Thermostable acid protease from sulfolobus acidocaldarius and ge

Details Thermostable acid protease from sulfolobus acidocaldarius and ge Thermostable acid protease from sulfolobus acidocaldarius and ge

1990-01-19

1992-12-22

Brown, Johnnie R.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

536 27, 935 6, 935 9, 435 23, C12N 1552, C12N 950, C12N 952

Patent

active

051734036

ABSTRACT:
A thermostable, very acidic protease, which has been named thermopsin, was purified to homogeneity from the culture medium of Sulfolobus acidocaldarius by a five-step procedure including column chromatographies on DEAE-Sepharose CL-6B, phenyl-Sepharose CL-4B, Sephadex G-100, MonoQ (FPLC), and gel filtration (HPLC). The enzyme is a single polypeptide chain having proteolytic activity over pH range 0 to 11 at temperatures between 0.degree. and 100.degree. C., with maximal activity at approximately pH 2 and 90.degree. C. Antibodies directed against thermopsin have been prepared. Through studies using various aspartic protease inhibitors, thiol and metalloprotease inhibitors, and serine protease inhibitors, it was determined that, although similar to some aspartic proteases, the active site of thermopsin is clearly not identical to that of other aspartic proteases. Thermopsin hydrolyzes the following bonds: Leu-Val, Leu-Tyr, Phe-Phe, Phe-Tyr, and Tyr-Thr, indicating that the specificity of thermopsin is similar to that of pepsin for the large hydrophobic residues at both sides of the scissile bond. In addition, thermospin is resistant to detergent inactivation, the protein retaining proteolytic activity even in the presence of high concentrations of sodium dodecyl sulfate.

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