Nitrogen Fixation and Ammonium Assimilation Pathway Expression of Geobacter sulfurreducens Changes in Response to the Anode Potential in Microbial Electrochemical Cells.

Nitrogen gas (N2) fixation in the anode-respiring bacterium Geobacter sulfurreducens occurs through complex, multistep processes. Optimizing ammonium (NH4+) production from this bacterium in microbial electrochemical technologies (METs) requires an understanding of how those processes are regulated in response to electrical driving forces. In this study, we quantified gene expression levels (via RNA sequencing) of G. sulfurreducens growing on anodes fixed at two different potentials (-0.15 V and +0.15 V versus standard hydrogen electrode). The anode potential had a significant impact on the expression levels of N2 fixation genes. At -0.15 V, the expression of nitrogenase genes, such as nifH, nifD, and nifK, significantly increased relative to that at +0.15 V, as well as genes associated with NH4+ uptake and transformation, such as glutamine and glutamate synthetases. Metabolite analysis confirmed that both of these organic compounds were present in significantly higher intracellular concentrations at -0.15 V. N2 fixation rates (estimated using the acetylene reduction assay and normalized to total protein) were significantly larger at -0.15 V. Genes expressing flavin-based electron bifurcation complexes, such as electron-transferring flavoproteins (EtfAB) and the NADH-dependent ferredoxin:NADP reductase (NfnAB), were also significantly upregulated at -0.15 V, suggesting that these mechanisms may be involved in N2 fixation at that potential. Our results show that in energy-constrained situations (i.e., low anode potential), the cells increase per-cell respiration and N2 fixation rates. We hypothesize that at -0.15 V, they increase N2 fixation activity to help maintain redox homeostasis, and they leverage electron bifurcation as a strategy to optimize energy generation and use. IMPORTANCE Biological nitrogen fixation coupled with ammonium recovery provides a sustainable alternative to the carbon-, water-, and energy-intensive Haber-Bosch process. Aerobic biological nitrogen fixation technologies are hindered by oxygen gas inhibition of the nitrogenase enzyme. Electrically driving biological nitrogen fixation in anaerobic microbial electrochemical technologies overcomes this challenge. Using Geobacter sulfurreducens as a model exoelectrogenic diazotroph, we show that the anode potential in microbial electrochemical technologies has a significant impact on nitrogen gas fixation rates, ammonium assimilation pathways, and expression of genes associated with nitrogen gas fixation. These findings have important implications for understanding regulatory pathways of nitrogen gas fixation and will help identify target genes and operational strategies to enhance ammonium production in microbial electrochemical technologies.

Structure, Function, and Thermal Adaptation of the Biotin Carboxylase Domain Dimer from Hydrogenobacter thermophilus 2-Oxoglutarate Carboxylase.

2-Oxoglutarate carboxylase (OGC), a unique member of the biotin-dependent carboxylase family from the order Aquificales, captures dissolved CO2 via the reductive tricarboxylic acid (rTCA) cycle. Structure and function studies of OGC may facilitate adaptation of the rTCA cycle to increase the level of carbon fixation for biofuel production. Here we compare the biotin carboxylase (BC) domain of Hydrogenobacter thermophilus OGC with the well-studied mesophilic homologues to identify features that may contribute to thermal stability and activity. We report three OGC BC X-ray structures, each bound to bicarbonate, ADP, or ADP-Mg2+, and propose that substrate binding at high temperatures is facilitated by interactions that stabilize the flexible subdomain B in a partially closed conformation. Kinetic measurements with varying ATP and biotin concentrations distinguish two temperature-dependent steps, consistent with biotin's rate-limiting role in organizing the active site. Transition state thermodynamic values derived from the Eyring equation indicate a larger positive ΔH⧧ and a less negative ΔS⧧ compared to those of a previously reported mesophilic homologue. These thermodynamic values are explained by partially rate limiting product release. Phylogenetic analysis of BC domains suggests that OGC diverged prior to Aquificales evolution. The phylogenetic tree identifies mis-annotations of the Aquificales BC sequences, including the Aquifex aeolicus pyruvate carboxylase structure. Notably, our structural data reveal that the OGC BC dimer comprises a "wet" dimerization interface that is dominated by hydrophilic interactions and structural water molecules common to all BC domains and likely facilitates the conformational changes associated with the catalytic cycle. Mutations in the dimerization domain demonstrate that dimerization contributes to thermal stability.

Temperature and solvent exposure response of three fatty acid peroxygenase enzymes for application in industrial enzyme processes.

