Thermophilic enzymes have advantages for their use in commercial applications and particularly for the production of chiral compounds to produce optically pure pharmaceuticals. They can be used as biocatalysts in the application of ‘green chemistry’. The thermophilic archaea contain enzymes that have already been used in commercial applications such as the L-aminoacylase from Thermococcus litoralis for the resolution of amino acids and amino acid analogues. This enzyme differs from bacterial L-aminoacylases and has similarities to carboxypeptidases from other archaeal species. An amidase/γ-lactamase from Sulfolobus solfataricus has been used for the production of optically pure γ-lactam, the building block for antiviral carbocyclic nucleotides. This enzyme has similarities to the bacterial signature amidase family. An alcohol dehydrogenase from Aeropyrum pernix has been used for the production of optically pure alcohols and is related to the zinc-containing eukaryotic alcohol dehydrogenases. A transaminase and a dehalogenase from Sulfolobus species have also been studied. The archaeal transaminase is found in a pathway for serine synthesis which is found only in eukaryotes and not in bacteria. It can be used for the asymmetric synthesis of homochiral amines of high enantioselective purity. The L-2-haloacid dehalogenase has applications both in biocatalysis and in bioremediation. All of these enzymes have increased thermostability over their mesophilic counterparts.

Introduction

The use of enzymes in industrial bioprocesses is becoming increasingly important. This is to some extent driven by the pharmaceutical sector for the development of new therapeutic agents that are required to be enantiomerically pure compounds. Drug molecules often have several chiral centres and are difficult to synthesize by conventional chemistry. Their production relies increasingly on individual enzymatic steps which can be combined with chemical steps to make a more rapid and cost-effective process. In addition, when using enzymes, the chemistry is carried out in an environmentally friendly way using less harmful chemicals that need to be disposed of after use. The use of a biocatalytic step shows high performance under mild conditions minimizing problems of isomerization, racemization, epimerization and rearrangements reducing the amount of side products that occur during the process. Enzymes are able to carry out reactions using both regio- and stereo-selectivity. The number of chiral drug intermediates produced by enzymes is predicted to reach 70% by the end of 2010. In fact, the number of industrialized biotransformations has doubled every decade since 1960.

One disadvantage of using enzymes rather than more traditional processes of chemical synthesis is that proteins are often not stable under the reaction conditions required for the industrial process. One approach to overcome this problem is to search for new novel enzymes from extremophiles, especially thermophilic and hyperthermophilic organisms that have increased stability not only to high temperatures, but also to organic solvents and proteolytic enzymes. Although the rate of reaction is optimal for these enzymes at high temperatures, they usually have the ability to work at reduced rates at ambient temperature. There is a need to discover new activities from ‘Nature's catalysts’ that are required for the chemical reactions that industry demands. The enzymes from thermophilic archaea often have novel activities that are distinct from their bacterial counterparts.

Several thermophilic archaeal enzymes have already been developed for industrial biocatalysis. The enzymes of interest can be cloned by screening of genomic libraries or by using direct PCR amplification from sequenced genomes. They can be overexpressed in Escherichia coli in order to produce quantities that are required for biochemical characterization and structural analysis. It is also possible to scale-up production for industrial applications.

Cartoon representations of the crystal structures of (a) A. pernix tetrameric ADH (PDB code 1H2B), (b) S. tokodaii dimeric L-haloacid dehalogenase (PDB code 2W11) and (c) S. solfataricus dimeric serine transaminase (PDB code to be deposited)

Figure 1
Cartoon representations of the crystal structures of (a) A. pernix tetrameric ADH (PDB code 1H2B), (b) S. tokodaii dimeric L-haloacid dehalogenase (PDB code 2W11) and (c) S. solfataricus dimeric serine transaminase (PDB code to be deposited)

The Figures were produced using PyMOL (Delano Scientific; http://www.pymol.org).

Figure 1
Cartoon representations of the crystal structures of (a) A. pernix tetrameric ADH (PDB code 1H2B), (b) S. tokodaii dimeric L-haloacid dehalogenase (PDB code 2W11) and (c) S. solfataricus dimeric serine transaminase (PDB code to be deposited)

The Figures were produced using PyMOL (Delano Scientific; http://www.pymol.org).

Thermococcus litoralisL-aminoacylase

Many pharmaceutically active structures are nitrogen-containing compounds which can be derived from either L- or D-amino acids. There has been large growth in the area of unnatural amino acids.

A thermostable L-aminoacylase from T. litoralis has been cloned, sequenced and overexpressed in E. coli [1]. The aminoacylase gene was found upstream of a gene pcp coding for a novel cysteine protease pyroglutamyl carboxypeptidase [24]. The aminoacylase enzyme is a homotetramer of 43 kDa monomers, and has an 82% sequence identity with an aminoacylase from Pyrococcus horikoshii [5] and 45% sequence identity with a carboxypeptidase from Sulfolobus solfataricus [6]. It contains one cysteine residue that is highly conserved among aminoacylases. Cell-free extracts of the recombinant enzyme have been characterized and were found to have optimal activity at 85°C in Tris/HCl (pH 8.0). The recombinant enzyme is thermostable, with a half-life of 25 h at 70°C. Aminoacylase inhibitors, such as mono-t-butylmalonate, had only a slight effect on activity. The enzyme was most specific for substrates containing N-benzoyl- or N-chloroacetyl-amino acids, preferring substrates containing hydrophobic, uncharged or weakly charged amino acids such as phenylalanine, methionine and cysteine [1].

