Molecular Cloning of the Genes Coding for Hyperthermostable Serine Proteases from Hyperthermophilic Archaebacteria and Its Functional Expression in Bacillus subtilis
Kiyozo ASADA*, Hikaru TAKAKURA, Mio MORISHITA, Katsuhiko YAMAMOTO, Masanori MITTA, Susumu TSUNASAWA, and Ikunoshin KATO
Biotechnology Research Laboratories, Takara Shuzo Co., Ltd., 3-4-1 Seta, Otsu, Shiga, 520- 21, Japan
Hyperthermophilic bacteria are very useful source for highly thermostable enzymes, which are of great interest from the aspect of industrial application as well as a target for basic research. Most of the enzymes derived from the organisms can be expected to have temperature optimum at around 100℃ or even higher temperature. A gene coding for a large serine protease of Pyrococcus furiosus, named PFUL, has been cloned from a cosmid library by monitoring its proteolytic activity at 95℃ in the lysate of each clone (1). PFUL has been expressed in Bacillus subtilis and Escherichia coli. Its presence in the P. furiosus has been confirmed by other group (2). PFUL has sequences with significant homology to subtilisin-like serine proteases around the catalytic residues. Mature form of PFUL consists of 1249 amino acid residues containing several insertion sequences and presumed anchor sequence in the C-terminal half. Finding of a subtilisin-like serine protease in P. furiosus led us to speculate that other hyperthermophilic archaebacteria could have a subtilisin-like protease. When genomic DNA of Thermococcus celer was used as a template, subtilisin-like sequence was amplified by use of consensus primers designed at the region around the catalytic residues. The amplified sequences was in turn used as a probe to isolate whole protease gene. Deduced amino acid sequence of the protease of T. celer, named TCES, revealed that it consists of 659 amino acid sequence, which is much smaller than PFUL protease having no insertion sequence and no C-terminal anchor sequence. TCES gene was introduced in B. subtilis and functionally expressed. Finding of a much smaller subtilisin-like protease in T. celer suggested the presence of second subtilisin-like protease in P. furiosus which has neither insertion sequence nor anchor sequence. DNA sequence coding for the second, much smaller protease, named PFUS was finally isolated by using TCES gene as a probe. Nucleotide sequence analysis of the PFUS gene revealed an open reading frame consisting of 654 amino acid residues. PFUS was also functionally expressed in B. subtilis as TCES.
The purified PFUS showed a molecular weight of 45 kDa. PFUS was active from 40℃ to 110℃ and exhibited optimal activity at 80℃ to 95℃ (pH 7.0) and at pH