Free fatty acids (FFAs) are a useful feedstock for a range of industrial chemical synthesis applications. However, efficiently converting FFAs to molecules for biofuel and other high-value chemicals requires more efficient and cost-effective catalysts. Cytochrome P450 fatty acid peroxygenases (CYP152) have a unique chemistry that allows use of the peroxide shunt pathway for biochemical conversion of FFAs. Known CYP152s are heat labile, however, requiring characterization of more thermotolerant versions for use in industrial applications. A fatty acid peroxygenase from Bacillus methanolicus (CYP152K6) was shown here to have a higher optimal reaction temperature than OleT (CYP152L1). CYP152K6 was stable up to 50 °C and showed great stability in 3% acetone and dimethylformamide. Stability in solvents helps the enzyme's substrates remain soluble in solution for more efficient catalysis, and heat stability allows enzymes to remain active longer during industrial processes.

Bacterial valorization of pulp and paper industry process streams and waste.

The pulp and paper industry is a major source of lignocellulose-containing streams. The components of lignocellulose material are lignin, hemicellulose, and cellulose that may be hydrolyzed into their smaller components and used as feedstocks for valorization efforts. Much of this material is contained in underutilized streams and waste products, such as black liquor, pulp and paper sludge, and wastewater. Bacterial fermentation strategies have suitable potential to upgrade lignocellulosic biomass contained in these streams to value-added chemicals. Bacterial conversion allows for a sustainable and economically feasible approach to valorizing these streams, which can bolster and expand applications of the pulp and paper industry. This review discusses the composition of pulp and paper streams, bacterial isolates from process streams that can be used for lignocellulose biotransformations, and technological approaches for improving valorization efforts. KEY POINTS: • Reviews the conversion of pulp and paper industry waste by bacterial isolates. • Metabolic pathways for the breakdown of lignocellulose components. • Methods for isolating bacteria, determining value-added products, and increasing product yields.

Thermostable endoglucanase gene derived by amplification from the genomic DNA of a cellulose-enriched mixed culture from mudspring water of Mt. Makiling, Laguna, Philippines.

Culture-independent molecular-based approaches can be used to identify genes of interest from environmental sources that have desirable properties such as thermo activity. For this study, a putative thermo stable endoglucanase gene was identified from a mixed culture resulting from the inoculation of Brock-CMcellulose (1%) broth with mudspring water from Mt. Makiling, Laguna, Philippines that had been incubated at 90 °C. Genomic DNA was extracted from the cellulose-enriched mixed culture and endo1949 forward and reverse primers were used to amplify the endoglucanase gene, which was cloned into pCR-script plasmid vector. Blastn alignment of the sequenced insert revealed 99.69% similarity to the glycosyl hydrolase, sso1354 (CelA1; Q97YG7) from Saccharolobus solfataricus. The endoglucanase gene (GenBank accession number MK984682) was determined to be 1,021 nucleotide bases in length, corresponding to 333 amino acids with a molecular mass of ~ 37 kDa. The endoglucanase gene was inserted into a pET21 vector and transformed in E. coli BL21 for expression. Partially purified recombinant Mt. Makiling endoglucanase (MM-Engl) showed a specific activity of 187.61 U/mg and demonstrated heat stability up to 80 °C. The thermo-acid stable endoglucanase can be used in a supplementary hydrolysis step to further hydrolyze the lignocellulosic materials that were previously treated under high temperature-dilute acid conditions, thereby enhancing the release of more glucose sugars for bioethanol production.

Methods for enhancing cyanobacterial stress tolerance to enable improved production of biofuels and industrially relevant chemicals.

Cyanobacteria are photosynthetic prokaryotes that can fix atmospheric CO2 and can be engineered to produce industrially important compounds such as alcohols, free fatty acids, alkanes used in next-generation biofuels, and commodity chemicals such as ethylene or farnesene. They can be easily genetically manipulated, have minimal nutrient requirements, and are quite tolerant to abiotic stress making them an appealing alternative to other biofuel-producing microbes which require additional carbon sources and plants which compete with food crops for arable land. Many of the compounds produced in cyanobacteria are toxic as titers increase which can slow growth, reduce production, and decrease overall biomass. Additionally, many factors associated with outdoor culturing of cyanobacteria such as UV exposure and fluctuations in temperature can also limit the production potential of cyanobacteria. For cyanobacteria to be utilized successfully as biofactories, tolerance to these stressors must be increased and ameliorating stress responses must be enhanced. Genetic manipulation, directed evolution, and supplementation of culture media with antioxidants are all viable strategies for designing more robust cyanobacterial strains that have the potential to meet industrial production goals.

