A novel subtilisin-like serine protease from Thermoanaerobacter yonseiensis KB-1: its cloning, expression, and biochemical properties.
A gene, tayI, encoding a novel subtilisin-like protease, designated thermicin, from the extremely thermophilic bacterium Thermoanaerobacter yonseiensis KB-1 (DSM 13777) was cloned by using a sequence tag containing the consensus sequence of proteases. The gene consisted of 1,239 nucleotides, and the deduced amino acid sequence indicated that it is a preproenzyme with a 311-residue mature protein composed of canonical catalytic residues (Asp29, His64, and Ser252). Thermicin was overproduced in E. coli as a fusion protein with a histidine tag and purified by nickel nitrilotriacetic acid affinity chromatography. Thermicin from E. coli showed maximum proteolytic activity at 92.5 degrees C and pH 9.0, and its half-life was 30 h at 80 degrees C. In order to determine cleavage specificity, thermicin was incubated with insulin beta chain, and the resulting peptides were analyzed by matrix assisted laser desorption/ionization-time of flight mass spectrometry. The carboxyl group side of the Val12, Leu15,17, Gly23, and Pro28 residues was cleaved. Thermicin is well known to hydrolyze Gly- and Pro-rich collagens. The K (m) and k (cat)/ K (m) values of thermicin for the hydrolysis of the synthetic substrate L-Gly-Pro- p-nitroaniline were 54.16 microM and 142.05 (10(5) s(-1) M(-1)), respectively, at 92.5 degrees C and pH 9.0. Amino acid sequence comparison and phylogenetic analysis indicated that this enzyme belongs to a new subgroup with respect to its molecular evolution, when compared with previously characterized subtilisins. This result indicates that thermicin is a novel enzyme different from other thermostable proteases.
Jang
Hyeung Jin
HJ
Program in Biomaterial Science and Engineering, Bioproducts Research Center, Department of Biochemistry, College of Science, Yonsei University, Seoul 120-749, Korea.
Kim
Byoung Chan
BC
Pyun
Yu Ryang
YR
Kim
Yu Sam
YS
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003094:Collagen / Q000378:metabolism; D004269:DNA, Bacterial; D005353:Fibronectins / Q000378:metabolism; D006094:Gram-Positive Bacteria / Q000201:enzymology*; D008969:Molecular Sequence Data; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity
D004269:DNA, Bacterial; D005353:Fibronectins; D003094:Collagen; D012697:Serine Endopeptidases