Archaeal Enzymes and Applications in Industrial Biocatalysts

Archaeal enzymes are playing an important role in industrial biotechnology. Many representatives of organisms living in “extreme” conditions, the so-called Extremophiles, belong to the archaeal kingdom of life. This paper will review studies carried by the Exeter group and others regarding archaeal enzymes that have important applications in commercial biocatalysis. Some of these biocatalysts are already being used in large scale industrial processes for the production of optically pure drug intermediates and amino acids and their analogues. Other enzymes have been characterised at laboratory scale regarding their substrate specificity and properties for potential industrial application. The increasing availability of DNA sequences from new archaeal species and metagenomes will provide a continuing resource to identify new enzymes of commercial interest using both bioinformatics and screening approaches.University of ExeterWellcome TrustBBSRCEPSRCTechnology Strategy Board, UKEU Framework

Improving the 'tool box' for robust industrial enzymes.

This is the final version of the article. Available from Springer Verlag via the DOI in this record.The speed of sequencing of microbial genomes and metagenomes is providing an ever increasing resource for the identification of new robust biocatalysts with industrial applications for many different aspects of industrial biotechnology. Using 'natures catalysts' provides a sustainable approach to chemical synthesis of fine chemicals, general chemicals such as surfactants and new consumer-based materials such as biodegradable plastics. This provides a sustainable and 'green chemistry' route to chemical synthesis which generates no toxic waste and is environmentally friendly. In addition, enzymes can play important roles in other applications such as carbon dioxide capture, breakdown of food and other waste streams to provide a route to the concept of a 'circular economy' where nothing is wasted. The use of improved bioinformatic approaches and the development of new rapid enzyme activity screening methodology can provide an endless resource for new robust industrial biocatalysts.This mini-review will discuss several recent case studies where industrial enzymes of 'high priority' have been identified and characterised. It will highlight specific hydrolase enzymes and recent case studies which have been carried out within our group in Exeter.The author would like to thank the University of Exeter, the Wellcome Trust, the BBSRC, EPSRC and Technology Strategy Board, UK, for sponsoring research in the JAL group at the Exeter Biocatalysis Centre. The BBSRC funded ERA-IB grant BB/L002035/1 and the University of Exeter are thanked for support. Thanks are also given to the EU Framework 7 grant ‘HOTZYME’ entitled Systematic Screening of Organisms from Hot Environments, Grant Number 265933

Enzymes from Extreme Environments and Their Industrial Applications.

Published onlineJournal ArticleReviewThis article will discuss the importance of specific extremophilic enzymes for applications in industrial biotechnology. It will specifically address those enzymes that have applications in the area of biocatalysis. Such enzymes now play an important role in catalyzing a variety of chemical conversions that were previously carried out by traditional chemistry. The biocatalytic process is carried out under mild conditions and with greater specificity. The enzyme process does not result in the toxic waste that is usually produced in a chemical process that would require careful disposal. In this sense, the biocatalytic process is referred to as carrying out "green chemistry" which is considered to be environmentally friendly. Some of the extremophilic enzymes to be discussed have already been developed for industrial processes such as an l-aminoacylase and a γ-lactamase. The industrial applications of other extremophilic enzymes, including transaminases, carbonic anhydrases, dehalogenases, specific esterases, and epoxide hydrolases, are currently being assessed. Specific examples of these industrially important enzymes that have been studied in the authors group will be presented in this review.The author would like to thank the University of Exeter, the Wellcome Trust, the BBSRC, the EPSRC, and the Technology Strategy Board, UK, for sponsoring research in the JL Laboratories at the Exeter Biocatalysis Centre. The EU Framework 7 grant “HotZyme” entitled Systematic Screening of Organisms from Hot Environments, Grant Number 265933, is thanked for supporting studies on the isolation of the metagenomes from Russia and China where the novel thermophilic limonene epoxide hydrolases were isolated and for the identification of the first thermophilic Planctomycetes species where the novel thermophilic esterase was identified. This EU project sponsored the research to clone, overexpress, and biochemically and structurally characterize these three new enzymes to allow their potential commercial exploitation. JL would like to thank all of the collaborators, postdoctoral fellows, and students who have contributed to the research work covered in this paper

Crystallization and preliminary x-ray diffraction studies of a novel bacterial esterase.

Journal ArticleResearch Support, Non-U.S. Gov'tA novel bacterial esterase has been crystallized in two forms suitable for X-ray diffraction studies. Crystals have been obtained by vapour-phase diffusion at 290 K using ammonium sulfate as precipitant. The first crystals grew in space group C2 with unit-cell parameters a = 134.7, b = 55.8, c = 110.3 A, beta = 125.1 degrees. A monoclinic data set has been collected to 2.0 A resolution. Microseeding yielded a second crystal form which grew in space group P212121 with unit-cell parameters a = 57.1, b = 115.4, c = 130.4 A. Native data from these crystals have been collected to 1.6 A resolution. A molecular envelope has been determined using an uranyl acetate derivative for phase calculation.BBSRCSmithKline Beecham PharmaceuticalsEuropean Unio

Crystallization and preliminary X-ray diffraction studies of pyrrolidone carboxyl peptidase from the hyperthermophilic archaeon Thermococcus litoralis.