Characterization of Clostridium ljungdahlii OTA1: a non-autotrophic hyper ethanol-producing strain.

A Clostridium ljungdahlii lab-isolated spontaneous-mutant strain, OTA1, has been shown to produce twice as much ethanol as the C. ljungdahlii ATCC 55383 strain when cultured in a mixotrophic medium containing fructose and syngas. Whole-genome sequencing identified four unique single nucleotide polymorphisms (SNPs) in the C. ljungdahlii OTA1 genome. Among these, two SNPs were found in the gene coding for AcsA and HemL, enzymes involved in acetyl-CoA formation from CO/CO2. Homology models of the respective mutated enzymes revealed alterations in the size and hydrogen bonding of the amino acids in their active sites. Failed attempts to grow OTA1 autotrophically suggested that one or both of these mutated genes prevented acetyl-CoA synthesis from CO/CO2, demonstrating that its activity was required for autotrophic growth by C. ljungdahlii. An inoperable Wood-Ljungdahl pathway resulted in higher CO2 and ethanol yields and lower biomass and acetate yields compared to WT for multiple growth conditions including heterotrophic and mixotrophic conditions. The two other SNPs identified in the C. ljungdahlii OTA1 genome were in genes coding for transcriptional regulators (CLJU_c09320 and CLJU_c18110) and were found to be responsible for deregulated expression of co-localized arginine catabolism and 2-deoxy-D-ribose catabolism genes. Growth medium supplementation experiments suggested that increased arginine metabolism and 2-deoxy-D-ribose were likely to have minor effects on biomass and fermentation product yields. In addition, in silico flux balance analysis simulating mixotrophic and heterotrophic conditions showed no change in flux to ethanol when flux through HemL was changed whereas limited flux through AcsA increased the ethanol flux for both simulations. In characterizing the effects of the SNPs identified in the C. ljungdahlii OTA1 genome, a non-autotrophic hyper ethanol-producing strain of C. ljungdahlii was identified that has utility for further physiology and strain performance studies and as a biocatalyst for industrial applications.

Metabolic response of Clostridium ljungdahlii to oxygen exposure.

Clostridium ljungdahlii is an important synthesis gas-fermenting bacterium used in the biofuels industry, and a preliminary investigation showed that it has some tolerance to oxygen when cultured in rich mixotrophic medium. Batch cultures not only continue to grow and consume H2, CO, and fructose after 8% O2 exposure, but fermentation product analysis revealed an increase in ethanol concentration and decreased acetate concentration compared to non-oxygen-exposed cultures. In this study, the mechanisms for higher ethanol production and oxygen/reactive oxygen species (ROS) detoxification were identified using a combination of fermentation, transcriptome sequencing (RNA-seq) differential expression, and enzyme activity analyses. The results indicate that the higher ethanol and lower acetate concentrations were due to the carboxylic acid reductase activity of a more highly expressed predicted aldehyde oxidoreductase (CLJU_c24130) and that C. ljungdahlii's primary defense upon oxygen exposure is a predicted rubrerythrin (CLJU_c39340). The metabolic responses of higher ethanol production and oxygen/ROS detoxification were found to be linked by cofactor management and substrate and energy metabolism. This study contributes new insights into the physiology and metabolism of C. ljungdahlii and provides new genetic targets to generate C. ljungdahlii strains that produce more ethanol and are more tolerant to syngas contaminants.

Characterization of recombinant glutathione reductase from the psychrophilic Antarctic bacterium Colwellia psychrerythraea.

Glutathione reductases catalyze the reduction of oxidized glutathione (glutathione disulfide, GSSG) using NADPH as the substrate to produce reduced glutathione (GSH), which is an important antioxidant molecule that helps maintain the proper reducing environment of the cell. A recombinant form of glutathione reductase from Colwellia psychrerythraea, a marine psychrophilic bacterium, has been biochemically characterized to determine its molecular and enzymatic properties. C. psychrerythraea glutathione reductase was shown to be a homodimer with a molecular weight of 48.7 kDa using SDS-PAGE, MALDI-TOF mass spectrometry and gel filtration. The C. psychrerythraea glutathione reductase sequence shows significant homology to that of Escherichia coli glutathione reductase (66 % identity), and it possesses the FAD and NADPH binding motifs, as well as absorption spectrum features which are characteristic of flavoenzymes such as glutathione reductase. The psychrophilic C. psychrerythraea glutathione reductase exhibits higher k cat and k cat/K m at lower temperatures (4 °C) compared to mesophilic Baker's yeast glutathione reductase. However, C. psychrerythraea glutathione reductase was able to complement an E. coli glutathione reductase deletion strain in oxidative stress growth assays, demonstrating the functionality of C. psychrerythraea glutathione reductase over a broad temperature range, which suggests its potential utility as an antioxidant enzyme in heterologous systems.

