Abstract
The goal of obtaining enzyme forms with higher catalytic activity, greater stability, and improved reusability has been pursued for the last few decades. Various novel biocatalyst designs have been created, and protein-coated microcrystals (PCMCs) are one of them. PCMC is an enzyme immobilization method based on simultaneous precipitation of protein and carrier, forming micron-sized enzyme-coated crystals. Highly active Rhizomucor miehei lipase (RML) PCMCs were prepared by immobilizing the protein onto K2SO4 as a carrier salt in acetone as a precipitating solvent. The formation of RML PCMCs was confirmed by scanning electron microscopy. Preparation of RML PCMCs was optimized by response surface methodology (RSM). Obtained PCMCs were found to be more active and stable during p-nitrophenyl palmitate hydrolysis in n-hexane, compared to liquid RML. The enzymatic activity and temperature optimum increased from 0.011 U/mgsoluble lipase to 8.70 U/mgimmobilized lipase and from 30 to 37 °C, respectively. Additionally, the ability of RML PCMCs to catalyze flavor ester 2-phenethyl octanoate synthesis was investigated. Some reaction parameters were optimized, resulting in 80 % conversion within 1 h with an enhanced reusability, compared to commercial immobilized RML preparation. Thus, PCMCs offer a cheap and effective technology for obtaining highly active lipase preparations for biocatalysis in organic media.
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Acknowledgments
This work was funded by the European Social Fund under the National Integrated Programme Biotechnology and Biopharmacy, Grant VP1-3.1-SMM-08-K01-005. Biopolis Ltd (Vilnius, Lihtuania), distributor of Novozymes, is also gratefully acknowledged for a kind supply of the enzymes.
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Simas Kazlauskas, is a Ph. D. Student, Department of Biochemistry and Molecular Biology, Faculty of Natural Sciences, Vilnius University, and holds a Msc. degree.
Vita Kiriliauskaitė, Department of Biochemistry and Molecular Biology, Faculty of Natural Sciences, Vilnius University, and holds a Ph. D. degree.
Lilija Kalėdienė, Faculty of Natural Sciences, Vilnius University, holds a Ph. D. degree.
Vida Bendikienė, Faculty of Natural Sciences, Vilnius University, holds a Ph. D. degree.
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Kazlauskas, S., Kiriliauskaitė, V., Kalėdienė, L. et al. High Performance Protein-Coated Microcrystals of Rhizomucor miehei Lipase: Preparation and Application for Organic Synthesis. Appl Biochem Biotechnol 176, 321–332 (2015). https://doi.org/10.1007/s12010-015-1576-4
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DOI: https://doi.org/10.1007/s12010-015-1576-4