title: Overexpression of a bacterial chymotrypsin: application for L-amino acid ester hydrolysis.
abstract: In this work, a reliable protocol was designed to rapidly express and purify a microbial chymotrypsin(ogen) as a useful alternative to using animal proteases. The cDNA encoding for chymotrypsinogen from the deuteromycete Metarhizium anisopliae (chy1) was overexpressed in an Origami2(DE3) E. coli strain deficient in thioredoxin reductase and glutathione reductase activities, thus possibly allowing disulfide exchange. By using a quick purification protocol, in which the hexahistidine tag was added at the C-terminal end of the protease, the recombinant CHY1 protein could be purified in a single step on an Ni-NTA column as a mixture of 19.5- and 15-kDa mature active forms and did not require further activation/maturation steps. This expression and purification procedure offers an easier and faster means of producing recombinant CHY1 chymotrypsin than that previously described for Pichia pastoris. The kinetic properties could be characterized and CHY1 chymotrypsin was demonstrated to efficiently catalyze N-acetylated L-phenylalanine and L-tyrosine methyl ester hydrolysis.
authors.affiliation: Dipartimento di Biotecnologie e Scienze Molecolari, Università degli Studi dell'Insubria, Varese, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Volontè
authors.forename: Ines
authors.initials: I
authors.lastname: Pisanelli
authors.forename: Paola
authors.initials: P
authors.lastname: D'Arrigo
authors.forename: Fiorenza
authors.initials: F
authors.lastname: Viani
authors.forename: Gianluca
authors.initials: G
authors.lastname: Molla
authors.forename: Stefano
authors.initials: S
authors.lastname: Servi
authors.forename: Loredano
authors.initials: L
authors.lastname: Pollegioni
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D002918:Chymotrypsin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004271:DNA, Fungal / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D004951:Esterification; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D052981:Metarhizium / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013237:Stereoisomerism
chemical_list: D000596:Amino Acids; D004271:DNA, Fungal; D005656:Fungal Proteins; D011994:Recombinant Proteins; D002918:Chymotrypsin