Ornithine carbamoyltransferase from psychrophiles to thermophiles: structural evolution of catalytic fold to accommodate physiological diversity.

Here, we have analyzed the enzyme ornithine carbamoyltransferase (OCTase) in different classes of microorganisms belonging to psychrophiles, mesophiles and thermophiles. This OCTase catalyzes the formation of citrulline from carbamoyl phosphate (CP) and ornithine (ORN) in arginine biosynthesis pathway and has certain unique adaptations to regulate metabolic pathways in extreme conditions. The tertiary structure of OCTase showed two binding domains, the CP domain and ORN-binding domain at N and C terminals, respectively. We propose general acid-base catalysis in Pseudomonas gessardii between His259 and Asp220 in which later may act as a recipient of proton in the process. The comparative docking analysis showed that substrate-binding loops have been evolved to accommodate their lifestyles across the physiological temperature range where two substrates bind on two distinct loops in psychrophiles and mesophiles, whereas both the substrates bind on a single-substrate-binding loop in thermophiles and bring down the flexibility of the active site pocket to improve its evolutionary fitness.

Microbial halophilic lipases: A review.

Microbial lipases are commercially significant due to their versatile catalytic function of hydrolysis triacylglycerol. Among these, lipases from extremophiles are optimal for industrial application. Halophilic microorganisms living in a high salinity environment, such as the ocean, salt lakes, salt wells, and so on, produce halophilic lipases. In recent decades, many remarkable achievements have been made related to the properties and application of halophilic lipases. This review offers information collected over the last decades on halophilic lipase sources as well as advances in production, factors influencing activity, stability under various conditions, structural characteristics, progress in industrial applications such as food flavor modification, biodiesel production, and waste treatment, to provide theoretical and methodological references for the research in this direction.

Modulation of the Biocatalytic Properties of a Novel Lipase from Psychrophilic Serratia sp. (USBA-GBX-513) by Different Immobilization Strategies.

In this work, the effect of different immobilization procedures on the properties of a lipase obtained from the extremophilic microorganism Serratia sp. USBA-GBX-513, which was isolated from Paramo soils of Los Nevados National Natural Park (Colombia), is reported. Different Shepharose beads were used: octyl-(OC), octyl-glyoxyl-(OC-GLX), cyanogen bromide (BrCN)-, and Q-Sepharose. The performance of the different immobilized extremophile lipase from Serratia (ESL) was compared with that of the lipase B from Candida antarctica (CALB). In all immobilization tests, hyperactivation of ESL was observed. The highest hyperactivation (10.3) was obtained by immobilization on the OC support. Subsequently, the thermal stability at pH 5, 7, and 9 and the stability in the presence of 50% (v/v) acetonitrile, 50% dioxane, and 50% tetrahydrofuran solvents at pH 7 and 40 °C were evaluated. ESL immobilized on octyl-Sepharose was the most stable biocatalyst at 90 °C and pH 9, while the most stable preparation at pH 5 was ESL immobilized on OC-GLX-Sepharose supports. Finally, in the presence of 50% (v/v) tetrahydrofuran (THF) or dioxane at 40 °C, ESL immobilized on OC-Sepharose was the most stable biocatalyst, while the immobilized preparation of ESL on Q-Sepharose was the most stable one in 40% (v/v) acetonitrile.

Computational Analysis of Thermal Adaptation in Extremophilic Chitinases: The Achilles' Heel in Protein Structure and Industrial Utilization.

