Analysis of Aspergillus sp. lipase immobilization for the application in organic synthesis.

Nowadays, for the industrial implementations, especially in the area of organic synthesis, immobilized enzymes are preferred over their soluble forms. Present study aimed to find fast, cost-efficient, and effective way of lipase immobilization for the use in organic media. Lipase from Aspergillus sp. (Resinase A 2X) was immobilized utilizing cross-linking of enzyme aggregates, covalent immobilization on magnetite particles and adsorption-immobilization using pyrolyzed sugar industry waste product as a novel type of carrier. Covalently- and adsorption-immobilized preparations exhibited greater specific activities (5.61±0.18U/mg and 14.2±0.63U/mg, respectively) in organic reaction media than the soluble form of the enzyme (0.06±0.01U/mg). Enzyme immobilized on the sugar industry waste pyrolyis product was determined as a best way to hyperactivate Resinase A 2X and was chosen for the synthesis of flavor and fragrance compound 2-phenylethyl butanoate. Furthermore, in order to optimize 2-phenylethyl butanoate synthesis conditions, central compositional experimental plan was designed using RSM. It showed that in optimal reaction conditions (4.5h at 40.7°C, with 0.1M of substrate) conversion higher than 90% can be achieved. Studies of the operational stability showed enhanced reusability of adsorption-immobilized lipase (with each cycle, efficiency of the 2-PB synthesis diminished by 20-30%). The use of the sugar industry waste pyrolysis product as a carrier provides a novel, cheap, fast, cost-efficient and eco-friendly way of immobilization with some crucial points to be noted for the best productivity.

High Performance Protein-Coated Microcrystals of Rhizomucor miehei Lipase: Preparation and Application for Organic Synthesis.

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/mg(soluble) lipase to 8.70 U/mg(immobilized) 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.