title: Expression and characterisation of chymosin pH optima mutants produced in Trichoderma reesei.
abstract: The production of chymosin mutants designed to have altered pH optima using the cellulolytic filamentous fungus Trichoderma reesei is described. The strong promoter of the gene encoding the major cellulase, cellobiohydrolase I (CBHI) has been used for the expression and secretion of active calf chymosin. Structural analysis of the hydrogen bonding network around the two active site aspartates 32 and 215 in chymosin have suggested that residues Thr 218 and Asp 303 may influence the rate and pH optima for catalysis. The chymosin mutants Thr218Ala and the double mutant Thr218Ala/Asp303Ala have been made by site-directed mutagenesis and expressed in T. reesei. Enzyme kinetics of the active enzyme T218A indicate a pH optimum of 4.2 compared to 3.8 for native chymosin B using a synthetic octa-peptide substrate, confirming the previous analysis undertaken in E. coli. The double mutant T218A/D303A exhibits a similar optimum of 4.4 to that reported for the D303A, indicating that the combination of these changes is not additive. The application of protein engineering in the rational design of specific modifications to tailor the properties of enzymes offers a new approach to the development of industrial processes.
authors.affiliation: Department of Crystallography, Birkbeck College, London, UK.
authors.forename: J E
authors.initials: JE
authors.lastname: Pitts
authors.forename: J M
authors.initials: JM
authors.lastname: Uusitalo
authors.forename: D
authors.initials: D
authors.lastname: Mantafounis
authors.forename: P G
authors.initials: PG
authors.lastname: Nugent
authors.forename: D D
authors.initials: DD
authors.lastname: Quinn
authors.forename: P
authors.initials: P
authors.lastname: Orprayoon
authors.forename: M E
authors.initials: ME
authors.lastname: Penttilä
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D012085:Chymosin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003460:Crystallization; D004247:DNA; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D014242:Trichoderma / Q000201:enzymology / Q000235:genetics*
chemical_list: D004247:DNA; D012085:Chymosin