Catalytic and physical features of a naturally immobilized Yarrowia lipolytica lipase in cell debris (LipImDebri) displaying high thermostability.

Lipase activity (337 U/g dry weight of cell debris) was detected in cell debris after ultrasound treatment of Yarrowia lipolytica cells cultivated in residual frying palm oil. It is a naturally immobilized lipase with protein content of 47%, herein called LipImDebri. This immobilized biocatalyst presents low hydrophobicity (8%), that can be increased adjusting pH and buffer type. Despite apparent intact cells, electron microscopy showed a shapeless and flat surface for LipImDebri and optical microscopy revealed no cell viability. Besides, an inferior mean diameter (3.4 mm) in relation to whole cells reveals structure modification. A high negative zeta potential value (- 33.86 mV) for pH 6 and 25 °C suggests that LipImDebri is a stable suspension in aqueous solution. Fourier Transform Infrared Spectra (FTIR) expose differences between LipImDebri and extracellular lipase extract signaling a physical interaction between enzyme and cell debris, which is possibly the reason for the high thermostability (k d = 0.246 h-1; t 1/2 = 2.82 h at 50 °C, pH 7.0). A good adjustment of LipImDebri kinetic data with Hill equation (R 2 = 0.95) exposes an allosteric behavior related to the presence of more than one lipase isoform. These features reveal that LipImDebri can be a good catalyst for industrial applications.

Yarrowia lipolytica Adhesion and Immobilization onto Residual Plastics.

Research in cell adhesion has important implications in various areas, such as food processing, medicine, environmental engineering, biotechnological processes. Cell surface characterization and immobilization of microorganisms on solid surfaces can be performed by promoting cell adhesion, in a relatively simple, inexpensive, and quick manner. The adhesion of Yarrowia lipolytica IMUFRJ 50682 to different surfaces, especially potential residual plastics (polystyrene, poly(ethylene terephthalate), and poly(tetrafluoroethylene)), and its use as an immobilized biocatalyst were tested. Y. lipolytica IMUFRJ 50682 presented high adhesion to different surfaces such as poly(tetrafluoroethylene) (Teflon), polystyrene, and glass, independent of pH, and low adhesion to poly(ethylene terephthalate) (PET). The adhesion of the cells to polystyrene was probably due to hydrophobic interactions involving proteins or protein complexes. The adhesion of the cells to Teflon might be the result not only of hydrophobic interactions but also of acid-basic forces. Additionally, the present work shows that Y. lipolytica cell extracts previously treated by ultrasound waves (cell debris) maintained their enzymatic activity (lipase) and could be attached to polystyrene and PET and used successfully as immobilized biocatalysts in hydrolysis reactions.

Use of Yarrowia lipolytica Lipase Immobilized in Cell Debris for the Production of Lipolyzed Milk Fat (LMF).

Lipase immobilized on Yarrowia lipolytica cell debris after sonication of yeast cells (LipImDebri) was used in hydrolysis reaction as a novel strategy to produce lipolyzed milk fat (LMF). Extracellular (4732.1 U/L), intracellular (130.0 U/g), and cell debris (181.0 U/g) lipases were obtained in a 4 L bioreactor using residual frying oil as inducer in 24 h fermentation process. LipImDebri showed a good operational stability retaining 70% of lipolytic activity after the second cycle and 40% after the fourth. The highest degree of hydrolysis (28%) was obtained with 500 mg LipImDebri for 6 h of lipolysis of anhydrous milk fat. LMF produced with LipImDebri presented high contents of oleic (35.2%), palmitic (25.0%), and stearic (15.4%) acids and considerable amounts of odor-active short and medium chain fatty acids (C:4⁻C:10) (8.13%).