Biochemical and genetic characterization of arazyme, an extracellular metalloprotease produced from Serratia proteamaculans HY-3.
Serratia proteamaculans HY-3 isolated from the digestive tract of a spider produces an extracellular protease named arazyme, with an estimated molecular mass of 51.5 kDa. The purified enzyme was characterized as having high activities at wide pH and temperature ranges. We further characterized biochemical features of the enzymatic reactions under various reaction conditions. The protease efficiently hydrolyzed a broad range of protein substrates including albumin, keratin, and collagen. The dependence of enzymatic activities on the presence of metal ions such as calcium and zinc indicated that the enzyme is a metalloprotease, together with the previous observation that the proteolytic activity of the enzyme was not inhibited by aspartate, cysteine, or serine protease inhibitors, but strongly inhibited by 1,10-phenanthroline and EDTA. The araA gene encoding the exoprotease was isolated as a 5.6 kb BamHl fragment after PCR amplification using degenerate primers and subsequent Southern hybridization. The nucleotide sequence revealed that the deduced amino acid sequences shared extensive similarity with those of the serralysin family of metalloproteases from other enteric bacteria. A gene (inh) encoding a putative protease inhibitor was also identified immediately adjacent to the araA structural gene.
Insect Resources Research Center Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Korea Insect BioTech Co., Ltd., Daejeon 305-811, Korea.
Jangyul
J
Kwak
Kieun
K
Lee
Dong-Ha
DH
Shin
Jin-Soo
JS
Maeng
Doo-Sang
DS
Park
Hyun Woo
HW
Oh
Kwang-Hee
KH
Son
Kyung-Sook
KS
Bae
Ho-Yong
HY
Park
D000418:Albumins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D003067:Coenzymes / Q000494:pharmacology; D003094:Collagen / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004492:Edetic Acid / Q000494:pharmacology; D004795:Enzyme Stability; D041981:Gastrointestinal Tract / Q000382:microbiology; D006863:Hydrogen-Ion Concentration; D007633:Keratins / Q000378:metabolism; D045726:Metalloproteases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010618:Phenanthrolines / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012705:Serratia / Q000201:enzymology* / Q000302:isolation & purification; D013112:Spiders / Q000382:microbiology; D013379:Substrate Specificity; D013696:Temperature
D000418:Albumins; D002413:Cations, Divalent; D003067:Coenzymes; D004269:DNA, Bacterial; D008670:Metals; D010618:Phenanthrolines; D011480:Protease Inhibitors; D007633:Keratins; D003094:Collagen; D004492:Edetic Acid; D045726:Metalloproteases; C025205:1,10-phenanthroline