Understanding protein stability is critical for the application of enzymes in biotechnological processes. The structural basis for the stability of thermally adapted chitinases has not yet been examined. In this study, the amino acid sequences and X-ray structures of psychrophilic, mesophilic, and hyperthermophilic chitinases were analyzed using computational and molecular dynamics (MD) simulation methods. From the findings, the key features associated with higher stability in mesophilic and thermophilic chitinases were fewer and/or shorter loops, oligomerization, and less flexible surface regions. No consistent trends were observed between stability and amino acid composition, structural features, or electrostatic interactions. Instead, unique elements affecting stability were identified in different chitinases. Notably, hyperthermostable chitinase had a much shorter surface loop compared to psychrophilic and mesophilic homologs, implying that the extended floppy surface region in cold-adapted and mesophilic chitinases may have acted as a "weak link" from where unfolding was initiated. MD simulations confirmed that the prevalence and flexibility of the loops adjacent to the active site were greater in low-temperature-adapted chitinases and may have led to the occlusion of the active site at higher temperatures compared to their thermostable homologs. Following this, loop "hot spots" for stabilizing and destabilizing mutations were also identified. This information is not only useful for the elucidation of the structure-stability relationship, but will be crucial for designing and engineering chitinases to have enhanced thermoactivity and to withstand harsh industrial processing conditions.

Two new ene-reductases from photosynthetic extremophiles enlarge the panel of old yellow enzymes: CtOYE and GsOYE.

Looking for new ene-reductases with uncovered features beneficial for biotechnological applications, by mining genomes of photosynthetic extremophile organisms, we identified two new Old Yellow Enzyme homologues: CtOYE, deriving from the cyanobacterium Chroococcidiopsis thermalis, and GsOYE, from the alga Galdieria sulphuraria. Both enzymes were produced and purified with very good yields and displayed catalytic activity on a broad substrate spectrum by reducing α,ß-unsaturated ketones, aldehydes, maleimides and nitroalkenes with good to excellent stereoselectivity. Both enzymes prefer NADPH but demonstrate a good acceptance of NADH as cofactor. CtOYE and GsOYE represent robust biocatalysts showing high thermostability, a wide range of pH optimum and good co-solvent tolerance. High resolution X-ray crystal structures of both enzymes have been determined, revealing conserved features of the classical OYE subfamily as well as unique properties, such as a very long loop entering the active site or an additional C-terminal alpha helix in GsOYE. Not surprisingly, the active site of CtOYE and GsOYE structures revealed high affinity toward anions caught from the mother liquor and trapped in the anion hole where electron-withdrawing groups such as carbonyl group are engaged. Ligands (para-hydroxybenzaldehyde and 2-methyl-cyclopenten-1-one) added on purpose to study complexes of GsOYE were detected in the enzyme catalytic cavity, stacking on top of the FMN cofactor, and support the key role of conserved residues and FMN cofactor in the catalysis.

Nature and bioprospecting of haloalkaliphilics: a review.

The haloalkaliphilics are an important subset of extremophiles that grow in salt [upto 33% (wt/vol) NaCl] and alkaline pH (> 9). They are found in hypersaline environments especially in the brines in arid, coastal and deep sea locations, and in alkaline environments, such as soda soils, lakes and deserts. Some authors have described haloalkaliphilic bacteria as moderate halophilic bacteria, but the molecular and classical studies revealed that they belong to moderately to extremely halophilic bacteria and archaea. Organic solutes, such as glycine, betaine and other amino acid derivatives, sugars such as, sucrose and trehalose, and sugar alcohols present in the haloalkaliphilics help for their osmoadaptation, and also serve as stabilizers. Haloalkalphilics secrete exoenzymes like proteases, amylases, xylanases, cellulases and peroxidases which have potential industrial applications. They also produce bacteriorhodopsin, compatible solutes, pigments, biopolymers, secondary metabolites like biosurfactants, polyhydroxyalkanoate (PHA) and exopolysaccharides and antimicrobial/anticancer compounds. They have unique metabolic pathways which can be used to treat industrial pollutants, heavy metals and waste water.

Salt bridge impact on global rigidity and thermostability in thermophilic citrate synthase.

It has been suggested that structural rigidity is connected to thermostability, e.g. in enzymes from thermophilic microorganisms. We examine the importance of correctly handling salt bridges, and interactions which we term 'strong polars', when constructing the constraint network for global rigidity analysis in these systems. Through a comparison of rigidity in citrate synthases, we clarify the relationship between rigidity and thermostability. In particular, with our corrected handling of strong polar interactions, the difference in rigidity between mesophilic and thermophilic structures is detected more clearly than in previous studies. The increase in rigidity did not detract from the functional flexibility of the active site in all systems once their respective temperature range had been reached. We then examine the distribution of salt bridges in thermophiles that were previously unaccounted for in flexibility studies. We show that in hyperthermophiles these have stabilising roles in the active site; occuring in close proximity to key residues involved in catalysis and binding of the protein.

