title: Purification, kinetic characterization and properties of a novel thermo-tolerant extracellular protease from Kluyveromyces marxianus IFO 0288 with potential biotechnological interest.
abstract: A novel extracellular hydrolase of ∼45kDa molecular mass was purified from Kluyveromyces marxianus IFO 0288 cultures and characterized as serine protease. The K(m)-value of protease (designated protease-KM-IFO-0288-A), which was found active in media containing elevated [NaCl] but lacking EDTAK(2), decreased with increasing [Ca(2+)]. The protease maintained considerable activity at the range of 10-60°C and pH 6.00-10.25, with optimum k(cat)/K(m)-value at 35.5°C and pH 7.75. It was strongly affected by specific irreversible inhibitors of serine proteases while was unaffected by inhibitors of cysteine proteases. Significant rate constants, activation energies, and proton inventories were estimated from the profiles of Michaelis-Menten parameters, versus pH, temperature and deuterium atom fraction, in the hydrolysis of Suc-AAPF-pNA showing that protease-KM-IFO-0288-A performs catalysis via a charge-relay system. The properties of protease-KM-IFO-0288-A suggest that K. marxianus represents a valuable source of extracellular protease of biotechnological interest which, given its GRAS status, could find several important applications.
authors.affiliation: University of Ioannina, Department of Chemistry, Ioannina 45110, Greece.
authors.forename: Athanasios
authors.initials: A
authors.lastname: Foukis
authors.forename: Panagiota-Yiolanda
authors.initials: PY
authors.lastname: Stergiou
authors.forename: Leonidas G
authors.initials: LG
authors.lastname: Theodorou
authors.forename: Maria
authors.initials: M
authors.lastname: Papagianni
authors.forename: Emmanuel M
authors.initials: EM
authors.lastname: Papamichael
mesh_terms: D000222:Adaptation, Physiological / Q000187:drug effects; D055162:Biocatalysis / Q000187:drug effects; D001709:Biotechnology; D002412:Cations; D004492:Edetic Acid / Q000494:pharmacology; D005110:Extracellular Space / Q000187:drug effects / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007554:Isotopes; D007700:Kinetics; D007716:Kluyveromyces / Q000187:drug effects / Q000201:enzymology*; D008670:Metals / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000302:isolation & purification* / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D011522:Protons; D012997:Solvents / Q000494:pharmacology; D013379:Substrate Specificity / Q000187:drug effects; D013501:Surface-Active Agents / Q000494:pharmacology; D013696:Temperature
chemical_list: D002412:Cations; D007554:Isotopes; D008670:Metals; D011480:Protease Inhibitors; D011522:Protons; D012997:Solvents; D013501:Surface-Active Agents; D004492:Edetic Acid; D010447:Peptide Hydrolases