron oxide nanoparticles (IONPs) by covalent multipoint attachment or hydrophobic physical adsorption upon previous tailored engineering of nanocarriers with poly-carboxylic groups (citric acid or succinic anhydride, CALB(EDA)@CA-NPs and CALB(EDA)@SA-NPs respectively) or hydrophobic layer (oleic acid, CALB(EDA)@OA-NPs) is described. After full characterization, the nanocatalysts have been assessed in the enantioselective kinetic resolution of racemic methyl mandelate. Depending on the immobilization strategy, each enzymatic nanoderivative permitted to selectively improve a specific property of the biocatalyst. In general, all the immobilization protocols permitted loading from good to high lipase amount (149 < immobilized lipase < 234 mg/g(Fe)). The hydrophobic CALB(EDA)@OA-NPs was the most active nanocatalyst, whereas the covalent CALB(EDA)@CA-NPs and CALB(EDA)@SA-NPs were revealed to be the most thermostable and also the most enantioselective ones in the kinetic resolution reaction (almost 90\% ee R-enantiomer). A strategy to maintain all these properties in long-time storage (up to 1 month) by freeze-drying was also optimized. Therefore, the nanocarrier surface engineering is demonstrated to be a key-parameter in the design and preparation of lipase libraries with enhanced catalytic properties.The CNIC is supported by Spanish Ministry for Economy and Competitiveness (MEyC) and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505). M.F. would like to thank MEyC for the research grant no. SAF2014-59118-JIN co-funded by Fondo Europeo de Desarrollo Regional (FEDER) and COST Action CA1520: `European Network on NMR Relaxometry-EURELAX'. M.F. would also thank the Community of Madrid for research contract num. 2017-T1/BIO-4992 ('Atraccion de Talentos' Action) also cofunded by Universidad Complutense de Madrid. The authors would thank CSIC for the contract of MV in the frame of project `Magnetic nanoparticles for environment and health applications' (Ref. 201760E007). The authors would like to thank Ramiro Martinez (Novozymes, Spain S.A) for kindly supplying the enzyme used in this research. The authors thank Dr. Jose Miguel Palomo (ICP-CSIC, Madrid, Spain) for his help in enzymatic kinetic resolution reaction development and Dr. M. Puerto Morales (ICMM-CSIC, Madrid, Spain) for her help in magnetic hyperthermia and VSM characterizations.S