Improved thermostability of lipase Lip2 from Yarrowia lipolytica through disulfide bond design for preparation of medium-long-medium structured lipids

Abstract

Lipase Lip2 from Yarrowia lipolytica with great potential value in the enzymatic preparation of medium-long-medium structured lipids (MLM-SLs), but the synthetic efficiency might be limited by its poor thermostability. We identified flexible regions of lipase Lip2 by molecular dynamics (MD) simulation, and then disulfide bonds were engineered into these regions to enhance its thermostability. Compared with the wild type, mutant 4sN with four disulfide bonds showed a 19.22 and 27.75 °C increase in the melting temperature (Tm) and the half-loss temperature at 15 min (T15 50), respectively. The half-life of the wild type at 50 °C was 1.66 min, while that of mutant 4sN at 60 °C was 40.52 min. MD simulation showed that the thermal stability improvement in the mutant was attributed to enhanced structural rigidity. Finally, the MLM-SLs were synthesized from olive oil and caprylic acid with immobilized wild-type and mutant 4sN. Mutant 4sN displayed a 5 °C higher optimum temperature and a higher reaction rate than the wild type. Meanwhile, residual activity of mutant 4sN after 7 consecutive cycles increased by 20.84% compared to the wild type. The increased reaction rate and recyclability of lipase Lip2 provide greater potential for industrial production of MLM-SLs.