Cell Surface Display of Yarrowia lipolytica Lipase Lip2p Using the Cell Wall Protein YlPir1p, Its Characterization, and Application as a Whole-Cell Biocatalyst

Abstract

The Yarrowia lipolytica lipase Lip2p was displayed on the yeast cell surface via N-terminal fusion variant using cell wall protein YlPir1p. The hydrolytic activity of the lipase displayed on Y. lipolytica cells reached 11,900 U/g of dry weight. However, leakage of enzyme from the cell wall was observed. The calculated number of recombinant enzyme displayed on the cell surface corresponds to approximately 6 × 10(5) molecules per cell, which is close to the theoretical maximum (2 × 10(6) molecules/cell). Furthermore, the leaking enzyme was presented as three N-glycosylated proteins, one of which corresponds to the whole hybrid protein. Thus, we attribute the enzyme leakage to the limited space available on the cell surface. Nevertheless, the surface-displayed lipase exhibited greater stability to short-term and long-term temperature treatment than the native enzyme. Cell-bound lipase retained 74 % of its original activity at 60 °C for 5 min of incubation, and 83 % of original activity after incubation at 50 °C during 5 h. Cell-bound lipase had also higher stability in organic solvents and detergents. The developed whole-cell biocatalyst was used for recycling biodiesel synthesis. Two repeated cycles of methanolysis yielded 84.1 and 71.0 % methyl esters after 33- and 45-h reactions, respectively.

Surface display system for probiotics and its application in aquaculture

“…Cell surface display of Yarrowia lipolytica lipase using the Pir1p can be applied as a whole‐cell biocatalyst (Yuzbasheva et al . 2015). Xylose reductase fused with Pir4 in the Saccharomyces cerevisiae cell wall had higher thermal and pH stability than the control (Hossain et al .…”

Biodiesel production using immobilized lipase: feasibility and challenges

“…These co‐displayed whole‐cell lipases also demonstrated high biodiesel conversion (95.4%) and good operational stability . Even though cell‐bound lipases have already been shown to display higher thermal tolerance and higher tolerance to the presence of detergents in contrast to free enzyme, Huang et al . still proposed approaches, such as stepwise addition of methanol or addition of hydrophilic solvent into the reaction media, to further prevent the occurrence of lipase inactivation …”

Enzyme-Catalyzed Production of Biodiesel as Alternative to Chemical- Catalyzed Processes: Advantages and Constraints

“…The whole cells showed good stability in isooctane system where methanol was provided in a three-stepwise addition procedure to reduce lipase inactivation. Under these conditions a methyl ester reaction yield of 83.1% was obtained after 72 h. Lipase 2 from Y. lipolytica is an extracellular lipase that was displayed on the surface of the yeast to test its applications as a whole cell catalyst [178]. Results showed that the cell-bound lipase was more thermostable than the free lipase and was capable of producing biodiesel with a yield of 84.1% after 33 h. Yan et al [179] fluorescens and Candida sp.…”