Bacterial biodegradation and bioconversion of industrial lignocellulosic streams.

Lignocellulose is a term for plant materials that are composed of matrices of cellulose, hemicellulose, and lignin. Lignocellulose is a renewable feedstock for many industries. Lignocellulosic materials are used for the production of paper, fuels, and chemicals. Typically, industry focuses on transforming the polysaccharides present in lignocellulose into products resulting in the incomplete use of this resource. The materials that are not completely used make up the underutilized streams of materials that contain cellulose, hemicellulose, and lignin. These underutilized streams have potential for conversion into valuable products. Treatment of these lignocellulosic streams with bacteria, which specifically degrade lignocellulose through the action of enzymes, offers a low-energy and low-cost method for biodegradation and bioconversion. This review describes lignocellulosic streams and summarizes different aspects of biological treatments including the bacteria isolated from lignocellulose-containing environments and enzymes which may be used for bioconversion. The chemicals produced during bioconversion can be used for a variety of products including adhesives, plastics, resins, food additives, and petrochemical replacements.

Biotechnological applications of halophilic lipases and thioesterases.

Lipases and esterases are enzymes which hydrolyze ester bonds between a fatty acid moiety and an esterified conjugate, such as a glycerol or phosphate. These enzymes have a wide spectrum of use in industrial applications where their high activity, broad substrate specificity, and stability under harsh conditions have made them integral in biofuel production, textile processing, waste treatment, and as detergent additives. To date, these industrial applications have mainly leveraged enzymes from mesophilic and thermophilic organisms. However, increasingly, attention has turned to halophilic enzymes as catalysts in environments where high salt stability is desired. This review provides a brief overview of lipases and esterases and examines specific structural motifs and evolutionary adaptations of halophilic lipases. Finally, we examine the state of research involving these enzymes and provide an in-depth look at an exciting algal-based biofuel production system. This system uses a recombinant halophilic lipase to increase oil production efficiency by cleaving algal fatty acids from the acyl carrier protein, which eliminates feedback inhibition of fatty acid synthesis.

Streptomyces spp. as biocatalyst sources in pulp and paper and textile industries: Biodegradation, bioconversion and valorization of waste.

Complex polymers represent a challenge for remediating environmental pollution and an opportunity for microbial-catalysed conversion to generate valorized chemicals. Members of the genus Streptomyces are of interest because of their potential use in biotechnological applications. Their versatility makes them excellent sources of biocatalysts for environmentally responsible bioconversion, as they have a broad substrate range and are active over a wide range of pH and temperature. Most Streptomyces studies have focused on the isolation of strains, recombinant work and enzyme characterization for evaluating their potential for biotechnological application. This review discusses reports of Streptomyces-based technologies for use in the textile and pulp-milling industry and describes the challenges and recent advances aimed at achieving better biodegradation methods featuring these microbial catalysts. The principal points to be discussed are (1) Streptomyces' enzymes for use in dye decolorization and lignocellulosic biodegradation, (2) biotechnological processes for textile and pulp and paper waste treatment and (3) challenges and advances for textile and pulp and paper effluent treatment.

Biochemical characterization of a psychrophilic and halotolerant α-carbonic anhydrase from a deep-sea bacterium, Photobacterium profundum.

Prokaryotic α-carbonic anhydrases (α-CA) are metalloenzymes that catalyze the reversible hydration of CO2 to bicarbonate and proton. We had reported the first crystal structure of a pyschrohalophilic α-CA from a deep-sea bacterium, Photobacterium profundum SS9. In this manuscript, we report the first biochemical characterization of P. profundum α-CA (PprCA) which revealed several catalytic properties that are atypical for this class of CA's. Purified PprCA exhibited maximal catalytic activity at psychrophilic temperatures with substantial decrease in activity at mesophilic and thermophilic range. Similar to other α-CA's, Ppr9A showed peak activity at alkaline pH (pH 11), although, PprCA retained 88% of its activity even at acidic pH (pH 5). Exposing PprCA to varying concentrations of oxidizing and reducing agents revealed that N-terminal cysteine residues in PprCA may play a role in the structural stability of the enzyme. Although inefficient in CO2 hydration activity under mesophilic and thermophilic temperatures, PprCA exhibited salt-dependent thermotolerance and catalytic activity under extreme halophilic conditions. Similar to other well-characterized α-CA's, PprCA is also inhibited by monovalent anions even at low concentrations. Finally, we demonstrate that PprCA accelerates CO2 biomineralization to calcium carbonate under alkaline conditions.