Biochemical and thermodynamic analyses of energy conversion in extremophiles.

A variety of extreme environments, characterized by extreme values of various physicochemical parameters (temperature, pressure, salinity, pH, and so on), are found on Earth. Organisms that favorably live in such extreme environments are called extremophiles. All living organisms, including extremophiles, must acquire energy to maintain cellular homeostasis, including extremophiles. For energy conversion in harsh environments, thermodynamically useful reactions and stable biomolecules are essential. In this review, I briefly summarize recent studies of extreme environments and extremophiles living in these environments and describe energy conversion processes in various extremophiles based on my previous research. Furthermore, I discuss the correlation between the biological system of electrotrophy, a third biological energy acquisition system, and the mechanism underlying microbiologically influenced corrosion. These insights into energy conversion in extremophiles may improve our understanding of the "limits of life". Abbreviations: PPi: pyrophosphate; PPase: pyrophosphatase; ITC: isothermal titration microcalorimetry; SVNTase: Shewanella violacea 5'-nucleotidase; SANTase: Shewanella amazonensis 5'-nucleotidase.

Properties and Applications of Extremozymes from Deep-Sea Extremophilic Microorganisms: A Mini Review.

The deep sea, which is defined as sea water below a depth of 1000 m, is one of the largest biomes on the Earth, and is recognised as an extreme environment due to its range of challenging physical parameters, such as pressure, salinity, temperature, chemicals and metals (such as hydrogen sulphide, copper and arsenic). For surviving in such extreme conditions, deep-sea extremophilic microorganisms employ a variety of adaptive strategies, such as the production of extremozymes, which exhibit outstanding thermal or cold adaptability, salt tolerance and/or pressure tolerance. Owing to their great stability, deep-sea extremozymes have numerous potential applications in a wide range of industries, such as the agricultural, food, chemical, pharmaceutical and biotechnological sectors. This enormous economic potential combined with recent advances in sampling and molecular and omics technologies has led to the emergence of research regarding deep-sea extremozymes and their primary applications in recent decades. In the present review, we introduced recent advances in research regarding deep-sea extremophiles and the enzymes they produce and discussed their potential industrial applications, with special emphasis on thermophilic, psychrophilic, halophilic and piezophilic enzymes.

A thermophilic microorganism from Deception Island, Antarctica with a thermostable glutamate dehydrogenase activity.

BACKGROUND: The Antarctic continent is a source of extreme microorganisms. Millions of years of isolation have produced unique biodiversity with adaptive responses to its extreme environment. Although the Antarctic climate is mainly cold, the presence of several geothermal sites, including thermal springs, fumaroles, hot soils and hydrothermal vents, provides ideal environments for the development of thermophilic and hyperthermophilic microorganisms. Their enzymes, called thermoenzymes, are the focus of interest in both academic and industrial research, mainly due to their high thermal activity and stability. Glutamate dehydrogenase, is an enzyme that plays a key role in the metabolism of carbon and nitrogen catalyzing reversibly the oxidative deamination of glutamate to alpha-ketoglutarate and ammonium. It belongs to the family of oxidoreductases, is widely distributed and it has been highly regarded for use as biosensors, particularly for their specificity and ability to operate in photochemical and electrochemical systems. However, the use of enzymes as biosensors is relatively problematic due to their instability to high temperatures, organic solvents and denaturing agents. The purpose of this study is to present the partial characterization of a thermophilic microorganism isolated from Deception Island, Antarctica, that displays glutamate dehydrogenase activity. RESULTS: In this work, we report the isolation of a thermophilic microorganism called PID15 from samples of Deception Island collected during the Antarctic Scientific Expedition ECA 46. This microorganism is a thermophile that grows optimally at 50 °C and pH 8.0. Scanning electron microscopy shows rod cells of 2.0 to 8.0 µm of length. Phylogenetic analysis of 16S rRNA gene revealed that this microorganism is closely related to Bacillus gelatini. This microorganism contains a thermostable glutamate dehydrogenase with optimal activity at pH 8.0 and temperatures for its activity from 37 to 50 °C, range of temperature of interest for biotechnological applications. This glutamate dehydrogenase is a highly thermostable enzyme. CONCLUSION: This is the first report of a microorganism from Antarctica containing a thermostable glutamate dehydrogenase that maintains its activity in a broad range of temperatures making it of potential interest for biotechnological applications.

