PURIFICATION AND PROPERTIES OF A HIGHLY THERMOSTABLE, SODIUM DODECYL SULFATE-RESISTANT AND STEREOSPECIFIC PROTEINASE FROM THE EXTREMELY THERMOPHILIC ARCHAEON THERMOCOCCUS-STETTERI

The cultivation of the extremely thermophilic archaeon Thermococcus st etteri in a dialysis membrane reactor was paralleled by the production of an extremely heat-stable proteinase(s). By applying preparative so dium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, an SDS- resistant proteinase was purified 67-fold in one step with a yield of 34%. The purified enzyme, which was composed of a single polypeptide c hain with a molecular mass of 68 kDa, showed a broad temperature and p H profile (50 to 100 degrees C; pH 5 to 11), The optimal activity with substantial thermal stability was measured with casein at 85 degrees C and pH 8.5 to 9. Inhibition by phenylmethylsulfonyl fluoride and dii sopropylfluorophosphate demonstrated that the enzyme was a serine prot einase, The enzyme displayed a relatively narrow substrate specificity , catalyzing the hydrolysis only of N-protected p-nitroanilides or p-n itrophenyl esters of basic (Arg or Lys) or hydrophobic (Phe or Tyr) L- amino acids, L-Phenylglycine amide was also attacked by the proteinase , but with a lower specificity constant, Within the detection limit, n o hydrolysis of D-amino acid derivatives was observed, The catalytic e fficiency of the enzyme at 80 degrees C (k(cat)/K-m for benzoyl-Arg-p- nitroanilide, 10(4)) is the same order of magnitude when compared with that of functionally similar mesophilic enzymes. The proteinase also acts as a transferase, catalyzing the acyl transfer from protected ami no acid ester or amide to amino acid amide, The observed thermostabili ty, SDS resistance, relatively narrow substrate specificity, high ster eospecificity, and limited catalytic efficiency probably reflect the t ighter packing of the thermostable protein molecule and its limited fl exibility, This was supported by fluorescence spectra of the enzyme, m ainly due to tryptophan residues, in the temperature range of 30 to 90 degrees C. Structural reorganization was observed at temperatures ove r 100 degrees C, The results obtained could be of relevance for the be tter understanding of the structure-function relationship of enzymes f rom extreme thermophiles and suggest possible biotechnological applica tion of the proteinase for resolution of racemic mixtures.