title: Organic solvent-tolerant elastase efficiently hydrolyzes insoluble, cross-linked, protein fiber of eggshell membranes.
abstract: Eggshell membrane is a mechanically stable and insoluble cross-linked fibrous protein. Pseudomonas aeruginosa strain ME-4 synthesizes a metalloprotease that degrades the eggshell membrane. We cloned the encoding gene in Escherichia coli. The recombinant protease, over-expressed in E. coli, was inactive but addition of acetone to crude cell extracts restored the activity and removed many E. coli proteins. We purified the active, acetone-treated protease to homogeneity in a single chromatography step with 57% recovery. The recombinant protease partially hydrolyzed eggshell membrane and produced more soluble peptides and proteins than commercial elastase, α-chymotrypsin, and collagenase. The soluble peptides produced from hydrolyzed eggshell membrane inhibited angiotensin-I-converting enzyme activity. The degradation of eggshell membrane by the recombinant elastase could be applied to the production of soluble bioactive peptides.
authors.affiliation: Department of Agrobioscience, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe 657-8501, Japan. hakko3@kobe-u.ac.jp
authors.forename: Shinji
authors.initials: S
authors.lastname: Takenaka
authors.forename: Shinpei
authors.initials: S
authors.lastname: Hano
authors.forename: Minyi
authors.initials: M
authors.lastname: Cheng
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Yoshida
authors.forename: Kenji
authors.initials: K
authors.lastname: Aoki
mesh_terms: D000096:Acetone / Q000494:pharmacology; D000818:Animals; D003001:Cloning, Molecular; D004527:Egg Proteins / Q000378:metabolism*; D004528:Egg Shell / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006868:Hydrolysis; D008566:Membranes / Q000737:chemistry; D010196:Pancreatic Elastase / Q000235:genetics / Q000378:metabolism*; D059748:Proteolysis; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012997:Solvents / Q000494:pharmacology
chemical_list: D004527:Egg Proteins; D011994:Recombinant Proteins; D012997:Solvents; D000096:Acetone; D010196:Pancreatic Elastase