Purification and characterization of a thermostable alkaline protease from Thermoactinomyces sp. E79 and the DNA sequence of the encoding gene.
A thermophilic Thermoactinomyces sp. E79 producing a highly thermostable alkaline protease was isolated from soil. The protease, produced extracellularly by Thermoactinomyces sp. E79, was purified by DEAE-Sepharose CL-6B and Butyl-Toyopearl 650M column chromatography. The relative molecular mass was estimated to be 31,000 by SDS-polyacrylamide gel electrophoresis. Enzyme activity was inhibited by phenylmethylsulfonyl fluoride, suggesting the enzyme to be a serine protease. The optimum temperature for the enzyme activity was 85 degrees C, and about 50% of the original activity remained after incubation at 90 degrees C for 10 min in the presence of Ca2+. The optimum pH for the enzyme activity was 11.0 and the enzyme was fairly stable from pH 5.0 to 12.0. The gene for this thermostable alkaline protease was cloned in Escherichia coli and the expressed intracellular enzyme was activated by heat treatment. Sequence analysis showed an open reading frame of 1,152 base pairs, coding for a polypeptide- of 384 amino acids. The polypeptide was composed of a signal sequence (25 amino acids), a prosequence (81 amino acids), and a mature protein of 278 amino acids. The deduced amino acid sequence of the mature protease had high similarity with thermitase, a serine protease from Thermoactinomyces vulgaris, and the extent of sequence identity was 76%.
Lee
J K
JK
Applied Microbiology Research Group, Korea Research Institute of Bioscience and Biotechnology, KIST, Taejon, Korea.
Kim
Y O
YO
Kim
H K
HK
Park
Y S
YS
Oh
T K
TK
D000595:Amino Acid Sequence; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D003902:Detergents / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D015698:Genomic Library; D006863:Hydrogen-Ion Concentration; D008848:Micromonosporaceae / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012984:Software; D012988:Soil Microbiology; D013696:Temperature
D004269:DNA, Bacterial; D003902:Detergents; D012697:Serine Endopeptidases; C032923:microbial serine proteinases