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Hyperthermophilic

A newly isolated bacterial strain SHJ was found to be capable of degrading diethyl phthalate (DEP) very efficiently. Its growth characteristics and 16S rDNA gene sequence were analyzed. Its whole genome was also sequenced. Strain SHJ was identified as Sphingobium yanoikuyae SHJ. Keywords: Sphingobium yanoikuyae, Gene sequence, Genome, Diethyl phthalate, Biodegradation

Microbial bio-alcohol production is achieved by fermentation mainly using carbohydrates as substrates, which are major components of biomass. Various types of microorganisms have the capacity to produce bio-alcohol and/or bio-hydrogen, however, for this chapter only hyperthermophiles will be discussed. Dependency and rapid consumption of fossil fuels result in significantly negative impacts on the environment. To combat such deteriorating trends and maintain sustainability, biofuels are rapidly considered to be a major alternative source of fuel. A biological or chemical process is needed to convert biomass into simple sugars that can be fermented by a particular microorganism. Depending on the microorganism, the type of alcohol fermentation can vary. Microbial hydrogen production has been found to be mainly from dark fermentation using complex biomass. Although they possess the metabolic pathways required for alcohol production, the yields are still quite low in comparison to other non-hyperthermophilic alcohol producing microorganisms.

Hyperthermophilic Thermotoga spp. are excellent candidates for the biosynthesis of cellulosic ethanol producing strains because they can grow optimally at 80 °C with ability to degrade and utilize cellulosic biomass. In T. neapolitana (Tne), a putative iron-containing alcohol dehydrogenase was, for the first time, revealed to be a bifunctional aldehyde/alcohol dehydrogenase (Fe-AAdh) that catalyzed both reactions from acetyl-coenzyme A (ac-CoA) to acetaldehyde (ac-ald), and from ac-ald to ethanol, while the putative aldehyde dehydrogenase (Aldh) exhibited only CoA-independent activity that oxidizes ac-ald to acetic acid. The biochemical properties of Fe-AAdh were characterized, and bioinformatics were analyzed. Fe-AAdh exhibited the highest activities for the reductions of ac-CoA and acetaldehyde at 80–85 °C, pH 7.54, and had a 1-h half-life at about 92 °C. The Fe-AAdh gene is highly conserved in Thermotoga spp., Pyrococcus furiosus and Thermococcus kodakarensis, indicating the existence of a fermentation pathway from ac-CoA to ethanol via acetaldehyde as the intermediate in hyperthermophiles.

Diethyl phthalate (DEP) is a representative of phthalate esters (PAEs) that are largely used as industrial additives and have become environmental contaminants and endocrine disruptors. A newly isolated bacterial strain Sphingobium yanoikuyae SHJ was found to be capable of degrading DEP very efficiently. Four intermediates during the DEP degradation process were identified to be ethyl methyl phthalate (EMP), monoethyl phthalate (MEP), monomethyl phthalate (MMP) and phthalic acid (PA) using high-performance liquid chromatography tandem high-resolution mass spectrometry (HPLC-HRMS). When there was limited oxygen under SSA conditions, the rates of the further degradation of MEP and PA were slower compared to that under aerobic conditions. Although all intermediates EMP, MEP and MMP eventually decreased or disappeared, the DEP degradation kinetics showed that EMP formed much faster than that of MEP, and MMP appeared after the decreasing of MEP. It is proposed that two DEP degradation pathways must be present in S. yanoikuyae SHJ. The first is DEP → MEP → PA through a sequential hydrolysis process. The second is DEP → EMP → MEP → MMP → PA through a combination of demethylation or trans-esterification with methanol and hydrolysis processes. It is concluded that S. yanoikuyae SHJ has a versatile capability of degrading DEP and will have a great application potential in bioremediation of PAEs-contaminated shallow aquifer.

Glutamine:fructose-6-phosphate aminotransferase (GFAT) catalyzes the formation of glucosamine-6-phosphate, and its gene is one of the genes essential for microbes. Using the GFAT-encoding gene can prevent the use of a drug-resistant gene as a selection marker in a bacterial system. Another unique property of the GFAT selection marker is that no particular compound is prohibited or required for creating a selective stress for a yeast. Filamentous fungi are major producers of industrial enzymes. However, there has been no report on the construction and application of the GFAT gene as a selection marker in filamentous fungi. To develop a new selection marker, the GFAT-encoding gene gfaA was deleted from the genome of the filamentous fungus Aspergillus nidulans, and the gfat gene of the straw mushroom Volvariella volvacea was used as the selection marker to mediate the transformation and overexpression of a thermostable bacterial laccase in A. nidulans. The GFAT-deficient strain A. nidulans ∆gfaA was not able to grow in the culture medium containing 0.5% yeast extract unless about 20 mM glucosamine was used to supplement to the medium. The gfat gene was amplified and inserted into the integration vector pAL5 and autonomous replication vector Prg3-AMA1-NotI for A. nidulans to generate the gfat vectors pALG and pAMAG, respectively. Using these gfat vectors, the laccase gene lcs from a hyperthermophilic bacterium was overexpressed intra- and extracellularly in A. nidulans ∆gfaA. Therefore, recombinant filamentous fungi can be constructed with gfat vectors, which can be maintained stably in host cells with the naturally occurred selective stress of a medium, forage, pulp, animal gut, wastewater, or soil.

