Enhancement of methanol resistance of Yarrowia lipolytica lipase 2 using β-cyclodextrin as an additive: Insights from experiments and molecular dynamics simulation

- Methanol resistance of lipase is increased by utilizing β-cyclodextrin.
- Experimental and computational results correspond well.
- Mechanism of methanol resistance of lipase with β-cyclodextrin is revealed.
- Conformation of lipase in methanol is stabilized by β-cyclodextrin.
- β-cyclodextrin and lipase interact through hydrogen bond.

The methanol resistance of lipase is a critical parameter in enzymatic biodiesel production. In the present work, the methanol resistance of Yarrowia lipolytica Lipase 2 (YLLIP2) was significantly improved using β-cyclodextrin (β-CD) as an additive. According to the results, YLLIP2 with β-CD exhibited approximately 7000 U/mg specific activity in 30 wt% methanol for 60 min compared with no activity without β-CD under the same conditions. Molecular dynamics (MD) simulation results indicated that the β-CD molecules weakened the conformational change of YLLIP2 and maintained a semi-open state of the lid by overcoming the interference caused by methanol molecules. Furthermore, the β-CD molecule could directly stabilize “pathway” regions (e.g., Asp61-Asp67) and indirectly stabilize “pathway” regions (e.g., Gly44-Phe50) by forming hydrogen bonds with “pathway” regions and nearby “pathway” regions, respectively. The regions stabilized by the β-CD molecule then prevented the closure of active pockets, thus retaining the enzymatic activity of YLLIP2 with β-CD in methanol solvent.

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