Thermophilic Microbes in Environmental and Industrial Biotechnology

The existence of life at high temperatures is quite fascinating. At both ends of the temperature range compatible with life, only microorganisms are capable of growth and survival. A great variety of microbes survives and grows at such elevated temperatures. Many thermophilic microbial genera have been isolated from man-made (acid mine effluents, biological wastes and waste treatment plants, and self-heated compost piles) and natural (volcanic areas, geothermal areas, terrestrial fumaroles, terrestrial hot springs, deep-sea hydrothermal vents, geothermally heated oil and petroleum reserves, sun-heated soils/sediments) thermal habitats throughout the world. Both culture-dependent and culture-independent approaches have been employed for understanding the diversity of microbes in hot environments. These organisms not only tolerate such high temperatures but also usually require these for their growth and survival and are known as thermophiles/thermophilic microbes, which include a wide variety of prokaryotes (bacteria and archaea) as well as eukaryotes. Interest in their diversity, ecology, and physiology and biochemistry has increased enormously during the past few decades. These organisms have evolved several structural and chemical adaptations that allow them to survive and grow at elevated temperatures.

The Great Artesian Basin (GAB) is the world’s largest subsurface aquifer, underlying approximately one-fifth of subarid regions of the Australian continent and covering an area of over 1.7 × 106 km

2

, with a water-storage capacity of 8.7 × 1012 m

3

. The GAB provides a vital water resource for rural semiarid communities and also contains the largest onshore oil and gas reserves in Australia. The GAB is composed of alternating layers of water-bearing permeable sandstone and non-water-bearing impermeable shale. These geological formations have an immense influence on the chemical composition of GAB groundwaters, which can be bicarbonate-, chloride, sulphate or iron rich. The depth of the aquifer is estimated to be 3,000 m, and the underground water flow from the recharge areas at the edge of the basin to the discharge areas in central Australia as mound springs is estimated to be 1–5 million year

−1

. The water is heated by the Earth’s magma due to its depth, and the age of the water is calculated to be over 2 Ma. Not only do more than 5,000 free-flowing bores, with source temperatures ranging between 100 and 30°C, depending on bore depth, provide an important water resource to the outback communities, but the GAB is also a favourable environment for the growth of a wide diversity of microbial life. Distinct thriving macroscopic microbial mat communities can be seen colonising specific temperatures along the temperature gradient of runoff channels formed by the free-flowing bores. In the last two decades, a range of thermophilic and mesophilic microorganisms have been characterised from the GAB waters which include sulphate reducers, carbohydrate fermenters, strict aerobes and dissimilatory metal-reducing microorganisms (DIRM). During recent years, there has been a significant drop in the GAB groundwater pressure and volume, largely due to water leakage from corroding bores, and this is a matter of great concern. The isolation of metal-reducing microorganisms from the GAB environment suggests that they could be colonising the metal casing of such bores, thereby contributing to bore corrosion and subsequent complete bore failure. It is widely accepted that metal-reducing microorganisms have a large impact on the geochemistry of subsurface environments through the cycling of metals and organic matter and thereby affect water quality and taste. Furthermore, metal-reducing microorganisms have potential applications in bioremediation, mineral leaching and energy generation processes and are of evolutionary interest as metal reduction is considered to be a very ancient form of respiration. In this report, we provide an insight into the microbial diversity of this unique subsurface aquifer.

Antarctica, far from being an exclusively cold continent, has many geothermal sites with volcanic activity. Different islands belonging to this continent harbour different geothermal sites as thermal springs, fumaroles, hot soils and hydrothermal vents, providing ideal environments for the growth of thermophilic and hyperthermophilic microorganisms. Hyperthermophiles are an important source of enzymes and bioactive compounds with biotechnological applications.

Deception Island, a horseshoe-shaped island, is the emergent part of a young active shield volcano, which is located at the southwestern part of the Bransfield Strait and has optimal and unique geological characteristics for supporting the growth of thermophilic microbes. Several thermophilic microorganisms have been isolated from this island, and several molecular studies are being carried out for obtaining bioactive compounds and enzymes of industrial interest. This review deals with thermophilic and hyperthermophilic microorganisms isolated from Antarctica and their biotechnological potential.

Compost is the environmentally safe and the most economical way to treat organic waste. Due to heat emitted by fermentation processes, the compost inside is hot, and therefore, a variety of thermophilic bacteria thrive in compost. The thermophiles play an important role in the degradation of biopolymers in compost. This chapter deals with microbial community in a newly developed aerobic, high-temperature compost, where temperature reaches 100°C or even higher. Physical, chemical, biochemical, and microbial analyses of the high-temperature compost are presented. The technical problems associated with the biochemical and microbial analyses are also discussed.

