The existence of life forms that not only survive but actually thrive at temperatures near and even above the normal boiling point of water is a very recent discovery in biology. This immediately led, with considerable success, to the search for organisms with similar properties.
However, an understanding of how biochemical processes are able to take place at such temperatures has been less forthcoming. Similarly, the first insights into the factors that lead to protein "hyperthermostability" are only just emerging Chapter 11 , as are potential mechanisms for stabilizing DNA Chapter The main objectives of this chapter are to describe the "extremely thermophilic" organisms known at present, and to summarize the properties of the enzymes and proteins that have been purified to date from the sulfur-dependent species.
As indicated, all but one of the extreme thermophiles have been classified within what is now termed the Archaea domain of life. The relationship between the Archaea and the Bacteria is shown in Figure 2. During the 's, two forms of life were recognized based on features at the cellular level. One was the Eucaryotae, which represented all "higher" life forms, and these were distinguished by the presence of a membrane-defined nucleus.
The other was the Procaryotae, which included all bacteria, and these lacked a nucleus. During the 's techniques were developed to classify organisms at the molecular level using sequences of proteins and nucleic acids. A fundamental dichotomy in the procaryotes was then shown to exist by Woese and coworkers on the basis of 16S rRNA sequences S, This confirmed in quantitative terms the close relatedness of the the vast majority of bacteria procaryotes and the fundamental difference between them and the eucaryotes.
However, two groups of organisms, methanogenic bacteria and extremely halophilic bacteria, were shown to be specifically related to each other, but widely separated from all other bacteria and also from eucaryotes. This separation was also reflected in the biochemistry of the two groups: the archaebacteria have different cell walls and cell membranes, and contain a range of enzymes and cofactors not present in other bacteria. Indeed, the genetic machinery of the archaebacteria in many ways more resembles that of eucaryotes rather than eubacteria. As more molecular sequences became available, it was possible to discern the relationship between these three domains of life, and a universal phylogenetic tree was proposed by Woese and colleagues in ii.
In fact, they recommended that the three domains of life be referred to as Bacteria, Archaea, and Eucarya, since, at the molecular level, the archaebacteria resembled other eu bacteria no more than they did eucarya otes , and so they should no longer be termed bacteria. These definitions will be used herein. A rather surprizing conclusion from the universal tree shown in Figure 2 is that the Eucarya and Archaea have a common ancestor that is not shared by the Bacteria. Thus, investigations into the molecular properties of the extreme thermophiles may give some insights into the development of the eucaryotic cell.
Furthermore, as shown in Figure 1, there is one extremely thermophilic genus that is not classified within Publication Date: July 7, doi: Thermophilic organisms isolated over the last twenty years. Data taken from Table I and Refs. The universal phylogenetic tree showing the three domains of life. It is proposed that the extent of extreme thermophily, shown by the heavy line, includes the Universal Ancestor. Evolutionary time is proportional to the distance along any one line.
Present time is represented by the ends of the lines. Adapted from reference As shown in Figure 2, in addition to being the most thermophilic, Thermotoga is also the most ancient bacterial genus currently known, the first to have diverged from the "Universal Ancestor". Of course, this is contrary to traditional wisdom, which has regarded thermophily as an adaptation. That is, thermophiles were thought to represent a few "conventional" organisms that originally grew at "normal" temperatures but had somehow adapted so that they couldflourishat higher temperatures.
However, the remarkable conclusion from the evolutionary analysis is that extreme thermophiles are the most ancient organisms currently known. Physiology of Extreme Thermophiles The genera of extremely thermophilic organisms currently known are depicted in Table I. Most of the genera shown in Table I are represented by only one or two species. The majority have been found in geothermally-heated marine environments, in both shallow several meters below sea level and deep water several kilometers below sea level. Both were recently isolated by Baross and colleagues near deep sea hydrothermal vents.
Stetter and coworkers have also recently extended the known habitats of extremely thermophilic bacteria. They isolated several different bacteria from within the crater and from the open sea plume of an erupting submarine volcano, located 40 m below sea level These organisms included relatives by DNA hybridization of species of Pyrodictium, Pyrococcus, Archaeglobus and Thermococcus, bacteria that had been previously found only in volcanic vents off the coast of Italy.
They also isolated a novel bacterium that showed no DNA homology with any of the extreme thermophiles tested. Some of the extreme thermophiles therefore appear to be spread in the open oceans, and are able to remain viable even under such cold and aerobic conditions. Accordingly, they grow optimally at low pH near 2.
