title: Purification, molecular cloning, and biochemical characterization of subtilisin JB1 from a newly isolated Bacillus subtilis JB1.
abstract: An extracellular gelatinolytic enzyme obtained from the newly isolated Bacillus subtilis JB1, a thermophilic microorganism relevant to the aerobic biodegradation process of fish-meal production, was purified via ammonium sulfate precipitation, Sephadex G-200 Gel filtration chromatography, and one-dimensional gel electrophoresis separation and subsequently identified via peptide mass fingerprinting and chemically assisted fragmentation matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The subtilisin JB1 gene was sequenced and its recombinant protein prosubtilisin JB1 was expressed in Escherichia coli, and the purified prosubtilisin JB1 (62 kDa) protein was digested with gelatin, bovine serum albumin, azocasein, fibrinogen, and the fluorogenic peptide substrate Ala-Ala-Phe-7-amido-4-methylcoumarin hydrochloride, whereas the serine protease inhibitors phenylmethylsulfonyl fluoride and chymostatin completely inhibited its enzyme activity at an optimal pH of 7.5. Thus, our results show that subtilisin JB1 may serve as a potential source material for use in industrial applications of proteolytic enzymes and microorganisms for fishery waste degradation and fish by-product processing.
authors.affiliation: Department of Biotechnology, Pukyong National University, Busan 608-737, South Korea.
authors.forename: Ji Hea
authors.initials: JH
authors.lastname: Sung
authors.forename: Sang Jung
authors.initials: SJ
authors.lastname: Ahn
authors.forename: Na Young
authors.initials: NY
authors.lastname: Kim
authors.forename: Soo-Kyoung
authors.initials: SK
authors.lastname: Jeong
authors.forename: Joong Kyun
authors.initials: JK
authors.lastname: Kim
authors.forename: Joon Ki
authors.initials: JK
authors.lastname: Chung
authors.forename: Hyung Ho
authors.initials: HH
authors.lastname: Lee
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020860:Subtilisin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D020860:Subtilisin