The booming of biodiesel industry all over the world has led to generate a large amount (10% v/v) of crude glycerol, created an oversupply problem. Herein, we compared the product concentrations of major metabolic products attained from pure and crude glycerol biotransformation process using an adapted mutant strain Klebsiella variicola SW3. Real-time qPCR and glycerol dehydrogenase (GDH) enzyme activity assay revealed that the overexpression of GDH gene resulted in an increased GDH enzyme activity, led to a markedly boosted 2,3-butanediol (2,3-BD) production. Based on these results, the SW3 strain obtained from wild type strain Klebsiella variicola SRP3 displayed a 1.39-fold increased 2,3-BD production of 82.5 g/L from 59.3 g/L, yielding 0.62 g/g using pure glycerol. However, in a batch culture, a final 33.5 g/L of 2,3-BD was accumulated within 96 h from 50 g/L glycerol. Moreover, the strain SW3 withstanding high concentration (200 g/L) of crude glycerol displayed 64.9 and 29.25 g/L 2,3-BD in fed-batch and batch cultures respectively. Therefore, this bioconversion of crude glycerol to 2,3-BD –a value-added green product with potential industrial applications as a liquid fuel or fuel additive would represent a remarkable alternative to add value to the biodiesel production helping biodiesel industries development.

Copper-catalyzed gem-difluoromethylenation of the C(sp2)–H bonds of alkenes has been developed. This protocol is the first to efficiently achieve gem-difluoromethylenation of C(sp2)–H bonds of unactivated aliphatic terminal olefins and is applied in gem-difluoromethylenation/cyclization of alkenes.

Biodiesel, a renewable fuel produced by transesterification of animal fats and vegetable oils, generates about 10% (v/v) of crude glycerol as a core byproduct. The high volume of this non bio-degradable glycerol is becoming of a great environmental and economical concern due to its worldwide ever-growing surplus. Herein we report a high production of 2,3-butanediol (2,3-BD) from pure and biodiesel derived crude glycerol using a mutant K. pneumoniae SRM2 obtained from a newly isolated strain Klebsiella pneumoniae SRP2. The mutant strain SRM2 with standing high glycerol concentration (220 g L-1 of medium) could rapidly convert glycerol aerobically to 2,3-BD, a versatile product extensively used in chemical, pharmaceutical and fuel industries Our study revealed that an increased GDH activity led to a substantially enhanced production of 2,3-BD. The mutant strain exhibited 1.3-fold higher activity of GDH than that of parent strain (500.08 vs. 638.6 µmol min -1 mg -1 protein), yielding of 32.3 g L-1 and 77.5 g L-1 2,3-BD with glycerol in batch and fed-batch process respectively. However, in batch culture with crude glycerol, cell growth and glycerol consumption were expressively boosted, and 2,3-BD production was 27.7 g L-1 from 75.0 g/L crude glycerol. In this report, the optimal conditions for high production of 2,3-BD were defined in a completely aerobic process, and 0.59 g g-1 product yield of 2,3-BD was attained by the mutated strain K. pneumoniae SRM2, which is the highest amount obtained from batch biotransformation process of glycerol metabolism till today. These results indicated that our newly developed mutant can tolerate high concentration of glycerol, have a high glycerol utilization rate, and high product yield of 2,3-BD. It is demonstrated that the mutant strain K. pneumoniae SRM2 has an ability to produce fewer co-products at trace concentrations at higher glycerol concentrations, and could be a potential candidate for 2,3-DB production in an industrial bioconversion process.