Journal ArticleResearch Support, Non-U.S. Gov'tPyrrolidone carboxyl peptidase from the hyperthermophilic archaeon Thermococcus litoralis has been crystallized in a form suitable for X-ray diffraction from ammonium sulfate or ammonium dihydrogen orthophosphate using the vapour-phase diffusion method. Crystals from both precipitants are of the orthorhombic space group P21212 with unit-cell dimensions a = 94.06, b = 149.06, c = 73.54 A. A complete data set to 2.8 A resolution has been collected from crystals grown from ammonium sulfate.BBSR

Crystallization and preliminary X-ray diffraction analysis of L-aminoacylase from the hyperthermophilic archaeon Thermococcus litoralis.

Journal ArticleResearch Support, Non-U.S. Gov'tThe enzyme L-aminoacylase catalyses the hydrolysis of N-acyl-L-amino acids from peptides or proteins. The recombinant enzyme from the hyperthermophilic archaeon Thermococcus litoralis has been purified to homogeneity. This zinc-containing enzyme has been crystallized from ammonium sulfate using the sitting-drop vapour-diffusion method. The crystals diffract to 2.8 A resolution and belong to the rhombohedral space group R32, with unit-cell parameters a = b = 102.4, c = 178.5 A, gamma = 120 degrees in a hexagonal lattice setting. The asymmetric unit contains one enzyme monomer, containing a single zinc ion. Two synchrotron data sets have been collected at a remote wavelength and at the maximum f'wavelength for zinc. This has allowed the position of the metal to be identified in anomalous Patterson maps.BBSRCTMR/LSF programme to the EMBL Hamburg Outstatio

Crystallization and preliminary X-ray diffraction studies of a novel alcohol dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix.

Journal ArticleResearch Support, Non-U.S. Gov'tA novel alcohol dehydrogenase enzyme has been cloned from the hyperthermophilic archaeon Aeropyrum pernix and overexpressed in Escherichia coli. This zinc-containing enzyme has been crystallized by the sitting-drop vapour-diffusion method using PEG 600 as precipitant. The crystals diffract to 1.5 A resolution and belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 100.7, b = 103.2, c = 67.5 A. The asymmetric unit contains two enzyme monomers. Two synchrotron data sets have been collected: one at a wavelength near the absorption edge of zinc and one at a remote wavelength. Three strong zinc-ion positions were visible in the anomalous Patterson map. Two additional weaker zinc ions have been identified by anomalous Fourier synthesis.BBSRCEuropean Community (Access to Research Infrastructure Action of the Improving Human Potential Programme to EMBL Hamburg Outstation

Crystallization and preliminary X-ray diffraction studies of the oxygenating subunit of 3,6-diketocamphane monooxygenase from Pseudomonas putida.

Journal ArticleResearch Support, Non-U.S. Gov'tThe oxygenating constituent of the 3,6-diketocamphane monooxygenase isozyme from Pseudomonas putida NCIMB 10007 has been crystallized under two different conditions. Crystals were initially grown from polyethylene glycol (PEG) 8000 and sodium acetate using the vapour-phase diffusion method. The crystals were of orthorhombic P212121 space group, with cell dimensions a = 55.8, b = 94.5 and c = 163.7 A and diffracted to 2.8 A resolution. More recently, improved crystals, which diffracted beyond 2 A, have been grown from ammonium sulfate. These crystals also belong to the orthorhombic P212121 space group, with cell dimensions of a = 54.6, b = 93.2 and c = 154. 1 A. A full native data set to 2.5 A resolution has been collected from the ammonium sulfate grown crystals.BBSR

An order-disorder twin crystal of L-2-haloacid dehalogenase from Sulfolobus tokodaii.

Journal ArticleResearch Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov'tThe L-2-haloacid dehalogenase enzymes catalyse the hydrolytic cleavage of a halogen from the C2 position of short-chain haloacids. The recombinant dehalogenase from the thermophilic archaeon Sulfolobus tokodaii has been cloned, overexpressed and purified to homogeneity. The 24 kDa enzyme was crystallized using the microbatch method in the monoclinic space group C2, with unit-cell parameters a = 127.6, b = 58.1, c = 51.2 A, beta = 97.2 degrees . Data were collected to 1.9 A resolution using synchrotron radiation and the structure was solved by molecular replacement. Analysis of the data and the preliminary refined model showed that the crystal was an order-disorder twin by reticular merohedry with a twin index of 10. It was possible to de-twin the experimental data utilizing the symmetry of the molecular layers from which the crystal is built.Engineering and Physical Sciences Research CouncilNIHBBSRCNWDA project awar

Crystallization and preliminary X-ray analysis of human thioredoxin peroxidase-B from red blood cells.

Journal ArticleResearch Support, Non-U.S. Gov'tTwo different crystal forms of human thioredoxin peroxidase-B have been grown by vapour diffusion using polyethylene glycol 400 as a precipitant. Monoclinic P21 crystals were grown from freshly purified protein, whilst orthorhombic P212121 crystals were grown from purified protein that had been stored in ammonium sulfate, but otherwise under the same conditions. The diffraction from both crystal forms was observed to extend to beyond 2.0 A resolution using synchrotron radiation. Complete native data sets to 1.8 and 3. 7 A have been collected from the monoclinic and orthorhombic crystals, respectively.EU (HCMP Access to Large Scale Facilities grant