CgYps1^D91A lacks proteolytic activity.

A. The Pichia pastoris GS115 strain expressing secretory forms of either catalytically active (rCgYps1) and inactive CgYps1 (rCgYps1^D91A) proteins was grown in the BMGY medium at 30°C for 24 h and incubated in the BMMY medium containing 2% methanol for 48 h. Methanol was used to induce the AOX1 promoter, which drives the expression of CgYps1 proteins. 2% Methanol was added every 24 h to maintain the induction and expression of CgYps1 proteins. After 48 h, 1 ml supernatant was precipitated with 80% ammonium sulphate, pellet was suspended in PBS, and CgYps1 protein induction was checked by SDS PAGE. Induced proteins were purified by anion exchange chromatography using DEAE cellulose resin. The eluate fractions were resolved on 12% SDS-PAGE. The orange asterisk marks CgYps1 band. The protein samples were also probed with anti-His antibody (1:5000 dilution) and shown underneath the gel images. M, protein marker. B. CgYps1-mediated proteolysis of gelatin. The YNB-agar medium containing 1% gelatin was incubated either with 100 μg of purified proteins (rCgYps1 and rCgYps1^D91A) or citrate buffer (pH 4.0; used for enzyme elution) for overnight at 37°C. The plate was stained with Coomassie Brilliant Blue G250 and imaged. The zone of hydrolysis was observed only with the CgYps1 enzyme. C. Proteolytic activity of DEAE cellulose-purified rCgYps1 and rCgYps1^D91A proteins (20 μg) was measured using 2.5% hemoglobin as a substrate in the citrate buffer (100 mM; pH 4.0) for 30 min at 37°C. TCA (10%) was added to stop the enzymatic reaction and precipitate the undigested hemoglobin and CgYps1 proteins. Cleaved peptides were collected from the supernatant and incubated with sodium bicarbonate (5 mM) and Folin-Ciocalteu reagent at 37°C for 30 min. The absorbance was read at 660 nm and converted into the μ moles of tyrosine released per mg of the protein. Control indicates the enzymatic reaction mixture containing buffer and substrate, but no CgYps1 protein. Data represent mean ± SEM of 4 independent samples. Of note, rCgYps1^D91A showed no proteolytic activity, indicating that Aspartate-91 in CgYps1 is required for enzyme activity. **, p < 0.01; unpaired two-tailed Student’s t test.

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