1887

Abstract

Summary: The extreme thermophile sp. strain Rt41A produces an extracellular alkaline serine proteinase during growth. This enzyme is stable for more than 24 h at 70C and has a pH optimum of 8-0. The proteinase gene was identified using primers designed to amplify a region between two highly conserved amino acid motifs in subtilisin-like proteinases and the PCR product was used to identify a genomic fragment containing the gene. The amino acid sequence deduced from the Rt41A gene contained a region identical to that obtained by amino-terminal sequencing of purified Rt41A proteinase. Comparison of the entire derived peptide sequence with other subtilisin-like serine proteinases revealed significant homologies, especially with aqualysin I from YT-1 and with exoprotease A from . The Rt41A proteinase was expressed in as a fusion protein with glutathione-S-transferase as an aid for purification and to overcome difficulties experienced with other plasmid vectors which produced inactive protein. The enzyme is inactive as synthesized and activation was shown to be temperature-dependent, with shorter incubation times required at higher temperatures; removal of the hydrophobic signal peptide from the start of the gene reduced the time required for activation to less than a third of that required if the signal peptide was present.

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1995-07-01
2021-08-05
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