A meta-analysis of the activity, stability, and mutational characteristics of temperature-adapted enzymes.

Understanding the characteristics that define temperature-adapted enzymes has been a major goal of extremophile enzymology in recent decades. In the present study, we explore these characteristics by comparing psychrophilic, mesophilic, and thermophilic enzymes. Through a meta-analysis of existing data, we show that psychrophilic enzymes exhibit a significantly larger gap (Tg) between their optimum and melting temperatures compared with mesophilic and thermophilic enzymes. These results suggest that Tg may be a useful indicator as to whether an enzyme is psychrophilic or not and that models of psychrophilic enzyme catalysis need to account for this gap. Additionally, by using predictive protein stability software, HoTMuSiC and PoPMuSiC, we show that the deleterious nature of amino acid substitutions to protein stability increases from psychrophiles to thermophiles. How this ultimately affects the mutational tolerance and evolutionary rate of temperature adapted organisms is currently unknown.

Discovery of Novel Cyclic Salt Bridge in Thermophilic Bacterial Protease and Study of its Sequence and Structure.

The plausible explanation behind the stability of thermophilic protein is still yet to be defined more clearly. Here, an in silico study has been undertaken by investigating the sequence and structure of protease from thermophilic (tPro) bacteria and mesophilic (mPro) bacteria. Results showed that charged and uncharged polar residues have higher abundance in tPro. In extreme environment, the tPro is stabilized by high number of isolated and network salt bridges. A novel cyclic salt bridge is also found in a structure of tPro. High number of metal ion-binding site also helps in protein stabilization of thermophilic protease. Aromatic-aromatic interactions also play a crucial role in tPro stabilization. Formation of long network aromatic-aromatic interactions also first time reported here. Finally, the present study provides a major insight with a newly identified cyclic salt bridge in the stability of the enzyme, which may be helpful for protein engineering. It is also used in industrial applications for human welfare.

Current perspectives for microbial lipases from extremophiles and metagenomics.

Microbial lipases are most broadly used biocatalysts for environmental and industrial applications. Lipases catalyze the hydrolysis and synthesis of long acyl chain esters and have a characteristic folding pattern of α/ß hydrolase with highly conserved catalytic triad (Serine, Aspartic/Glutamic acid and Histidine). Mesophilic lipases (optimal activity in neutral pH range, mesophilic temperature range, atmospheric pressure, normal salinity, non-radio-resistant, and instability in organic solvents) have been in use for many industrial biotransformation reactions. However, lipases from extremophiles can be used to design biotransformation reactions with higher yields, less byproducts or useful side products and have been predicted to catalyze those reactions also, which otherwise are not possible with the mesophilic lipases. The extremophile lipase perform activity at extremes of temperature, pH, salinity, and pressure which can be screened from metagenome and de novo lipase design using computational approaches. Despite structural similarity, they exhibit great diversity at the sequence level. This diversity is broader when lipases from the bacterial, archaeal, plant, and animal domains/kingdoms are compared. Furthermore, a great diversity of novel lipases exists and can be discovered from the analysis of the dark matter - the unexplored nucleotide/metagenomic databases. This review is an update on extremophilic microbial lipases, their diversity, structure, and classification. An overview on novel lipases which have been detected through analysis of the genomic dark matter (metagenome) has also been presented.

Amylomaltases in Extremophilic Microorganisms.