The thermostable archaeal L-aminoacylase from T. litoralis has been used in immobilization trials to optimize its application in industrial biotransformation reactions [7]. Immobilization techniques used included direct adsorption and cross-linking of the enzyme on to solid supports, bioencapsulation and covalent bonding on to a variety of activated matrices. The most successful immobilization methods were covalent binding of the enzyme on to glyoxyl-Sepharose and Amberlite XAD7. These methods yielded an average of 15 and 80 mg of protein bound per g of support (wet weight for glyoxyl-Sepharose) respectively, with nearly 80% activity recovery in both cases. Enzyme immobilized on to glyoxyl-agarose was stabilized 106-fold under aqueous conditions and 142-fold in 100% acetonitrile when activity was measured after 24 h at 90°C. A column bioreactor containing the recombinant L-aminoacylase immobilized on to Sepharose beads was constructed with the substrate N-acetyl-DL-tryptophan, continuously flowing at 60°C for 10 days. No loss of activity was detected over 5 days, with 32% activity remaining after 40 days at 60°C.

More recently the enzyme has been immobilized on to monoliths formed in microreactor channels [8]. The use of this microfluidic system is ideal for rapid substrate screening and overcomes any problems with substrate or product inhibition.

The recombinant T. litoralisL-aminoacylase has been purified to homogeneity. This zinc-containing enzyme has been crystallized in a form suitable for X-ray structural analysis. The crystals diffract to 2.8 Å (1 Å=0.1 nm) resolution and belong to the rhombohedral space group R32 with unit cell parameters a=b=102.4 Å, c=178.5 Å, γ=120°. The asymmetric unit contains one enzyme monomer. Two synchrotron datasets have been collected at remote and maximum f′ wavelengths. The single zinc ion position has been identified in both the anomalous and isomorphous difference Patterson maps [9]. The crystal structure of this enzyme has now been determined and will be published elsewhere.

S. solfataricus γ-lactamase

The resolution of the bicyclic synthon (racemic)–γ-lactam (2-azabicyclo[2.2.1]hept-5-en-3-one) is an important step in the synthesis of a group of chemotherapeutic agents known as carbocyclic nucleosides. The archaeon S. solfataricus MT4 produces a thermostable γ-lactamase that was cloned, sequenced and overexpressed in E. coli [10]. It has high sequence homology with the signature amidase family of enzymes. The enzyme was thermostable, with a half-life of 14 min at 80°C in the absence of the substrate. The γ-lactamase was found to selectively cleave the (+)-enantiomer of γ-lactam. It also exhibits general amidase activity by cleaving linear and branched aliphatic and aromatic amides. The enzyme catalyses the synthesis of benzoic hydrazide from benzamide preferentially to benzamide cleavage in the presence of excess hydrazine. This enzyme has potential for use in industrial biotransformations in the production of both carbocyclic nucleosides and hydrazides.

Alignment of the amino acid sequences of the γ-lactamase from S. solfataricus MT4 with four other amidases from Pseudomonas chlororaphis B23, Rhodococcus sp. N-771, Rhodococcus erythropolis N-774 and Rhodococcus rhodochrous J1 shows it has a 41–44% sequence identity. These amidases belong to the signature amidase family as they all contain the sequence GGSS(S/G)GS. This family of enzymes hydrolyse amide bonds other than peptide bonds producing carboxylic acid and ammonia. The amino acid sequence of γ-lactamase contains the highly conserved putative catalytic residues aspartate and serine, but not the highly conserved cysteine residue [11]

This archaeal γ-lactamase has been purified to homogeneity [12]. The molecular mass of the monomer was estimated to be 55 kDa by SDS/PAGE, which is consistent with the calculated molecular mass of 55.7 kDa. The native molecular mass was determined to be 110 kDa by gel filtration, indicating the enzyme exists as a dimer. The purified enzyme has been crystallized with a view to determining its three-dimensional structure.

The cross-linked polymerized enzyme has also recently been packed into microreactors [13]. The thermophilic (+)-γ-lactamase retained 100% of its initial activity at the assay temperature, 80°C, for 6 h and retained 52% activity after 10 h, indicating the advantage of immobilization. This high stability of the immobilized enzyme provided the advantage that it could be utilized to screen many compounds in the microreactor system.

