Identification of a serine protease from a Bacillus sp. using multiple loading of O'Farrell-type isoelectric focusing slab two-dimensional gel.
A protease was purified from Bacillus sp. DJ isolated from Doenjang, a traditional Korean fermented food. Its molecular weight (MW) and isoelectric point (pI) were 18-19 kDa and 6.0-6.5 using 1- or 2-D fibrin zymography, respectively. The protease was optimally active at pH 9 and 55 degrees C. Activity was inhibited by 1 mM PMSF, but not by EDTA, EGTA, aprotinin, or leupeptin, indicating that the protease is a serine protease. By using a new electrophoretic technique, multiple loading of O'Farrell-type isoelectric focusing (IEF) slab gel, the first amino acid residues of the N-terminal sequence of the protease were determined as HPLVLVDPIL, which is 80% identical with serine proteases of the subtilase family.
Choi
Nack-Shick
NS
Enzyme Based Fusion Technology Research Team, Molecular Bioprocess Research Center, Korea Research Institute of Bioscience and Biotechnology, Jeonbuk, 580-185, Korea.
Choi
Jong Hyun
JH
Yoon
Jung-Hoon
JH
Lee
Seung-Goo
SG
Song
Jae Jun
JJ
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000302:isolation & purification / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005516:Food Microbiology; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D013696:Temperature
D004791:Enzyme Inhibitors; D012697:Serine Endopeptidases