Lyticase from arthrobacter luteus

Total genomic DNA from the yeast isolates was extracted according to the protocol described by Ercolini et al. (2001) (link) modified by adding lyticase at 2.5 U/mL (Lyticase from Arthrobacter luteus, Sigma–Aldrich, Germany) for yeast cell lysis (Bonatsou et al., 2018 (link)). Moreover, quantification and quality control of DNA extract was performed by spectrophotometer (Epoch, Biotek, USA) at wavelengths of 260, 280, and 230 nm.

Total membranes vesicles from Y. lipolytica cells were isolated as described previously (Okorokov and Lehle, 1998 (link); Lobão et al., 2007 (link)) with some modifications. Briefly, the cells grown in YED medium pH 4.5 were transformed to the spheroplasts by incubation with lyticase from Arthrobacter luteus (Sigma-Aldrich, ≥200 units mg⁻¹) at 37°C using 1.2 M sorbitol in 10 mM Tris-HCl, pH 7.2. Spheroplasts were homogenated in buffer containing 12.5% sucrose, 20 mM MOPS-Na, pH 7.6, 1 mM DTT, 1 mM benzamidine, 1 mM phenylmethanesulphonyl fluoride, a cocktail of protease inhibitors and 0.3% BSA, and total membranes were precipitated for 45 min at 100,000× g. The total membranes were resuspended in buffer containing 12.5% sucrose, 20 mM MOPS-Na, pH 7.6, 1 mM DTT, 1 mM benzamidine, 1 mM phenylmethanesulphonyl fluoride and a cocktail of protease inhibitors, aliquoted and stored at −70°C.

For the determination of mitochondrial complexes activity, mitochondria of S. cerevisiae were isolated from cultures grown in liquid medium YPD at 30°C in a shaking incubator, using a previously described method with light modifications [22] , Lyticase from Arthrobacter luteus (Sigma-Aldrich) was used instead of zymolyase. Yeast cells were harvested in late exponential growth phase by centrifugation at 2,750× g for 15 min at 4°C and washed thrice using distilled water and suspended in digestion solution (sorbitol 1.2 M, EGTA 1 mM, Tris-HCl 50 mM, DTT 10 mM, at pH 7.5); Lyticase was added at 2 mg g⁻¹ weight for spheroplast generation. Yeast suspensions were incubated for 60 min at 30°C. Spheroplasts were washed twice with spheroplast washing buffer (sorbitol 1.2 M, EGTA 1 mM, Tris-HCl 50 mM, DTT 10 mM, at pH 7.5). Then, spheroplasts were suspended in homogenizing buffer (sorbitol 0.6 M, HEPES-KOH 20 mM, DTT 10 mM, at pH 7.4) and lysed in a Potter-Elvehjem pestle and glass tube and washed thrice with the same buffer. The unruptured cells were removed by centrifugation at 2,500× g for 10 min at 4°C, and yeast mitochondria were harvested from the supernatant by centrifugation at 9,600× g for 10 min at 4°C and suspended in homogenizing buffer.