Aeropyrum pernix ADH (alcohol dehydrogenase)

A. pernix is one of the most thermophilic aerobic archaeal species. The ADH enzyme was amplified by PCR and overexpressed in E. coli. The A. pernix ADH enzyme is a tetrameric zinc-containing type I ADH with a monomer size of 39.5 kDa [14,15]. The sequence identity with horse liver ADH is 24%. The highest identity with a known structure is 39% to a medium-chain ADH from the hyperthermophilic archaeon S. solfataricus [16]. The A. pernix enzyme is highly specific for the cofactor NAD(H), and displays activity towards a broad range of alcohols, aldehydes and ketones, while appearing to show a preference for cyclic substrates. The enzyme is very thermostable, with a half-life of 2 h at 90°C. The maximal activity is beyond 75°C; however, there is still 10% activity at 20°C. The enzyme is solvent-stable with over 50% activity retained after incubation, with 60% acetonitrile or dioxan. The enzyme is stabilized by an ion-pair cluster at the subunit interface and a disulfide bond formed at a second zinc-binding site in the enzyme. It has been predicted that disulfide bonds do exist to stabilize many cytoplasmic proteins from A. pernix [17].

The enzyme is active against primary and secondary alcohols with optimum chain length of C4–C5. It is most active with large cyclic alcohols such as cycloheptanol and cyclo-octanol.

Sulfolobus tokodaiiL-haloacid dehalogenase

L-Haloacid dehalogenase from S. tokodaii has been cloned and overexpressed in E. coli. It has been characterized biochemically and structurally [18,19]. The enzyme monomer has two domains. The core domain has a Rossmann fold with a six-stranded parallel β-strand bundle surrounded by five α-helices and three 310 helices. The subdomain is composed of α-helices. The active site is located between the two domains and the native enzyme forms a dimer.

This enzyme has applications for chiral halocarboxylic acid production and bioremediation. Chiral halocarboxylic acids are important intermediates in the fine chemical/pharmaceutical industries. Many drugs are halogenated, and this is difficult to achieve chemically and can be carried out by haloperoxidase enzymes. Removal of the halogen group can be carried out by a dehalogenase. The Sulfolobus enzyme has the potential to resolve racemic mixtures of bromocarboxylic acids. The L-bromoacid dehalogenase catalyses the conversion of 2-halocarboxylic acids into the corresponding hydroxyalkanoic acid. The S. tokodaii dehalogenase has been shown to display activity towards longer-chain substrates than the bacterial Xanthomonas autotrophicus dehalogenase with activity seen towards 2-chlorobutyric acid which is due to a more accessible active site. The enzyme has a maximum activity at 60°C and a half-life of over 1 h at 70°C. It is stabilized by salt bridge and hydrophobic interactions on the subunit interface, helix capping, a more compact subdomain than related enzymes and shortening of surface loops. Other thermophilic enzymes of this family have addressed the problem of thermostability in different ways. Pyrococcus dehalogenase (29% sequence identity) solved from a structural genomics project is a monomeric structure stabilized by a disulfide bond [20].

S. solfataricus serine transaminase

Serine transaminase from S. solfataricus is a pyridoxal phosphate-containing enzyme involved in the non-phosphorylated pathway for serine synthesis which is not found in bacteria and is found in animals and plants [21]. The transaminase reaction that the enzyme carries out is the conversion of L-serine and pyruvate into 3-hydroxypyruvate and alanine. Activity is also shown towards methionine, asparagine, glutamine, phenylalanine, histidine and tryptophan. The enzyme can be used in combination with transketolase for the synthesis of chiral intermediates [22]. The enzyme structure has been solved in the holo form of the enzyme and in complex with an inhibitor gabaculine and in a substrate complex with phenolpyruvate, the keto product of phenylalanine [23]. These studies have given some insight into the conformational changes around the active site of the enzyme that occur during catalysis and help to understand substrate specificity. The most related enzyme is the mesophilic yeast alanine:glyoxylate aminotransferase, which shares 37% amino acid identity [24]. The yeast enzyme has ten salt bridges compared with 21 salt bridges in the Sulfolobus serine transaminase enzyme which includes three or four amino acid networks. There is a C-terminal extension in the Sulfolobus enzyme and shorter surface loops. The Sulfolobus transaminase dimer interface is hydrophobic in nature with few salt bridges. The Sulfolobus serine transaminase is the first example of a thermophilic archaeal α-transaminase enzyme to be studied structurally and has implications for the commercial application of the enzyme for biotransformation reactions (C. Sayer, M.J.S. Bommer, J.M. Ward and J.A. Littlechild, unpublished work).

Conclusion

The present article gives some insight into the novelty and applications of thermophilic archaeal enzymes in the industrial environment. It concentrates on several specific enzymes studied by myself and colleagues at the Exeter Biocatalysis Centre.

Molecular Biology of Archaea II: A Biochemical Society Focused Meeting held at Robinson College, Cambridge, U.K., 16–18 August 2010. Organized and Edited by Stephen Bell (Oxford, U.K.) and Finn Werner (University College London, U.K.).

Abbreviations

     
  • ADH

    alcohol dehydrogenase

Funding

This work was supported by grants from the Biotechnology and Biological Sciences Research Council, Engineering and Physical Science Research Council, and the Technology Strategy Board. I am also thankful for industrial support from Chirotech and TMO Renewables.

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