Composting is a process that converts organic waste to a humus-like end product and is not only a waste treatment technique but also a recycling method as the end product can be used in agriculture as fertiliser, in gardening or in landscaping. Microbes play a key role as degraders during the composting process; mesophilic microflora constitutes the pioneer component, while thermophilic microflora, the climax and also the dominant component contributes significantly to the quality of compost. The dynamics of microbial community in different compost ecosystems change in context with qualitative and quantitative changes in physico-chemical conditions of compost. Optimisation of compost quality is directly linked to composition and succession of microbial communities in compost ecosystem. This necessitates the monitoring and characterisation of the microbial community composition, patterns and dynamics of species diversity at spatial scales. The biasedness and time-consuming nature of cultivation-based approaches severely limits spatial and temporal intensity of sampling, but with the advent of culture-independent molecular techniques, new insights into the composition of uncultivable communities have been gained. It has also been now possible to define the causes of time-dependent changes in the health of microbial community on the basis of observed 16S and 18S rDNA diversity. The genomic information represented by such a ‘community genome’ offers a tremendous resource for examining the extent and patterns of microbial genetic diversity and metabolic capabilities in the natural ecosystem. These studies also provide basic knowledge on mechanisms that control species coexistence, which have fundamental applications since they offer the framework that serves in maintaining, restoring and manipulating the diversity in natural compost ecosystem and hastening the process of composting besides improving the quality of compost.

Environmental pollution with toxic metals is a severe threat to biota and human health. Microbe-mediated bioremediation of such contaminants has emerged as a potential alternative to conventional treatment methods. Thermophilic microorganisms, owing to their natural ability to survive and flourish under elevated temperatures along with other stressful environmental conditions including high concentrations of heavy metals, have developed various adaptation strategies to cope with harsh environments, which may offer enormous opportunities for bioremediation of heavy metals at higher temperatures. Thermophilic microorganisms, being common in geological and anthropogenic thermal environments with high concentrations of dissolved metal, possess unique cell wall structures and metabolic and enzymatic properties that may contribute in metals–thermophiles interactions. Biosorption/bioaccumulation of metals is most effective and widely used approach for the bioremediation. The nature and extent of metal biosorption onto thermophilic bacteria may differ greatly from the mesophilic organisms. Microbial transformation of metal through oxidation/reduction reactions plays a critical role in metal speciation, distribution, and thus altered toxicity in the ecosystems, which may be implemented in metal recovery and remediation. Both sulfate- and metal-reducing bacteria have profound application in metal bioremediation. Thermophilic bacteria with higher metal tolerance and metabolic characteristics at high temperature may exhibit enhanced metal solubilization through sulfur- or iron-oxidizing processes. Thermophilic microbial community can perform both degradative and productive functions through coupling of metal reduction with oxidation of a variety of organic and inorganic substrates. Thermophilic bacteria are also able to reduce a wide spectrum of metals including Mn (IV), Cr (VI), U (VI), Tc (VII), Co (III), Mo (VI), Au (I, III), and Hg (II) which can be used for the immobilization of toxic metals/radionuclides during bioremediation of hot wastewater of disposal sites of radioactive wastes having temperature range favorable for thermophiles for a long period of time. This chapter discusses various modes of metal-microbe interactions in thermophilic bacteria for their promising application in bioremediation.

Being a potent electron donor (E

0'

CO/CO2

= −520 mV), CO may serve as an energy source for anaerobic prokaryotes. The main sources of CO in hot environments inhabited by anaerobic thermophiles are volcanic exhalations and thermal degradation of organic matter. A number of phylogenetically diverse anaerobic prokaryotes, both Bacteria and Archaea, are known to metabolize CO. CO transformation may be coupled to methanogenesis, acetogenesis, hydrogenogenesis, and sulfate or ferric iron reduction. This review will mainly focus on the diversity, ecology, physiology, and certain genomic features of the hydrogenogenic species, which are most numerous among the currently recognized thermophilic anaerobic carboxydotrophs and many of which were isolated and described in recent years. Among them are diverse Firmicutes, Dictyoglomi, and Eury- and Crenarchaeota. Despite their phylogenetic diversity, they employ similar enzymatic mechanisms of the СО + Н

2

О → СО

2

+ Н

2

process. The key enzyme of anaerobic CO utilization, the Ni-containing CO dehydrogenase, forms in hydrogenogens an enzymatic complex with the energy-converting hydrogenase, and genomic analysis shows this enzymatic complex to be encoded by a single-gene cluster.

