Deactivation of microbial lipases with acetaldehyde after lyophilization at pH 6 and pH 10, respectively. A) R. oryzae lipase (ROL). B) C. rugosa lipase (CRL). C) P. fluorescens lipase (PFL). D) B. subtilis lipase A (BSL-A). E) B. subtilis lipase B (BSL-B). Lipases from C. rugosa, R. oryzae, and P. fluorescens were dissolved in citrate/NaOH-buffer (100 mM, pH 6) or Na2CO3/NaHCO3-buffer (100 mM, pH 10), respectively, at a final concentration of 10 mg/mL. BSL-A and BSL-B were stored in glycine/NaOH-buffer (10 mM, pH 10) and diluted 1:2 with glycine/NaOH (10 mM, pH 10) and citrate/NaOH-buffer (100 mM, pH 6), respectively to a final concentration of 0.5 mg/mL. Samples were incubated in screw-capped vials for 1 h at - 80°C prior to lyophilization carried out overnight at 0.011 mbar. The lyophilized enzymes were treated with toluene, sonicated for 3 min to obtain homogenous dispersions, than incubated with acetaldehyde (0-1 M for BSL-A, BSL-B, and PFL, and 0-2.5 M for CRL and ROL, respectively) and incubated for 24 h at room temperature. Lipases were extracted from the organic phase with 800 μL Na2HPO4/KH2PO4-buffer (50 mM, pH 8), and the centrifuged solution was assayed for residual activity using the p-nitrophenylpalmitate assay .
Franken et al. BMC Biochemistry 2011 12:10 doi:10.1186/1471-2091-12-10