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

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/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.

Enzyme-Coated Micro-Crystals: An Almost Forgotten but Very Simple and Elegant Immobilization Strategy

“…The biocatalyst was used to produce 2-phenethyl octanoate. After optimization, a yield of 80% in 1 h was obtained, clearly surpassing the results obtained with the commercial Lipozyme RM-IL [136]. In another contribution, lipase from Candida rugosa was used to produce PCMCs [137].…”

“…In another research effort, PCMCs of the lipase from Rhizomucor miehei were prepared and characterized [136]. The crystal forming agent employed was K 2 SO 4 , and the dehydrating solved was acetone.…”

Novel Hybrid Gel–Fiber Membranes as Carriers for Lipase Catalysis Based on Electrospinning and Gelation Technology

“…Holding Capacity (mg/g) = M 1 − C T M so −(M T −M sa ) ρ so M sa (2) where M 1 and M so are the mass (mg) of lipases dissolved in PBS initially and the mass (g) of this lipase solution, respectively. M sa is the original mass (g) of PVP/PLMA-HGFMs.…”

“…Lipase, a triacylglycerol hydrolase, has been increasingly studied and applied in biochemistry, pharmaceutical development, food engineering, and biosensors [1][2][3][4][5][6][7]. Unlike other enzymes, most lipases are compounds that are incompatible or only minimally compatible in terms of polarity with their substrates, and the catalytic reaction takes place at the interfacial junction of the two media, which is known as the interfacial catalytic mechanism of lipases [8,9].…”