Biomineralization is the process in which various organisms internally or externally produce inorganic minerals as biominerals such as bones, teeth, shells, and invertebrate exoskeletons. The magnetites, iron deposits, gold deposits, calcium carbonates, calcium phosphates, and silicates are well-known examples of the ­biominerals. Although silica is the most abundant compound in the earth’s crust and its precipitation is an important geological process in many geothermal environments, it is not useful for microorganisms. Recent research efforts revealed that both inorganic chemical reactions and microbial activity can be implicated in the formation of siliceous deposits. The extremely thermophilic bacteria within the genus

Thermus

are predominant component in the indigenous microbial community in siliceous deposits formed in pipes and equipment of geothermal power plants, which contributes to the rapid formation of huge siliceous deposits. In vitro examination suggested that

Thermus

cells induced precipitation of supersaturated amorphous silica during the exponential growth phase. A silica-induced protein (Sip) was isolated from the cell envelope fraction. The amino acid sequence of Sip was similar to that of the solute-binding protein of the Fe

3+

-binding ABC transporter. Furthermore, Sip promotes silica deposition on the surfaces of cells, after which the silicified outer membrane may serve as a “suit of armor” that confers resistance to peptide antibiotics. Dissolved silica in geothermal hot water may be a significant ­component in the maintenance and survival of microorganisms in nutrient-limited niches. And thus, thermophilic bacteria may use biosilicification for their own survival. This chapter reviews the formation of siliceous deposits by thermophilic bacteria in geothermal environments.

Thermophilic prokaryotes constitute a quite large group among the currently known prokaryotic species. At present, more than 220 genera and 580 species of the thermophiles have been isolated from various thermal and non-thermal environments and characterized. They are classified in more than 16 phyla of the two domains, Archaea and Bacteria. This chapter provides an overview of their phylogenetic relationships and their phenotypic features in the light of the current prokaryote systematics. Possible reasons for the phylogenetic diversity of the thermophiles in geothermal habitats are also discussed.

Where there is life, there are viruses. Thermophilic viruses are essential in regulating the structure and composition of microbial communities in the terrestrial and marine hot environments. Moreover, they constitute an important source of novel enzymes of high biotechnological and industrial potential. This chapter focuses on the application potential of thermophilic viruses. Central to this topic are the many novel genetic and morphological features discovered in thermophilic viruses. In order to fully appreciate the huge potential these viruses have in basic research and biotechnology, we will introduce recent results derived from studies of structural adaptations, functional genomics, metagenomics, virus–host interactions and virus life cycle, all areas with remarkable characteristics. In the end, a section will cover more specific application areas including thermophilic viruses as nanobuilding blocks.

An overview of the genome analyses of (hyper)thermophilic archaea and bacteria has been provided in this chapter. Basic information such as the species with published genome sequences, their genome size, and predicted gene number are presented. Genes that are specifically present on the genomes of (hyper)thermophiles are described. Various strategies to utilize the genome sequences for novel enzyme discovery are discussed with several examples. A brief overview of the wide range of omics research that has been performed with (hyper)thermophiles is also briefly dealt with.

Elucidation of the origin and the early evolution of life is fundamental to our understanding of ancient living systems and of the ancient global environment where early life evolved. A number of molecular phylogenetic trees have been constructed by comparing the homologous gene sequences.

In this chapter, we have reviewed the universal trees constructed based on different types of genetic information. The tree topology was different depending on the type of the gene analyzed as well as the method used. The root of the universal tree is most likely placed between the bacterial branch and the common ancestor of Archaea and Eucarya. However, there are possibilities that the root may be within the bacterial branches.

Monophyly of Archaea is rather controversial. Though the rRNA tree suggested the monophyly, other types of the tree are also reported. The conclusive result where the Eucarya originated within/outside of the branch of Archaea is yet to come.

The growth temperature of the ancient organism has long been a topic that has interested many scientists. Theoretical works suggested mesophilic, thermophilic, and hyperthermophilic origin of life, depending on the report. Experimental test analyzing the effect of each or combination of ancestral amino acid residues suggested the hyperthermophilic origin of life. However, we cannot totally deny the possible artifact based on the method used for the estimation of ancestral sequences possessed by the ancestral organisms.

The potential application of thermophiles and their enzymes for industry is enormous where the development of host-vector systems in thermophiles is essential. Compared to mesophilic organisms, the host-vector systems in thermophiles are lagging behind. However, in recent years a number of host-vector systems were developed for thermophilic bacteria and also for some archaea. Host-vector systems in thermophiles are more advanced than is commonly believed.

In this chapter, firstly, we introduce basic genetic methodology for host-vector systems in thermophiles including transformation, selectable markers, vectors, and hosts (recipient cells). We show transformation methods used for thermophiles, selectable markers that are effective for thermophiles, some

Escherichia coli

-host shuttle vectors, and important property of the host.

Secondly, we show the actual application systems for the host-vector systems in thermophiles, expression vectors, reporter gene systems, and targeted gene disruption (replacement) method.

