Protein engineering of lipase A from Candida antarctica to improve esterification of tertiary alcohols.

Chiral tertiary alcohols are important organic compounds in science as well as in industry. However, their preparation in enantiomerically pure form is still a challenge due to their complex structure and steric hindrances compared with primary and secondary alcohols, so kinetic resolution could be an attractive approach.  Lipase A from Candida antarctica (CAL-A) has been shown to catalyze the enantioselective esterification of various tertiary alcohols with excellent enantioselectivity but low activity. Here we report a mutagenesis study by rational design to improve CAL-A activity against tertiary alcohols. Single mutants of CAL-A were selected, expressed, immobilized and screened for esterification of the tertiary alcohol 1,2,3,4-tetrahydronaphthalene-1-ol. A double mutant V278S+S429G showed a 1.5-fold higher reaction rate than that of the wild type CAL-A, while maintaining excellent enantioselectivity.

Candida antarctica Lipase A-Based Enantiorecognition of a Highly Strained 4-Dibenzocyclooctynol (DIBO) Used for PET Imaging.

The enantiomers of aromatic 4-dibenzocyclooctynol (DIBO), used for radiolabeling and subsequent conjugation of biomolecules to form radioligands for positron emission tomography (PET), were separated by kinetic resolution using lipase A from Candida antarctica (CAL-A). In optimized conditions, (R)-DIBO [(R)-1, ee 95%] and its acetylated (S)-ester [(S)-2, ee 96%] were isolated. In silico docking results explained the ability of CAL-A to differentiate the enantiomers of DIBO and to accommodate various acyl donors. Anhydrous MgCl2 was used for binding water from the reaction medium and, thus, for obtaining higher conversion by preventing hydrolysis of the product (S)-2 into the starting material. Since the presence of hydrated MgCl26H2O also allowed high conversion or effect on enantioselectivity, Mg2+ ion was suspected to interact with the enzyme. Binding site predictions indicated at least two sites of interest; one in the lid domain at the bottom of the acyl binding pocket and another at the interface of the hydrolase and flap domains, just above the active site.

Immobilization of Lipase A from Candida antarctica onto Chitosan-Coated Magnetic Nanoparticles.

In this communication, lipase A from Candida antarctica (CALA) was immobilized by covalent bonding on magnetic nanoparticles coated with chitosan and activated with glutaraldehyde, labelled CALA-MNP, (immobilization parameters: 84.1% ± 1.0 for immobilization yield and 208.0 ± 3.0 U/g ± 1.1 for derivative activity). CALA-MNP biocatalyst was characterized by X-ray Powder Diffraction (XRPD), Fourier Transform Infrared (FTIR) spectroscopy, Thermogravimetry (TG) and Scanning Electron Microscope (SEM), proving the incorporation of magnetite and the immobilization of CALA in the chitosan matrix. Besides, the immobilized biocatalyst showed a half-life 8-11 times higher than that of the soluble enzyme at pH 5-9. CALA showed the highest activity at pH 7, while CALA-MNP presented the highest activity at pH 10. The immobilized enzyme was more active than the free enzyme at all studied pH values, except pH 7.

Ethyl Butyrate Synthesis Catalyzed by Lipases A and B from Candida antarctica Immobilized onto Magnetic Nanoparticles. Improvement of Biocatalysts' Performance under Ultrasonic Irradiation.

The synthesis of ethyl butyrate catalyzed by lipases A (CALA) or B (CALB) from Candida antarctica immobilized onto magnetic nanoparticles (MNP), CALA-MNP and CALB-MNP, respectively, is hereby reported. MNPs were prepared by co-precipitation, functionalized with 3-aminopropyltriethoxysilane, activated with glutaraldehyde, and then used as support to immobilize either CALA or CALB (immobilization yield: 100 ± 1.2% and 57.6 ± 3.8%; biocatalysts activities: 198.3 ± 2.7 Up-NPB/g and 52.9 ± 1.7 Up-NPB/g for CALA-MNP and CALB-MNP, respectively). X-ray diffraction and Raman spectroscopy analysis indicated the production of a magnetic nanomaterial with a diameter of 13.0 nm, whereas Fourier-transform infrared spectroscopy indicated functionalization, activation and enzyme immobilization. To determine the optimum conditions for the synthesis, a four-variable Central Composite Design (CCD) (biocatalyst content, molar ratio, temperature and time) was performed. Under optimized conditions (1:1, 45 °C and 6 h), it was possible to achieve 99.2 ± 0.3% of conversion for CALA-MNP (10 mg) and 97.5 ± 0.8% for CALB-MNP (12.5 mg), which retained approximately 80% of their activity after 10 consecutive cycles of esterification. Under ultrasonic irradiation, similar conversions were achieved but at 4 h of incubation, demonstrating the efficiency of ultrasound technology in the enzymatic synthesis of esters.