Biotechnological applications of proteins and enzymes are often hampered by their low stability to heat, pH, organic solvents, and proteolysis. With the aid of protein engineering, however, many attempts are being made to improve the operational stability of current commercial enzymes, and, in a more general sense, to establish guidelines for improving the thermostability of proteins and enzymes (Mozhaev et al. 1988).

This paper reports the coenzymatic properties of low molecular weight functionalized N(1)- and N 6 -(2-aminoethyl)-NAD(P), and macromolecular polyethylene glycol (M r 20,000)-N(1)-(2-aminoethyl)-NAD(P) and −N 6 -(2-aminoethyl)-NAD(P) in aqueous as well as in water-organic reaction media for the thermostable NAD(P)-dependent malic enzyme and NAD-dependent alcohol dehydrogenase from the archaebacterium Sulfolobus solfataricus. The enzymes recognize all of the derivatives tested, with V max values ranging from 30 to 250% of those for the native coenzymes. Remarkably, the malic enzyme shows higher (2.5 times) V max values for the N(1)-derivatives of NADP than for the native coenzyme. Most of the K m values were in the micromolar range which is suitable for practical use. On the basis of the k cat /K m ratios, the derivatives at the N 6 -position have the best coenzyme activity for both enzymes.

This paper describes the determination of stereospecificity of hydride transfer reaction of an alcohol dehydrogenase isolated from the archaebacterium Sulfolobus solfataricus. The 1H-NMR and EI-MS data indicate that the enzyme transfers the pro-R hydrogen from coenzyme to substrate and is therefore an A-specific dehydrogenase.

A thermophilic and thermostable β‐galactosidase activity was purified to homogeneity from crude extracts of the archaebacterium Sulfolobus solfataricus , by a procedure including ion‐exchange and affinity chromatography. The homogeneous enzyme had a specific activity of 116.4 units/mg at 75°C with o ‐nitrophenyl β‐galactopyranoside as substrate. Molecular mass studies demonstrated that the S. solfataricus β‐galactosidase was a tetramer of 240 ± 8 kDa composed of similar or identical subunits. Comparison of the amino acid composition of β‐galactosidase from S. solfataricus with that from Escherichia coli revealed a lower cysteine content and a lower Arg/Lys ratio in the thermophilic enzyme. A rabbit serum, raised against the homogeneous enzyme did not crossreact with β‐galactosidase from E. coli. The enzyme, characterized for its reaction requirements and kinetic properties, showed a thermostability and thermophilicity notably greater than those reported for β‐galactosidases from other mesophilic and thermophilic sources.

A DNA-dependent DNA polymerase was obtained in homogenous form from the thermoacidophilic archaebacterium Sulfolobus solfataricus. The enzyme, purified 706-fold, has a molecular mass of about 110000 daltons as determined by gel filtration and by glycerol gradient centrifugation. It requires Mg++ for its activity and has a pH optimum of 7.7. The activity is sharply dependent on the ionic strength. The enzyme is thermostable; its properties and activity requirements were characterized. The features of this enzyme are compared to those of other DNA polymerases isolated either from prokaryotes or eukaryotes.

An NADP-preferring malic enzyme ((S)-malate:NADP oxidoreductase (oxalacetate-decarboxylating) EC 1.1.1.40) with a specific activity of 36.6 units per mg of protein at 60 degrees C and an isoelectric point of 5.1 was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4. The purification procedure employed ion exchange chromatography, ammonium sulfate fractionation, affinity chromatography, and gel filtration. Molecular weight determinations demonstrated that the enzyme was a dimer of Mr 105,000 +/- 2,000 with apparently identical Mr 49,000 +/- 1,500 subunits. Amino acid composition of S. solfataricus enzyme was determined and found to be significantly higher in tryptophan content than the malic enzyme from Escherichia coli. In addition to the NAD(P)-dependent oxidative decarboxylation of L-malate, S. solfataricus malic enzyme was able to catalyze the decarboxylation of oxalacetate. The enzyme absolutely required divalent metal cations and it displayed maximal activity at 85 degrees C and pH 8.0 with a turnover number of 376 s-1. The enzyme showed classical saturation kinetics and no sigmoidicity was detected at different pH values and temperatures. At 60 degrees C and in the presence of 0.1 mM MnCl2, the Michaelis constants for malate, NADP, and NAD were 18, 3, and 250 microM, respectively. The S. solfataricus malic enzyme was shown to be very thermostable.

An NAD+-dependent alcohol dehydrogenase (alcohol: NAD+ oxidoreductase, EC 1.1.1.1) was detected in cellular extracts of the extreme thermophilic archaebacterium Sulfolobus solfataricus. The enzyme was purified to homogeneity and shown to be a dimer with a native molecular mass of 71 kDa by sucrose gradient centrifugation and SDS electrophoresis. The enzyme has a broad substrate specificity that includes linear and branched primary alcohols, linear and cyclic secondary alcohols, linear and cyclic ketones and anisaldehyde. The enzyme has an extraordinary thermophilicity and a remarkable thermostability, and appears to have some properties and a structure different from those previously described for thermophilic alcohol dehydrogenases.

A DNA-dependent DNA polymerase activity was purified to homogeneity from the archaebacterium Sulfolobus solfataricus, grown at 87°C and pH 3.5. This activity was the most abundant (80–85%) of two chromatographically distinguishable DNA polymerases. The enzyme purified about 1000-fold had a Mr of 210,000 ± 10,000 as determined by gel filtration. SDS gel electrophoresis revealed the presence of three peptides with a Mr of 116,000, 53,000 and 37,000, respectively, of which only the 116,000 subunit showed activity after elution from the gel and renaturation. However, by glycerol gradient centrifugation a Mr of 115,000 ± 5,000 was obtained. The DNA polymerase, assayed at 75°C and pH 6.8, required activated DNA and Mg++ or Mn++ for its activity and was thermophilic and thermostable. The temperature at which the activity was optimal depended on the type of DNA used as template-primer. It was concluded that the activity decreased at high temperature because of the melting of the template-primer, not as a result of enzyme inactivation. The DNA polymerase was also characterized with respect to its behaviour with inhibitors used to discriminate between enzymes isolated from prokaryotes or eukaryotes.

The 5-mercury derivative of dCMP is a substrate of deoxycytidylate aminohydrolase in the presence of mercaptoethanol. With this substrate a reversal of the effect of the allosteric ligands of the enzyme is observed. dCTP, which is an allosteric activator for aminohydrolysis of dCMP, becomes an inhibitor for the mercury substrate, whilst dTTP, an allosteric inhibitor for dCMP, becomes an activator for the mercury substrate.