Finally, we introduce some commercial and potential application of thermophile host-vector systems. Homologous and heterologous expression of the thermophilic proteins which were difficult to produce in full active form from mesophilic hosts was successfully developed in thermophiles, especially in

T. thermophilus

, and several hyperthermophilic archaea using their host-vector systems, “genetic and metabolic engineering,” were developed for biofuel production in thermophiles, especially in thermophilic ethanologens. And, directed evolution methods were developed for thermo-adaptation of mesophilic proteins in thermophiles such as

T. thermophilus

.

Thermophilic organisms tolerate or adapt to high temperatures by making their proteins thermostable or thermophilic. Even though, thermophilic bacteria have their own optimal growth temperatures, and heat-shock responses are still induced at the temperatures higher than optimal temperatures. The molecular chaperone systems of thermophilic eubacteria are very similar to those of mesophilic eubacteria. On the contrary, the molecular chaperone system of hyperthermophilic archaea is much simpler than those of other organisms. Within the hyperthermophilic archaea, only the following six kinds of chaperones have so far been identified: group II chaperonins, prefoldin, small heat-shock proteins, peptidyl-prolyl cis-trans isomerases, AAA proteins, and NAC. These archaea lack the Hsp70 chaperone system as well as Hsp90 and Hsp100, though these are thought to be indispensable chaperones in all other organisms. Since group II chaperonins are highly induced at elevated temperatures and related to the stress response of hyperthermophilic archaea, this manuscript focuses on the group II chaperonin and its cofactor, prefoldin, in addition to sHsps that are ubiquitous in thermophilic eubacteria and archaea. The limited number of molecular chaperones in hyperthermophilic archaea might be due to the relatively high stability of their proteins. The molecular chaperones in hyperthermophilic archaea might contribute to the protection of only a limited number of relatively unstable proteins.

In the last decade, the genes encoding hyperthermophilic proteins and enzymes have been extensively expressed in heterologous organisms such as

Escherichia coli

, and their good productions have been achieved. However, some difficulties are often encountered when attempting to produce these proteins in the mesophilic hosts. This chapter focuses on the recent efforts made to overcome problems in heterologous production of hyperthermophilic enzymes: (1) successful production of hetero-oligomeric dye-linked

l

-proline dehydrogenases by use of effective promoters, (2) a typical procedure for the in vitro refolding of inclusion bodies composed of several hyperthermophilic enzymes (malate dehydrogenase, lysine dehydrogenase, and agmatinase), and (3) heat-induced structural conversion of hyperthermophilic glutamate dehydrogenase. This information could be useful in successful production of hyperthermophilic proteins and enzymes.

Besides continuous progress in molecular biological techniques, metagenomic approach has become one of the routine experiments to examine the microbial diversity, ecology, and enzymes. This approach basically does not require the cultivation of microbes, which has a strong bias to a population of microbes and allows to have a glimpse of uncultured microbes and to find novel biomolecules. In the last 15 years, nearly 20 papers describing properties of thermostable enzymes discovered from hot environments by metagenomic strategies have been published. Most of them attempted to find and analyze hydrolytic enzymes such as cellulases, amylase, xylanase, esterase, lipase, and others because such enzymes are relatively easy to detect their activities in the function-based screening procedure. On the other hand, sequencing-based approach does not rely on the activity measurement and aids in discovering novel thermostable enzymes employing recently improved DNA sequencing technology and cloning vectors. The spectacular progress made in molecular biological techniques facilitates comprehensive metagenomic analysis for discovering novel biomolecules from the major fraction of the uncultured microbes in nature.

DNA polymerase and DNA ligase are ubiquitous enzymes that synthesize complementary DNA strands according to the template DNA and ligate breaks in the backbone structure of DNA, respectively, in living organisms. Multiple enzymes have been identified from each organism, and the functional sharing of these enzymes has been investigated for both DNA polymerase and DNA ligase. In addition to their fundamental role in maintaining genome integrity during replication and repair, DNA polymerases and DNA ligases are widely used for genetic engineering techniques, including DNA cloning, dideoxy sequencing, DNA labeling, mutagenesis, and other in vitro DNA manipulations. Thermostable DNA polymerases and DNA ligases are especially important in PCR and LDR/LCR, which are indispensable techniques for gene manipulation. In this chapter, we have summarized the recent developments in DNA polymerases and DNA ligases from thermophilic microbes.