They are also autotrophs and use C O 2 as a carbon source. Remarkably, these organisms are facultative aerobes, and are able to switch to an anaerobic growth mode using H 2 as an electron donor and In Biocatalysis at Extreme Temperatures; Adams, M. Modified from refs. Phylogenetically, both genera belong to the archaeal order Sulfolobales, which includes the less thermophilic T t Publication Date: July 7, doi: Extremely thermophilic bacteria and their thermostable enzymes have numerous biotechnological advantages over their mesophilic counterparts 5,8,46,47 , and the notion of hyperthermophilic enzymes presents some intriguing commercial possibilities.
They may provide an opportunity to bridge the gap between biochemistry and a great deal of industrial chemistry. However, as shown in Table I, all of the extremely thermophilic and hyperthermophilic S -dependent bacteria were isolated in the 80's, and most in just the last few years. Hence the few biochemical characterizations reported with these organisms have focused mainly on their genetic machinery, e.tigesenro.gq
Some like it cold: biocatalysis at low temperatures | FEMS Microbiology Reviews | Oxford Academic
RNA's and polymerases, to try and gain insight into their relationship with the rest of the microbial world 4,7,12,48, Consequently, little is known about the metabolisms of these organisms, and even less about the enzymes involved. Three unique research opportunities are therefore presented by these bacteria, and particularly with the hyperthermophilic species: a elucidating the novel metabolic pathways that are likely to be present in these remarkable organisms, b determining the mechanisms by which these organisms stabilize biomolecules, especially proteins, and c understanding how their enzymes are specifically adapted to catalyze reactions at extreme temperatures.
Let us briefly consider all three aspects. The unusual natural environments of these organisms may also be a factor. For example, hydrothermal vent fluids contain minerals such as iron and manganese, and gases such as methane, C O 2 and H 2 , at concentrations many orders of magnitude above those in normal sea water. Thus, the availability of both nutrients and minerals may have severely limited the metabolic options of extreme thermophiles, or may have enabled them to develop novel catalytic systems because of the availability of a particular nutrient, not normally found in more conventional ecosystems.
Are the metabolic pathways of the hyperthermophiles minor variants of those of mesophilic organisms, or do completely different pathways exist? Coupled to this, of course, are the unique biochemical aspects of life at extreme temperatures. How have the hyperthermophiles circumvented, or even taken advantage of, such basic problems? As described below, at least one hyperthermophile, P. Moreover, this pathway is Publication Date: July 7, doi: Metabolic Enzymes from Thermophilic Organisms 11 dependent upon tungsten, a rarely used element in biological systems, but one that could be prevalent in deep sea hydrothermal vent fluids.
Elucidation of all of the factors which contribute to the enhanced thermostability of some proteins has, and continues to be, one of the most challenging problems in both biotechnology and biochemistry, e. Significant insight into stabilizing mechanisms should be obtained by comparisons of homologous hyperthermophilic and mesophilic proteins at the molecular level. However, as will be shown below, amino acid sequence information is not sufficient and three-dimensional structural information is required. In addition, although most of the proteins that have been purified from extreme thermophiles are remarkably thermostable, some of them, such as the hydrogenase of T.
The other intriguing and potentially useful property of the extremely thermophilic enzymes is that they have no or very low activity at ambient temperatures. This is in agreement with the idea that thermostable proteins are much more "rigid" at ambient temperatures than mesophilic proteins 55 , and presumably there is insufficient "flexibility" to allow interactions between adjacent redox centers or between substrates and redox centers.
Potential and utilization of thermophiles and thermostable enzymes in biorefining
This has enormous utility in that one can, in essence, carry out "cryoenzymology" at and above ambient temperatures, without the complications of cryosolvents. Some of these factors will be considered in the following description of the enzymes and proteins that have been purified so far from two extreme thermophilic organisms. These are the hyperthermophilic archaeon, Pyrococcus furiosus, and the novel extremely thermophilic bacterium, Thermotoga maritima.
Pyrococcus furiosus P.
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It is a strictly anaerobic heterotroph, and utilizes both simple maltose and complex starch carbohydrates for growth with the production of organic acids, C O 2 and H 2 as products. It will not use ammonia or free amino acids as an N-source, instead, proteins or peptides tryptone or yeast extract are required. The organism will also grow, albeit poorly, with these complex substrates as the sole N and C source Growth rates and cells yields appear to be similar when either method is used, In Biocatalysis at Extreme Temperatures; Adams, M.
We routinely obtain cell yields of approximately g wet weight from a liter culture 2. This enzyme was recently purified 56 , as was the amylase from the related organism, P.