A thermophilic and thermostable P-galactosidase activity was purified to homogeneity from crude extracts of the archaebacterium Suljiulobus sovuturicus, 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 P-galactopyranoside as substrate. Molecular mass studies demonstrated that the S. solfataricus P-galactosidase was a tetramer of 240 Ifr S kDa composed of similar or identical subunits. Comparison of the amino acid composition of pgalactosidase from S. solfataricus with that from Eschevichia culi 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 P-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 P-galactosidases from other mesopbilic and thermophilic sources. More recently, a new P-galactosidase activity has been identified in E. colicells with LacZ deletion selected for growth on lactose [6]. This enzyme, termed ebg for evolved pgalactosidases, has a subunit molecular mass of 120 kDa, very close to that of the LacZ enzyme. However, the ebg protein has a hexameric and not a tetrameric structure, and the two enzymes are not immunologically related.In recent years, p-galactosidase activities from various microbial sources have been purified and characterized for their physicochemical properties, reaction requirements and substrate specificities [l, 71. Thermostable p-galactosidases [S ~ 1 I] have received considerable attention because of their possible utilization in the industrial processing of lactose-contain-4,51.

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The NAD(+)-dependent alcohol dehydrogenase (EC 1.1.1.1) from the thermoacidophilic archaebacterium Sulfolobus solfataricus, DSM1617 strain (SSADH), has been purified and characterized. Its gene has been isolated by screening two S. Solfataricus genomic libraries using oligonucleotide probes. The encoding sequence consists of 1041 base pairs, and it shows a high preference for codons ending in T or A. The primary structure, determined by peptide and gene analysis, consists of 347 amino acid residues, yielding a molecular weight of 37,588. A level of identity of 24-25% was found with the amino acid sequences of horse liver, yeast, and Thermoanaerobium brockii alcohol dehydrogenases. The coenzyme-binding and catalytic and structural zinc-binding residues typical of eukaryotic alcohol dehydrogenases were found in SSADH with the difference that one out of the four structural zinc-binding Cys residues is substituted by Glu. The protein contains four zinc atoms per dimer, two of which are removed by chelating agents with a concomitant loss of structural stability.

The gene encoding a novel alcohol dehydrogenase (ADH) that belongs to the short-chain dehydrogenase/ reductase (SDR) superfamily was identified in the extremely thermophilic, halotolerant gram-negative eubacterium Thermus thermophilus HB27. The T. thermophilus ADH gene (adh Tt ) was heterologously overexpressed in Escherichia coli, and the protein (ADH Tt ) was purified to homogeneity and characterized. ADH Tt is a tetrameric enzyme consisting of identical 26,961-Da subunits composed of 256 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to ϳ73°C and a 30-min half-inactivation temperature of ϳ90°C, as well as good tolerance to common organic solvents. ADH Tt has a strict requirement for NAD(H) as the coenzyme, a preference for reduction of aromatic ketones and ␣-keto esters, and poor activity on aromatic alcohols and aldehydes. This thermophilic enzyme catalyzes the following reactions with Prelog specificity: the reduction of acetophenone, 2,2,2-trifluoroacetophenone, ␣-tetralone, and ␣-methyl and ␣-ethyl benzoylformates to (S)-(؊)-1-phenylethanol (>99% enantiomeric excess [ee]), (R)-␣-(trifluoromethyl)benzyl alcohol (93% ee), (S)-␣-tetralol (>99% ee), methyl (R)-(؊)-mandelate (92% ee), and ethyl (R)-(؊)-mandelate (95% ee), respectively, by way of an efficient in situ NADH-recycling system involving 2-propanol and a second thermophilic ADH. This study further supports the critical role of the D37 residue in discriminating NAD(H) from NADP(H) in members of the SDR superfamily.Alcohol dehydrogenases (ADHs) are of interest for the synthesis of the (S) or (R) enantiomers of alcohols from prochiral ketones (11). Since their early application in asymmetric synthesis using horse liver and yeast ADHs (13) and Thermoanaerobacter brockii ADH (ADH Tb ) (15), screening efforts have been directed at various species of microorganisms, which has resulted in new ADHs that have distinctive substrate specificity, good efficiency, and high enantioselectivity. Representative examples of enzymes from mesophilic microorganisms are the NADP-dependent (R)-specific ADH from Lactobacillus brevis (RADH Lb ), which is active on aryl ketones and whose crystal structure has recently been solved (29), the NAD-dependent (S)-specific 1-phenylethanol dehydrogenase from the denitrifying bacterium strain EbN1 (PED), which was characterized and crystallized (10), and the NAD-dependent ADH from Leifsonia sp. strain S749 (ADH Ls ), which was found to be active on (R)-sec alcohols, aryl ketones, aldehydes, and keto esters and whose gene has recently been cloned for protein expression in Escherichia coli (12). These enzymes are homotetrameric and belong to the short-chain dehydrogenase/ reductase (SDR) superfamily (14), which is characterized by ϳ250-residue subunits, a Gly motif in the coenzyme-binding regions, and a catalytic triad formed by the highly conserved residues Tyr, Lys, and Ser, to which an Asn residue has been added based on the proposal of Filling et al. (2), which was...