Extremophilic organisms have attracted significant attention of the research community during the recent past, not only due to their survival and growth at extreme conditions but also due to their huge potential in various fields of biotechnology. Among the various groups of extremophiles, thermophilic actinomycetes have been less explored due to the difficulties in their isolation and maintenance in pure culture. Therefore, it largely remains to explore their diversity, molecular phylogeny, adaptive features, and biocatalytic and other biotechnological potentials. In order to study actinomycetes, morphological features and morphogenesis, antibiotic sensitivity and resistance, biochemical characteristics, and certain key molecular features have been taken into account to get insight into the actinomycetes, in general. The molecular approaches include sequence homology of 16S rRNA genes, nucleic acid hybridization, G + C% ratio, protein profiling, RFLP, DGGE, TGGE and ARDRA for the assessment of diversity, taxonomic status, and molecular phylogeny. The thermophilic actinomycetes reflect quite appealing and unique applications in various fields of biotechnology, viz., production of thermostable enzymes, antibiotics, and hormones and their role in bioremediation processes of recalcitrant compounds. Further studies on the diversity and phylogeny would enhance understanding of the unexplored thermophilic actinobacteria that will promote their applications.

Considerable interest has been generated in the mechanism which nature utilises to increase the stability of enzymes found in thermophilic and hyperthermophilic species. This has been the subject of many reviews, and our understanding has been enhanced by the increasing number of high-resolution thermostable enzyme structures that have been determined. Different species of bacteria and Archaea have used different mechanisms to achieve stability. A comparative approach has been used to carry out a detailed study of specific enzymes from a range of organisms in order to understand acquired stability at a structural level. This chapter will discuss the rules to increase protein thermostability that have been obtained from protein structural studies that are currently available. It will also examine other ways to stabilise existing proteins by lessons learnt from nature and by protein immobilisation.

Thermostable enzymes find applications in ‘white biotechnology’ including the biosynthesis of fine chemicals. This chapter will discuss specific examples of thermophilic enzymes already adopted for industrial applications. These include alcohol dehydrogenases for chiral alcohol production, aminoacylases for optically pure amino acids and amino acid analogues, transaminases for chiral amine production and gamma-lactamases for chiral gamma-lactam building blocks which are subsequently incorporated into carbocyclic nucleotides. A brief overview of other applications in biorefining, biofuel cells and detergents are also presented.

Starch represents a ubiquitous molecule in plants and is composed of linear polymer amylose and branched polymer amylopectin. Due to its complex structure, it is insoluble in water and needs to be liquefied at high temperatures to make it a useable substrate for hydrolyzing biocatalysts. Hyper-/thermophilic microorganisms belonging to Archaea and Bacteria have been isolated from volcanically and geothermal-heated hydrothermal vent systems and were shown to be capable of utilizing natural polymeric compounds such as starch and cellulose as energy and carbon sources. During the last 25 years, considerable efforts have been made to shed light on structure-function relationships of starch-degrading thermoactive enzymes (extremozymes) and exploit these in various industrial processes. Mostly derived from Bacteria or Archaea, these biocatalysts are stable and highly active at temperatures up to 120°C even in the presence of high concentrations (99%) of organic solvents. A great portfolio of amylolytic enzymes enables these microorganisms to degrade polysaccharides into oligo- and monosaccharides. Such enzymes (e.g. amylases, glucoamylases, pullulanases and CGTases) have been employed in producing a series of valuable products. In this chapter, we will focus on starch-converting enzymes from thermophiles and their application in food, feed, textile, chemical, pharmaceutical and other industrial sectors.

Pullulanase is one of the industrially important debranching enzymes, capable of hydrolyzing α-1, 6-glucosidic linkages in pullulan, starch, amylopectin, and other related oligosaccharides. It is widely used in starch industry for the production of various sugar syrups. Type I pullulanases specifically attack α-1, 6 linkages in branched oligosaccharides such as pullulan, starch, amylopectin, and glycogen, forming linear α-1, 4-linked oligomers, while the type II pullulanases (amylopullulanases) hydrolyze α-1, 6-glycosidic linkages in pullulan and branched substrates besides α-1, 4-glycosidic linkages in polysaccharides. With the advancements in biotechnology, the application of pullulanase has been extended to pharmaceutical chemistry as well as in automatic dishwashing detergents, baking industry, and production of cyclodextrins. Although pullulanases are ubiquitous in their occurrence in plants, animals, as well as microbes, the microbial sources are the most preferred ones for large-scale production and application. This chapter deals with the developments in production, characteristics, molecular aspects, and applications of microbial pullulanases and amylopullulanases.

Although some of the enzymes involved in carbohydrate metabolism in thermophilic archaea have been identified from genomic data, much less is known about the metabolic pathways in thermophilic archaea than in bacteria such as

Escherichia coli

. This is because many gaps still remain in most of the metabolic pathways constructed using data predicted from the genomic sequences of thermophilic archaea. In order to understand carbohydrate metabolism in thermophilic archaea, the proteins predicted to be carbohydrate metabolic enzymes from their genomic data have been expressed in

E. coli

, and the expressed proteins have been functionally analyzed. These analyses have suggested that the activities of novel and not seen in mesophiles could be detected in the thermostable enzymes from archaea. These observations enable novel pathways to be constructed based on the actual activities or functions of the enzymes obtained from thermophilic archaea. Furthermore, these investigations have confirmed that functional genomics is a powerful tool for studying the detailed features of microorganisms.

