title: Production of barley endoprotease B2 in Pichia pastoris and its proteolytic activity against native and recombinant hordeins.
abstract: Barley (Hordeum vulgare L.) cysteine proteases are of fundamental biological importance during germination but may also have a large potential as commercial enzyme. Barley cysteine endoprotease B2 (HvEPB2) was expressed in Pichia pastoris from a pPICZαA based construct encoding a HvEPB2 C-terminal truncated version (HvEPB2ΔC) and a proteolytic resistant His6 tag. Maximum yield was obtained after 4 days of induction. Recombinant HvEPB2ΔC (r-HvEPB2ΔC) was purified using a single step of Ni(2+)-affinity chromatography. Purified protein was evaluated by SDS-PAGE, Western blotting and activity assays. A purification yield of 4.26 mg r-HvEPB2ΔC per L supernatant was obtained. r-HvEPB2ΔC follows first order kinetics (Km=12.37 μM) for the substrate Z-Phe-Arg-pNA and the activity was significantly inhibited by the cysteine protease specific inhibitors E64 and leupeptin. The temperature optimum for r-HvEPB2ΔC was 60°C, thermal stability T50 value was 44°C and the pH optimum was 4.5. r-HvEPB2ΔC was incubated with native purified barley seed storage proteins for up to 48 h. After 12h, r-HvEPB2ΔC efficiently reduced the C and D hordeins almost completely, as evaluated by SDS-PAGE. The intensities of the B and γ hordein bands decreased continuously over the 48 h. No degradation occurred in the presence of E64. Recombinant hordeins (B1, B3 and γ1) were expressed in Escherichia coli. After 2h of incubation with r-HvEPB2ΔC, an almost complete degradation of γ1 and partial digests of hordein B1 and B3 were observed.
authors.affiliation: Aarhus University, Faculty of Science and Technology, Dept. of Molecular Biology and Genetics, Research Centre Flakkebjerg, DK-4200 Slagelse, Denmark.
authors.forename: Anne Lind
authors.initials: AL
authors.lastname: Rosenkilde
authors.affiliation: Aarhus University, Faculty of Science and Technology, Dept. of Molecular Biology and Genetics, Research Centre Flakkebjerg, DK-4200 Slagelse, Denmark.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Dionisio
authors.affiliation: Aarhus University, Faculty of Science and Technology, Dept. of Molecular Biology and Genetics, Research Centre Flakkebjerg, DK-4200 Slagelse, Denmark.
authors.forename: Preben B
authors.initials: PB
authors.lastname: Holm
authors.affiliation: Aarhus University, Faculty of Science and Technology, Dept. of Molecular Biology and Genetics, Research Centre Flakkebjerg, DK-4200 Slagelse, Denmark. Electronic address: Henrik.BrinchPedersen@agrsci.dk.
authors.forename: Henrik
authors.initials: H
authors.lastname: Brinch-Pedersen
mesh_terms: D057056:Cysteine Proteases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000032:analysis / Q000378:metabolism*; D005285:Fermentation; D005983:Glutens / Q000378:metabolism*; D001467:Hordeum / Q000201:enzymology*; D010449:Peptide Mapping; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D005983:Glutens; D057056:Cysteine Proteases; D010450:Endopeptidases
keywords: DTT; Endo H; Heterologous expression; Hordein degradation; Hordeum vulgare; Hordeum vulgare endoprotease B1; Hordeum vulgare endoprotease B2; Hordeum vulgare endoproteaseB2; HvEPB1; HvEPB2; Pichia pastoris; Proteolytic resistant tag; Trx; dithiothreitol; endo-β-N-acetylglucosaminidase H; thioredoxin