Amylomaltases (4-α-glucanotransferases, E.C. 2.4.1.25) are enzymes which can perform a double-step catalytic process, resulting in a transglycosylation reaction. They hydrolyse glucosidic bonds of α-1,4'-d-glucans and transfer the glucan portion with the newly available anomeric carbon to the 4'-position of an α-1,4'-d-glucan acceptor. The intramolecular reaction produces a cyclic α-1,4'-glucan. Amylomaltases can be found only in prokaryotes, where they are involved in glycogen degradation and maltose metabolism. These enzymes are being studied for possible biotechnological applications, such as the production of (i) sugar substitutes; (ii) cycloamyloses (molecules larger than cyclodextrins), which could potentially be useful as carriers and encapsulating agents for hydrophobic molecules and also as effective protein chaperons; and (iii) thermoreversible starch gels, which could be used as non-animal gelatin substitutes. Extremophilic prokaryotes have been investigated for the identification of amylomaltases to be used in the starch modifying processes, which require high temperatures or extreme conditions. The aim of this article is to present an updated overview of studies on amylomaltases from extremophilic Bacteria and Archaea, including data about their distribution, activity, potential industrial application and structure.

CRISPR/Cas9-Mediated Genome Engineering Reveals the Contribution of the 26S Proteasome to the Extremophilic Nature of the Yeast Debaryomyces hansenii.

The marine yeast Debaryomyces hansenii is of high importance in the food, chemical, and medical industries. D. hansenii is also a popular model for studying molecular mechanisms of halo- and osmotolerance. The absence of genome editing technologies hampers D. hansenii research and limits its biotechnological application. We developed novel and efficient single- and dual-guide CRISPR systems for markerless genome editing of D. hansenii. The single-guide system allows high-efficiency (up to 95%) mutation of genes or regulatory elements. The dual-guide system is applicable for efficient deletion of genomic loci. We used these tools to study transcriptional regulation of the 26S proteasome, an ATP-dependent protease complex whose proper function is vital for all cells and organisms. We developed a genetic approach to control the activity of the 26S proteasome by deregulation of its essential subunits. The mutant strains were sensitive to geno- and proteotoxic stresses as well as high salinity and osmolarity, suggesting a contribution of the proteasome to the extremophilic properties of D. hansenii. The developed CRISPR systems allow efficient D. hansenii genome engineering, providing a genetic way to control proteasome activity, and should advance applications of this yeast.

[L-asparaginases of extremophilic microorganisms in biomedicine]. / L-asparaginazy ékstremofil'nykh mikroorganizmov v biomeditsine.

L-asparaginase is extensively used in the treatment of acute lymphoblastic leukemia and several other lymphoproliferative diseases. In addition to its biomedical application, L-asparaginase is also of prospective use in food industry to reduce the formation of acrylamide, which is classified as probably neurotoxic and carcinogenic to human, and in biosensors for determination of L-asparagine level in medicine and food chemistry. The importance of L-asparaginases in different fields, disadvantages of commercial ferments, and the fact that they are widespread in nature stimuli the search for biobetter L-asparaginases from new producing microorganisms. In this regard, extremofile microorganisms exhibit unique physiological properties such as thermal stability, adaptability to extreme cold conditions, salt and pH tolerance and so provide one of the most valuable sources for novel L-asparaginases. The present review summarizes the recent results on studying the structural, functional, physicochemical and kinetic properties, stability of extremophilic L-asparaginases in comparison with their mesophilic homologues.

Production and Characterization of Extremophilic Proteinases From a New Enzyme Source, Barrientosiimonas sp. V9.