Restriction endonucleases (REases) are enzymes that recognize and cleave DNA in a sequence specific manner. The recognition site consists of a sequence of nucleotides in the DNA duplex, typically four to eight base pairs long. Most of the commercially produced REases are isolated from the mesophilic bacteria. But the disadvantage of REases from mesophilic sources is that these enzymes are usually denatured at ambient and high temperature. As temperature produces opposite effects on both enzyme activity and stability, it is therefore a key variable in any biocatalytic process. Also, mesophilic enzymes are unstable, have low reactivity, lose activity during purification, and require refrigerated transport and storage. So, thermostable REases are preferred to circumvent these problems. This chapter deals mainly with thermophilic REases. The increasing interest in this field is reflected by the growing information on the discovery, purification, and characterization of REases from thermophilic sources. The properties associated with these enzymes offer additional advantages over their mesophilic counterparts.

Chitin is the second most abundant polysaccharide, next to cellulose, occurring nature in the fungal cell walls, insect exoskeletons, while the shells of crustaceans contribute significantly to the availability of renewable biopolymer. Several enzymes are known to degrade different forms of chitin mostly produced by bacteria, fungi, and plants. Deacetylated polymer of chitosan and chitin is chemically hydrolyzed to generate oligomers and monomers for variety of applications that include pharmaceutical, environmental, agricultural, and cosmetic sectors. It would be possible to select from natural sources or modify the natural sources of chitinases to develop industrial processes that could replace the chemical processes for production of the chitooligomers, dimers, and monomers. Thermostable chitinases would give an added advantage for such industrial processes, and therefore there is a need to identify sources of such chitinases. In this chapter we have examined the availability of microbial sources of chitinases with a special attention to the thermostable chitinases. The approaches used in modifying chitinases and other related enzymes have been discussed to present the possible biotechnological approaches to generate novel thermostable enzymes. However, there was limited information available for chitinases indicating the need to focus research in that direction.

Thermophilic microbes have been considered as good sources of ­thermostable enzymes with high catalytic activity, greater resistance to denaturing agents and lower incidence of contamination. Thermostable enzymes are receiving considerable attention because of their usefulness in high-temperature catalysis of various enzymatic industrial processes. Phytases (

myo

-inositol hexakisphosphate phosphohydrolase) are the phosphatases, which catalyse the hydrolysis of phytic acid to inorganic phosphate and

myo

-inositol phosphate derivatives, while phosphatases are able to hydrolyse a wide variety of esters and anhydride phosphoric acids, releasing phosphate, and are also able to perform transphosphorylation reactions. The phosphorus thus liberated is used in metabolic pathways. The phosphatases have been considered to be one of the most versatile groups of hydrolases because of their adaptability under different environmental extremes such as high-temperature regimes and regulate phosphate metabolism for maintaining phosphorus economy of the cell for fulfilling its growth as well as bioenergetic requirements. The reduction of phytic acid content in the foods and feeds by enzymatic hydrolysis using phytase is desirable, because the physical and chemical methods of phytate removal negatively affect their nutritional value. These enzymes, therefore, have potential applications in food and feed industries for mitigating their phytic acid content to liberate available inorganic phosphate and improve digestibility as a result of elimination of antinutrient characteristics. In this review, the attention is focused on the production, characteristics and potential biotechnological aspects of phytases and phosphatases from thermophilic microbes.

Represented by archeal, bacterial, and fungal species, thermophilic organisms have been isolated from all types of terrestrial and marine hot environment. Pectinase from these organisms (thermophilic pectinase) developed unique structure–function properties of high thermostability and optimal activity at higher temperature. The advantage of using thermostable enzymes for various industrial applications is of course the intrinsic thermostability, and hence low activity losses during the raw material pretreatment at the elevated temperatures. Industrial applications of thermophilic pectinolytic enzyme have drawn a great deal of attention for use as biocatalysts because most of the industrial processes are carried out at higher temperature zone. Their potential to carry out myriads of biochemical reactions even at stringent conditions makes their use eco-friendly and best alternative to polluting chemical technologies. The role of acidic pectinases in extraction and clarification of fruit juices is well established. Recently, these have emerged as suitable candidate for biobleaching of wood pulp, desizing and bioscouring of cotton, degumming of plant fibers and biomass conversion, etc.

