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 beta-cyclodextrin (beta-CD) as an additive. According to the results, YLLIP2 with (beta-CD exhibited approximately 7000 U/mg specific activity in 30 wt% methanol for 60 min compared with no activity without beta-CD under the same conditions. Molecular dynamics (MD) simulation results indicated that the beta-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 beta-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 beta-CD molecule then prevented the closure of active pockets, thus retaining the enzymatic activity of YLLIP2 with beta-CD in methanol solvent. (C) 2016 Published by Elsevier Inc.