A versatile method for preparation of hydrated microbial-latex biocatalytic coatings for gas absorption and gas evolution.

We describe a latex wet coalescence method for gas-phase immobilization of microorganisms on paper which does not require drying for adhesion. This method reduces drying stresses to the microbes. It is applicable for microorganisms that do not tolerate desiccation stress during latex drying even in the presence of carbohydrates. Small surface area, 10-65 µm thick coatings were generated on chromatography paper strips and placed in the head-space of vertical sealed tubes containing liquid to hydrate the paper. These gas-phase microbial coatings hydrated by liquid in the paper pore space demonstrated absorption or evolution of H2, CO, CO2 or O2. The microbial products produced, ethanol and acetate, diffuse into the hydrated paper pores and accumulate in the liquid at the bottom of the tube. The paper provides hydration to the back side of the coating and also separates the biocatalyst from the products. Coating reactivity was demonstrated for Chlamydomonas reinhardtii CC124, which consumed CO2 and produced 10.2 ± 0.2 mmol O2 m⁻² h⁻¹, Rhodopseudomonas palustris CGA009, which consumed acetate and produced 0.47 ± 0.04 mmol H2 m⁻² h⁻¹, Clostridium ljungdahlii OTA1, which consumed 6 mmol CO m⁻² h⁻¹, and Synechococcus sp. PCC7002, which consumed CO2 and produced 5.00 ± 0.25 mmol O2 m⁻² h⁻¹. Coating thickness and microstructure were related to microbe size as determined by digital micrometry, profilometry, and confocal microscopy. The immobilization of different microorganisms in thin adhesive films in the gas phase demonstrates the utility of this method for evaluating genetically optimized microorganisms for gas absorption and gas evolution.

Improving the catalytic activity of hyperthermophilic Pyrococcus horikoshii prolidase for detoxification of organophosphorus nerve agents over a broad range of temperatures.

Prolidases hydrolyze Xaa-Pro dipeptides and can also cleave the P-F and P-O bonds found in organophosphorus (OP) compounds, including the nerve agents soman and sarin. Ph1prol (PH0974) has previously been isolated and characterized from Pyrococcus horikoshii and was shown to have higher catalytic activity over a broader pH range, higher affinity for metal, and increased thermostability compared to P. furiosus prolidase, Pfprol (PF1343). To obtain a better enzyme for OP nerve agent decontamination and to investigate the structural factors that may influence protein thermostability and thermoactivity, randomly mutated Ph1prol enzymes were prepared. Four Ph1prol mutants (A195T/G306S-, Y301C/K342N-, E127G/E252D-, and E36V-Ph1prol) were isolated which had greater thermostability and improved activity over a broader range of temperatures against Xaa-Pro dipeptides and OP nerve agents compared to wild type Pyrococcus prolidases.

Hydrolysis of organophosphorus compounds by microbial enzymes.

There are classes of microbial enzymes that have the ability to degrade harmful organophosphorus (OP) compounds that are present in some pesticides and nerve agents. To date, the most studied and potentially important OP-degrading enzymes are organophosphorus hydrolase (OPH) and organophosphorus acid anhydrolase (OPAA), which have both been characterized from a number of organisms. Here we provide an update of what is experimentally known about OPH and OPAA to include their structures, substrate specificity, and catalytic properties. Current and future potential applications of these enzymes in the hydrolysis of OP compounds are also addressed.

Validation of the H2S method to detect bacteria of fecal origin by cultured and molecular methods.