Thermostable carbohydrate-degrading enzymes are of special interest for many industrial applications as the solubility of carbohydrates at elevated temperatures sharply increases. Gellan is among the microbial exopolysaccharides found recently extensive use in food, microbial cultivation media, and pharmaceutical industries. Enzyme modification of gellan could change its molecular weight, hardness of its gel, and its elasticity and in such a way might broaden its current spectrum of application. As gellan is soluble at temperatures higher than 60°C, an industrial need in a thermostable gellan lyase is clearly outlined. Several reports on mesophilic bacterial strains producing gellan lyases are known, and only one thermophilic bacterial producer,

Geobacillus stearothermophilus

98, was reported up to now. In this chapter, the source microorganism and properties of the thermostable gellan lyase are discussed in relation to those of mesophilic producers. Even though the accumulated knowledge on the structural and catalytic properties of the gellan lyase is still very limited, the results obtained clearly demonstrate that it is a new enzyme with interesting characteristics, which could add to the commercial value of gellan as an emulsifier, stabilizer, gel agent, thickener and suspending agent, and application in the future are also suggested.

Thermophilic fungi and actinomycetes have been extensively studied in vegetal biomass bioconversion processes for the formulation of industrial enzymatic pools and as gene donors for the heterologous expression of thermostable enzymes. The production of second-generation biofuels and the application in industries such as the textile are of particular interest. In this chapter, we have reviewed the gene structure, gene regulation, biochemical properties, and biotechnological applications of lignocellulolytic enzymes and other potential industrial hydrolases of thermophilic fungi and actinobacteria. Besides

Humicola grisea

var.

thermoidea

, the object of study of our group for several years, we focus the following fungi:

Humicola insolens

,

Aureobasidium pullulans

,

Candida peltata

,

Chaetomium thermophilum

,

Coprinopsis cinerea

,

Ganoderma colossum

,

Malbranchea pulchella var. sulfurea

,

Melanocarpus albomyces

,

Rhizomucor pusillus

,

Myceliophthora thermophila

,

Myriococcum thermophilum

,

Penicillium duponti

,

Sporotrichum pulverulentum

,

Sporotrichum thermophile

,

Stilbella thermophila

,

Talaromyces emersonii

,

Thermoascus aurantiacus

,

Thermomyces lanuginosus

, and

Thielavia terrestris

. Among the actinomycetes, we explored

Acidothermus cellulolyticus

,

Cellulomonas

spp.,

Streptomyces

spp.,

Thermobifida fusca

, and

Thermomonospora curvata

.

The renewed interest in cellulase biotechnology is drawing the attention of researchers globally due to their diverse range of applications. The major applications of cellulases (E.C.3.2.1.4) are in textile and detergent industry. Additionally, they are in huge demand in food and feed sector for the improvement in digestibility, in nutritional quality of food/feed material, and in paper industries as de-inking agents. Another most promising application of cellulases is in the bioconversion of renewable lignocellulosic biomass into fermentable sugar constituents that are subsequently used for the production of value-added chemicals after the fermentation reaction with appropriate microorganisms. The success of ethanol-based biorefinery truly depends upon the efficiency of cellulase titers stable at high temperature and their cost at shop floor. Looking at the applications of cellulases, stable and active thermostable cellulases at high pH range would be more advantageous as compared to thermolabile enzymes in terms of time, cost savings, and getting the suitable product with desired yields/productivities. Recent developments on the proteomics, genomics, and fermentation strategies have paved the way for searching more efficient and novel thermostable cellulase titers from thermophilic microorganisms of different habitats.

Xylanases are an important category under the glycoside hydrolase families and together with cellulases constitute nearly 25% market in enzyme sector. Some of the major applications of this enzyme are in bleaching of pulp and paper, food and feed sector, etc. For several of these applications, enzymes from thermophilic sources are preferred. In this chapter, we present information on classification of family 11 xylanases, used in pulp and paper industry. Factors underlying thermostability, such as the length and composition of the N-terminus, Ser/Thr ratio, presence of Arg on enzyme surface, core packing, and hydrophobic interactions, have been described. Based on these principles, protein engineering approaches to achieve thermostability of fungal xylanases are reviewed. Our own work on development of hyper-xylanase-producing mutant and process strategies adopted to enhance production of this enzyme from a thermophilic fungus

Melanocarpus albomyces

is summarized. Role of nitrogen source, pH, temperature, aeration, and agitation is emphasized through this case study whereby productivities of 22,000 IU/L/h have been achieved. Additives, currently in use, to make stable xylanase preparation are also described. Special emphasis is laid on downstream processing, which includes role of carriers and binders in producing the product of desired quality.