An NAD+-dependent alcohol dehydrogenase (alcohol: NAD' oxidoreductase, EC 1.1 .l .l) 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.Alcohol dehydrogenase is widely distributed in nature and has been found in many microorganisms, plant and animal tissues [l]. In addition to being present in multiple forms [2], alcohol dehydrogenases isolated from different sources show different substrate specificities. For example, the yeast enzyme can catalyse oxidation of a very limited range of acyclic primary and secondary alcohols [3], while horse liver alcohol dehydrogenase exhibits a broader specificity and catalyses even the oxidation of cyclic, aromatic and steroid alcohols [4].The potential biotechnological applications of these enzymes in stereospecific organic synthesis and in the production of high-added-value products [5 -91 has been limited by several factors and so investigation has continued in search of more stable enzymes with a broader specificity.The interest in enzymes from thermophilic and extreme thermophilic bacteria is justified by the fact that many enzymes isolated from these organisms are thermostable and capable of acting at high temperatures. On the other hand, their catalytic activity is low at moderate temperatures at which conventional enzymes are optimally active. Besides, these enzymes have been found to be not only stable to heat but also to common protein denaturants and organic solvents [lo, 111. An NADP +-dependent alcohol dehydrogenase, thermostable and exhibiting a high tolerance towards organic solvents, was isolated from the extreme thermophilic bacterium Thermoanaerobium brockii by Lamed and Zeikus 1121.In this paper we report the purification to homogeneity and the characterization of an NAD+-dependent alcohol deCorrespondence to M. Rossi,

A carboxypeptidase was purified to electrophoretic homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus. Molecular masses assessed by SDS/PAGE and gel filtration were 42 kDa and 170 kDa, respectively, which points to a tetrameric structure for the molecule. An isoelectric point of 5.9 was also determined. The enzyme was proven to be a metalloprotease, as shown by the inhibitory effects exerted by EDTA and o-phenanthroline; furthermore, dialysis against EDTA led to a complete loss of activity, which could be restored by addition of Zn2+ in the micromolar range, and, to a lesser extent, by Co2+. The enzyme was endowed with a broad substrate specificity, as shown by its ability to release basic, acidic and aromatic amino acids from the respective benzoylglycylated and benzyloxycarbonylated amino acids. An esterase activity of the carboxypeptidase was also demonstrated on different esterified amino acids and dipeptides blocked at the N-terminus. The enzyme displayed broad pH optima ranging over 5.5-7.0, or 5.5-9.0, when using an acidic or a basic benzyloxycarbonylated amino acid, respectively. With regard to thermostability, it was proven to be completely stable on incubation for 15 min at 85°C. Furthermore, thanks to its relatively low activation energy, i.e. 31.0 kJ/mol, it was still significantly active at room temperature. At 40°C, the enzyme could withstand 0.1% SDS and different organic solvents: particularly ethanol up to 99%. Amino acid and N-terminal sequence analyses did not evidence any similarity to carboxypeptidases A nor thermolysin. A weak similarity was only found with bovine carboxypeptidase B.

The crystal structure of a ternary complex of the alcohol dehydrogenase from the archaeon Sulfolobus solfataricus (SsADH) has been determined at 2.3 A. The asymmetric unit contains a dimer with a NADH and a 2-ethoxyethanol molecule bound to each subunit. The comparison with the apo structure of the enzyme reveals that this medium chain ADH undergoes a substantial conformational change in the apo-holo transition, accompanied by loop movements at the domain interface. The extent of domain closure is similar to that observed for the classical horse liver ADH, although some differences are found which can be related to the different oligomeric states of the enzymes. Compared to its apo form, the SsADH ternary complex shows a change in the ligation state of the active site zinc ion which is no longer bound to Glu69, providing additional evidence of the dynamic role played by the conserved glutamate residue in ADHs. In addition, the structure presented here allows the identification of the substrate site and hence of the residues that are important in the binding of both the substrate and the coenzyme.

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