Using biochemical and molecular methods, this research determined whether or not the H(2)S test did correctly identify sewage-contaminated waters by being the first to use culturing and molecular methods to identify the types and numbers of fecal indicator organisms, pathogens, and other microbes present in sewage samples with positive H(2)S test results. For the culture-based method, samples were analyzed for the presence of fecal bacteria by spread plating the sewage sample onto differential and selective media for Aeromonas spp., Escherichia coli, sulfite-reducing clostridia, H(2)S-producing bacteria, and Salmonella/Shigella spp. The isolates were then: (1) tested to determine whether they were H(2)S-producing organisms and (2) identified to the genus and species level using biochemical methods. The molecular method used to characterize the microbial populations of select samples was terminal restriction fragment length polymorphisms. These experiments on sewage provided evidence that positive H(2)S tests consistently contained fecal bacteria and pathogens. There were strong relationships of agreement between the organisms identified by both methods tested. This study is an important advance in microbial water quality detection since it is focused on the evaluation of a novel, low-cost, water microbiology test that has the potential to provide millions of people worldwide access to water quality detection technology. Of prime consideration in evaluating water quality tests is the determination of the test's accuracy and specificity, and this article is a fundamental step in providing that information.

In situ H2O2 generation methods in the context of enzyme biocatalysis.

Hydrogen peroxide is a versatile oxidant that has use in medical and biotechnology industries. Many enzymes require this oxidant as a reaction mediator in order to undergo their oxygenation chemistries. While there is a reliable method for generating hydrogen peroxide via an anthraquinone cycle, there are several advantages for generating hydrogen in situ. As highlighted in this review, this is particularly beneficial in the case of biocatalysts that require hydrogen peroxide as a reaction mediator because the exogenous addition of hydrogen peroxide can damage their reactive heme centers and render them inactive. In addition, generation of hydrogen peroxide in situ does not dilute the reaction mixture and cause solution parameters to change. The environment would also benefit from a hydrogen peroxide synthesis cycle that does not rely on nonrenewable chemicals obtained from fossil fuels. Generation of hydrogen peroxide in situ for biocatalysis using enzymes, bioelectrocatalyis, photocatalysis, and cold temperature plasmas are addressed. Particular emphasis is given to reaction processes that support high total turnover numbers (TTNs) of the hydrogen peroxide-requiring enzymes. Discussion of innovations in the use of hydrogen peroxide-producing enzyme cascades for antimicrobial activity, wastewater effluent treatment, and biosensors are also included.

Production of biofuels from synthesis gas using microbial catalysts.

World energy consumption is expected to increase 44% in the next 20 years. Today, the main sources of energy are oil, coal, and natural gas, all fossil fuels. These fuels are unsustainable and contribute to environmental pollution. Biofuels are a promising source of sustainable energy. Feedstocks for biofuels used today such as grain starch are expensive and compete with food markets. Lignocellulosic biomass is abundant and readily available from a variety of sources, for example, energy crops and agricultural/industrial waste. Conversion of these materials to biofuels by microorganisms through direct hydrolysis and fermentation can be challenging. Alternatively, biomass can be converted to synthesis gas through gasification and transformed to fuels using chemical catalysts. Chemical conversion of synthesis gas components can be expensive and highly susceptible to catalyst poisoning, limiting biofuel yields. However, there are microorganisms that can convert the CO, H(2), and CO(2) in synthesis gas to fuels such as ethanol, butanol, and hydrogen. Biomass gasification-biosynthesis processing systems have shown promise as some companies have already been exploiting capable organisms for commercial purposes. The discovery of novel organisms capable of higher product yield, as well as metabolic engineering of existing microbial catalysts, makes this technology a viable option for reducing our dependency on fossil fuels.

Characterization of two proline dipeptidases (prolidases) from the hyperthermophilic archaeon Pyrococcus horikoshii.

Prolidases hydrolyze the unique bond between X-Pro dipeptides and can also cleave the P-F and P-O bonds found in organophosphorus compounds, including the nerve agents, soman and sarin. The advantages of using hyperthermophilic enzymes in biodetoxification strategies are based on their enzyme stability and efficiency. Therefore, it is advantageous to examine new thermostable prolidases for potential use in biotechnological applications. Two thermostable prolidase homologs, PH1149 and PH0974, were identified in the genome of Pyrococcus horikoshii based on their sequences having conserved metal binding and catalytic amino acid residues that are present in other known prolidases, such as the previously characterized Pyrococcus furiosus prolidase. These P. horikoshii prolidases were expressed recombinantly in the Escherichia coli strain BL21 (lambdaDE3), and both were shown to function as proline dipeptidases. Biochemical characterization of these prolidases shows they have higher catalytic activities over a broader pH range, higher affinity for metal and are more stable compared to P. furiosus prolidase. This study has important implications for the potential use of these enzymes in biotechnological applications and provides further information on the functional traits of hyperthermophilic proteins, specifically metalloenzymes.