Hemicellulose is the second most abundant component in lignocellulosics available in nature. It is a storage polymer occurring in seeds and a prominent structural component of cell walls in plants. Hemicelluloses in agricultural residues constitute up to 40%. Monomers of various hemicelluloses are useful in various biotechnological processes like the production of different antibiotics, alcohols, animal feeds, and biofuels. Xylan is the most abundant of all hemicelluloses. It has a linear backbone of β-1,4-linked

D

-xylopyranose residues. Immense interest in the enzymatic hydrolysis of xylan has been due to the applications of hydrolysates in feedstocks, production of biochemicals, and paper pulp bleaching. Biodegradation of xylan requires action of several enzymes, among which xylanases play a key role. A wide variety of microorganisms are known to produce xylanases. The interest in thermostable xylanases has markedly increased due to their potential applications in pulping and bleaching processes, in food and feed industry, textile processing, enzymatic saccharification of lignocellulosic materials, and waste treatment. Since elevated temperatures have a significant influence on the bioavailability and solubility of organic compounds, most of these processes are carried out at high temperatures. The elevation in temperature is accompanied by a decrease in viscosity and an increase in the diffusion coefficient of organic compounds, and thus, higher rates of reactions are expected. Thermophilic organisms are of special interest as sources of thermostable xylanases. The development of new analytical techniques and the commercial availability of new matrices have led to the purification and characterization of a large number of xylan-degrading bacterial enzymes. The recombinant DNA technology has permitted selection and overproduction of xylanolytic enzymes that are suitable for industrial applications. The developments in cloning and expression, directed evolution, physicochemical and functional characteristics, and biotechnological applications and commercialization of thermostable xylanases of bacterial origin have been reviewed.

Proteases are one of the largest selling enzymes in the world. This is rationalised by their extensive usage in the detergent, food, pharmaceutical, leather and textile industries. Thermostability in industrial enzymes remains a desirable attributes for (1) achieving faster conversion rates, (2) greater catalytic efficiencies and (3) protection from microbial contamination while operating at higher temperatures. Proteases endowed with such characteristics are all the more needed for baking and textile processing. In general, most of the industrial proteases are sourced from

Bacillus

sp. Thermostability in protease is accorded by protein engineering or appropriate immobilisation methods. Proteases from hyperthermophiles and thermophiles are natural choice for exploring the inherent heat stability. Few classical thermostable proteases especially those from

Pyrococcus

and

Thermococcus

have generated considerable interest. Heat stability in these cases has been attributed to large proportion of hydrophobic residue, extensive hydrogen bonding and increased share of disulphide bonds. Extensive screening of large range of unexplored thermophiles is well called for. Understanding their protein architecture may enable rationale design for heat-stable proteases in future.

This chapter highlights the enzymatic characteristics and novel properties of known thermostable proteases and focuses on their structure–function relationship. Recent developments and future perspectives in screening new proteases from hyperthermophiles/thermophiles, metagenomic studies, directed evolution, site-directed mutagenesis, modern immobilisation methods such as CLEC, CLEA and PCMC and immobilisation on nanoparticles are comprehensively covered.

Keratinases are unique proteases that are capable of degrading recalcitrant, “hard-to-degrade” keratin residues. Diverse microorganisms that belong to Eukarya, bacteria, and Archaea produce these enzymes. A large number of Bacilli, Actinobacteria, and fungi are reported to produce keratinases. Microbial keratinases present great diversity in their biochemical and biophysical properties. They are robust enzymes with wide temperature and pH activity range. They are optimally active at neutral to alkaline and 40–60°C, but examples of microbial keratinolysis at alkalophilic and thermophilic conditions have been well documented. Studies with specific substrates and inhibitors indicated that keratinases preferentially act on hydrophobic and aromatic residues at P1 position. Keratinases have several current and potential applications in agro-industrial, pharmaceutical, and biomedical fields. These enzymes are useful in processes related with the bioconversion of keratin waste into feed and fertilizers. Other promising applications are enzymatic dehairing for leather and cosmetic industry, detergent industry, and development of biopolymers from keratin fibers. The use of keratinases to enhance drug delivery in some tissues and hydrolysis of prion proteins arises as novel outstanding applications. Their use in biomass conversion into biofuels may address the increasing concern on energy conservation and recycling. Looking into their biotechnological impetus, they are being cloned and expressed in a variety of heterologous hosts.

In this technology-oriented world, when every phase is going green, enzymes have found tremendous applications. Thousands of enzymes have been identified and are being used commercially. However, microbial origin enzymes have gained more relevance than those from other sources. Among others, lipases are spectacular enzymes known for their unique attributes and significant industrial potential. They are one of the most important biocatalysts known for their applications in the biotechnology industry. Since these enzymes find massive applications, with the passage of time, the trend has shifted towards the identification of thermostable lipases which could be used in the industries which require harsh conditions to work in. Thermostable lipases have found applications in various areas such as in pharmaceuticals, food, and chemical industries. The following article talks about the thermophilic lipases derived from various microorganisms and their applications.