Purification and Properties of a Highly Thermostable, Sodium Dodecyl Sulfate-Resistant and Stereospecific Proteinase from the Extremely Thermophilic Archaeon Thermococcus stetteri.

Applied and Environmental Microbiology
M KlingebergG Antranikian

Abstract

The cultivation of the extremely thermophilic archaeon Thermococcus stetteri in a dialysis membrane reactor was paralleled by the production of an extremely heat-stable proteinase(s). By applying preparative sodium 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 chain with a molecular mass of 68 kDa, showed a broad temperature and pH profile (50 to 100(deg)C; pH 5 to 11). The optimal activity with substantial thermal stability was measured with casein at 85(deg)C and pH 8.5 to 9. Inhibition by phenylmethylsulfonyl fluoride and diisopropylfluorophosphate demonstrated that the enzyme was a serine proteinase. The enzyme displayed a relatively narrow substrate specificity, catalyzing the hydrolysis only of N-protected p-nitroanilides or p-nitrophenyl 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, no hydrolysis of d-amino acid derivatives was observed. The catalytic efficiency of the enzyme at 80(deg)C (k(infcat)/K(infm) for benzoy...Continue Reading

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Citations

Mar 10, 2001·Microbiology and Molecular Biology Reviews : MMBR·C Vieille, G J Zeikus
Mar 4, 2014·Applied Biochemistry and Biotechnology·M Papagianni, D Sergelidis
Jun 1, 2015·Journal, Genetic Engineering & Biotechnology·Aysha KamranMustafa Kamal
Nov 4, 2008·Applied and Environmental Microbiology·Ilya V KublanovElizaveta A Bonch-Osmolovskaya
Jun 23, 2009·International Journal of Systematic and Evolutionary Microbiology·I V KublanovE A Bonch-Osmolovskaya
Nov 27, 2010·Journal of Industrial Microbiology & Biotechnology·Sreeja ChellappanM Chandrasekaran
Aug 3, 2010·Current Microbiology·Rafael UzarragaYannick Combet-Blanc
Jan 1, 1995·World Journal of Microbiology & Biotechnology·C Leuschner, G Antranikian
Dec 1, 2018·Emerging Topics in Life Sciences·Julie A Maupin-Furlow
Aug 1, 1996·Applied and Environmental Microbiology·A B Friedrich, G Antranikian
Nov 1, 2005·Current Opinion in Microbiology·Ksenia Egorova, Garabed Antranikian

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