Microbial proteases are widely used as commercial enzymes, which have an active role in several industrial processes. The aim of this study was to investigate the production and properties of extracellular proteases from Barrientosiimonas sp. strain V9. The cultivation conditions for protease production were studied using different carbon and nitrogen sources. Maximum protease production was obtained in medium containing 25 g L-1 sucrose, 7 g L-1 KNO3, and initial pH 7.0 at 35 °C and 150 rpm during 72 h. Under these conditions, maximum proteolytic activity reached 1200 U mL-1. The enzyme extract showed optimum activity at 60 °C, pH 9.0, and was stable from 30 to 50 °C within a pH range from 4.0 to 10.0 and NaCl concentration up to 2.5 M. The enzyme was stable in the presence of EDTA, urea, Triton X-100 and laundry detergent (sodium lauryl sulfate as main component). The addition of 1% sodium dodecyl sulfate, Tween-80, or Tween-20 increased the activity by 183% and 119% respectively, while 2-mercaptoethanol reduced the activity to 71%. Casein zymogram analysis revealed three hydrolysis zones suggesting that Barrientosiimonas sp. V9 expresses proteases with molecular weights about 60, 45, and 35 kDa, which were inhibited in the presence of phenylmethylsulfonyl fluoride. Barrientosiimonas sp. V9 produces halotolerant serine proteases with great biotechnological potential.

Crystal structure of adenylate kinase from an extremophilic archaeon Aeropyrum pernix with ATP and AMP.

The crystal structure of an adenylate kinase from an extremophilic archaeon Aeropyrum pernix was determined in complex with full ligands, ATP-Mg2+ and AMP, at a resolution of 2.0 Å. The protein forms a trimer as found for other adenylate kinases from archaea. Interestingly, the reacting three atoms, two phosphorus and one oxygen atoms, were located almost in line, supporting the SN2 nucleophilic substitution reaction mechanism. Based on the crystal structure obtained, the reaction coordinate was estimated by the quantum mechanics calculations combined with molecular dynamics. It was found that the reaction undergoes two energy barriers; the steps for breaking the bond between the oxygen and γ-phosphorus atoms of ATP to produce a phosphoryl fragment and creating the bond between the phosphoryl fragment and the oxygen atom of the ß-phosphate group of ADP. The reaction coordinate analysis also suggested the role of amino-acid residues for the catalysis of adenylate kinase.

Efficient One-Pot Synthesis of Cytidine 5'-Monophosphate Using an Extremophilic Enzyme Cascade System.

A rapid in vitro enzymatic biosynthesis system has been developed as a biological manufacturing platform with potential industrial uses. Cytidine 5'-monophosphate (5'-CMP) is a key intermediate in the preparation of several nucleotide derivatives and is widely used in food and pharmaceutical industries. In this study, a highly efficient biosynthesis system was constructed for manufacturing 5'-CMP in vitro. Cytidine kinase (CK) was used for the biotransformation of cytidine to 5'-CMP, while polyphosphate kinase (PPK) was coupled for adenosine triphosphate regeneration. Both CK and PPK were selected from extremophiles, possessing great potential for biocatalytic synthesis. The effects of temperature, substrate concentration, and enzyme ratios were investigated to enhance the titer and yield of 5'-CMP. After optimization, 96 mM 5'-CMP was produced within 6 h, and the yield reached nearly 100%. This work highlights the ease of 5'-CMP production by an in vitro biomanufacturing platform and provides a green and efficient approach for the industrial synthesis of 5'-CMP.

New Nuclease from Extremely Psychrophilic Microorganism Psychromonas ingrahamii 37: Identification and Characterization.

Nucleases are an important group of hydrolases that degrade nucleic acids, with broad spectrum of applications in science and industry. In this paper, we report the identification and characterization of the nuclease from extremely psychrophilic bacterium Psychromonas ingrahamii that grows exponentially at 5 °C, but may also grow at even lower temperatures (down to - 12 °C). The putative endonuclease I gene, identified in the genome of P. ingrahamii, was cloned and expressed in Pichia pastoris. The recombinant protein was purified and its nucleolytic features were studied. The new enzyme, named by us as PinNuc, displays the features characteristic for the nonselective endonucleases, and has the ability to degrade different forms of nucleic acids. It is very active at room temperature in low ion-strength buffer and in the presence of low concentrations of magnesium ions. The enzyme, which possesses six cysteine residues, the most likely all engaged in disulphide bridges, is active only in oxidized form, and can be efficiently inactivated by the addition of low amounts of a reducing agent. According to our knowledge, it is the first nuclease, belonging to endonuclease I family, isolated from such extremely psychrophilic organism.