title: Isolation of a protease-deficient mutant of Bacillus brevis and efficient secretion of a fungal protein disulfide isomerase by the mutant.
abstract: The efficient production of a thermostable protein disulfide isomerase (PDI) was successfully achieved using the newly isolated protease-deficient mutant, Bacillus brevis 31-OK. Extracellular protease (exoprotease) activity was about a quarter of that in the parent, and the mutant was deficient in at least one of the major exoproteases. The cDNA encoding the fungal PDI was inserted downstream of the signal peptide-encoding region in an expression-secretion vector for B. brevis. Efficient production of PDI was feasible using B. brevis 31-OK as a host and modified signal sequences composed of three leucine residues inserted in the hydrophobic region of the MWP (middle wall protein) signal sequence. The maximal secretion of PDI into the culture medium was 1.1 g/l, which is about twice that by the parent strain and fifty times greater than the amount of rat and murine PDIs produced by Escherichia coli. The enzymatic properties such as the specific activity and thermal stability of the recombinant PDI are similar to those of natural PDI derived from Humicola insolens mycelia. B. brevis 31-OK was able to maintain its exoprotease activity at a low level throughout the cultivation and is considered to be useful host for production of a protease-sensitive protein and for increase of protein productivity due to stable accumulation.
authors.affiliation: Toyota Central Research & Development Labs., Inc., Nagakute, Aichi 480-1192 Japan.
authors.forename: T
authors.initials: T
authors.lastname: Kajino
authors.forename: K
authors.initials: K
authors.lastname: Kato
authors.forename: C
authors.initials: C
authors.lastname: Miyazaki
authors.forename: O
authors.initials: O
authors.lastname: Asami
authors.forename: M
authors.initials: M
authors.lastname: Hirai
authors.forename: Y
authors.initials: Y
authors.lastname: Yamada
authors.forename: S
authors.initials: S
authors.lastname: Udaka