A robust whole-cell biocatalyst that introduces a thermo- and solvent-tolerant lipase into Aspergillus oryzae cells: Characterization and application to enzymatic biodiesel production

• Aspergillus oryzae (r-BTL) harboring lipase gene (BTL2) was constructed.
• r-BTL showed higher thermo- and solvent-tolerance lipase activity.
• Enzymatic production of biodiesel from palm oil was performed.
• r-BTL efficiently catalyzed methanolysis at elevated temperature (40–50 °C).
• r-BTL efficiently catalyzed methanolysis at a high methanol concentration

To develop a robust whole-cell biocatalyst that works well at moderately high temperature (40–50 °C) with organic solvents, a thermostable lipase from Geobacillus thermocatenulatus (BTL2) was introduced into an Aspergillus oryzae whole-cell biocatalyst. The lipase-hydrolytic activity of the immobilized A. oryzae (r-BTL) was highest at 50 °C and was maintained even after an incubation of 24-h at 60 °C. In addition, r-BTL was highly tolerant to 30% (v/v) organic solvents (dimethyl carbonate, ethanol, methanol, 2-propanol or acetone). The attractive characteristics of r-BTL also worked efficiently on palm oil methanolysis, resulting in a nearly 100% conversion at elevated temperature from 40 to 50 °C. Moreover, r-BTL catalyzed methanolysis at a high methanol concentration without a significant loss of lipase activity. In particular, when 2 molar equivalents of methanol were added 2 times, a methyl ester content of more than 90% was achieved; the yield was higher than those of conventional whole-cell biocatalyst and commercial Candida antarctica lipase (Novozym 435). On the basis of the results regarding the excellent lipase characteristics and efficient biodiesel production, the developed whole-cell biocatalyst would be a promising biocatalyst in a broad range of applications including biodiesel production.