Acetohydroxyacid synthase (AHAS) catalyzes the production of acetolactate from pyruvate. The enzyme from the hyperthermophilic bacterium Thermotoga maritima has been purified and characterized ( k cat ~100 s −1 ). It was found that the same enzyme also had the ability to catalyze the production of acetaldehyde and CO 2 from pyruvate, an activity of pyruvate decarboxylase (PDC) at a rate approximately 10% of its AHAS activity. Compared to the catalytic subunit, reconstitution of the individually expressed and purified catalytic and regulatory subunits of the AHAS stimulated both activities of PDC and AHAS. Both activities had similar pH and temperature profiles with an optimal pH of 7.0 and temperature of 85 °C. The enzyme kinetic parameters were determined, however, it showed a non-Michaelis-Menten kinetics for pyruvate only. This is the first report on the PDC activity of an AHAS and the second bifunctional enzyme that might be involved in the production of ethanol from pyruvate in hyperthermophilic microorganisms.

The Acinetobacter sp. strain M isolated from a contaminated soil sample in Jiangsu Province of China was found to be able to degrade perfluorooctane sulfonamide (PFOSA) effectively. Fluoride anion (F- ) released from PFOSA degradation was detected by ion chromatography, and showed positive correlation to the growth curve of Acinetobacter sp. strain M. The PFOSA degradation efficiency of strain M was approximately 27 %, as assessed by GC analysis. It was shown that enzymes localized outside of cells of Acinetobacter sp. strain M catalyzed the degradation of PFOSA. This further indicates a possibly new (multi-step/pathway) mechanism for PFOSA degradation. It revealed that the extracellular enzyme of the Acinetobacter strain M preferentially cleaves carbon-carbon and carbon-fluorine bonds instead of destroying the carbon-sulfur bond. The growth condition for Acinetobacter sp. strain M was optimized at 30 °C and pH 7.0 in the presence of 2000 mg L-1 of PFOSA and 0.5 % (v/v) of Tween-20. The optimal PFOSA degradation time was found to be 12 h, with a degradation efficiency of 76 % by extracellular enzymes in strain M as determined by GC analysis. The result may provide potential applications for biodegradition of perfluoro organic compounds, such as derivatives of perfluorooctane (C8).

We sequenced wastewater effluent from the Agricultural Research Council–Animal Production in South Africa that conducts studies on livestock health and farm ecology. Thauera, Oscillibacter, and Pseudomonas were the most abundant genera within the community., We sequenced wastewater effluent from the Agricultural Research Council–Animal Production in South Africa that conducts studies on livestock health and farm ecology. Thauera, Oscillibacter, and Pseudomonas were the most abundant genera within the community. Thirty-one different antibiotic resistance genes were identified, 10 of which are associated with tetracycline resistance.

His-Asn-His (HNH) proteins are a very common family of small nucleic acid-binding proteins that are generally associated with endonuclease activity and are found in all kingdoms of life. Although HNH endonucleases from mesophiles have been widely investigated, the biochemical functions of HNH endonucleases from thermophilic bacteriophages remain unknown. Here, we characterized the biochemical properties of a thermostable HNH endonuclease from deep-sea thermophilic bacteriophage GVE2. The recombinant GVE2 HNH endonuclease exhibited non-specific cleavage activity at high temperature. The optimal temperature of the GVE2 HNH endonuclease for cleaving DNA was 60-65 °C, and the enzyme retained its DNA cleavage activity even after heating at 100 °C for 30 min, suggesting the enzyme is a thermostable endonuclease. The GVE2 HNH endonuclease cleaved DNA over a wide pH spectrum, ranging from 5.5 to 9.0, and the optimal pH for the enzyme activity was 8.0-9.0. Furthermore, the GVE2 HNH endonuclease activity was dependent on a divalent metal ion. While the enzyme is inactive in the presence of Cu(2+), the GVE2 HNH endonuclease displayed cleavage activity of varied efficiency with Mn(2+), Mg(2+), Ca(2+), Fe(2+), Co(2+), Zn(2+), and Ni(2+). The GVE2 HNH endonuclease activity was inhibited by NaCl. This study provides the basis for determining the role of this endonuclease in life cycle of the bacteriophage GVE2 and suggests the potential application of the enzyme in molecular biology and biotechnology.

The data provide additional support of the characterization of the biophysical and biochemical properties of the enzyme acetohydroxyacid synthase from the hyperthermophilic bacterium Thermotoga maritima (Eram et al., 2015) [1]. The genes encoding the enzyme subunits have been cloned and expressed in the mesophilic host Escherichia coli. Detailed data include information about the optimization of the expression conditions, biophysical properties of the enzyme and reconstitution of the holoenzyme from individually expressed and purified subunits.

We report here the draft genome sequences of two novel strains of Streptomyces (NWU339 and NWU49) isolated from South African rhizosphere soils. Both strains were found to possess strong cellulolytic activity and contain numerous putative cellulase genes., We report here the draft genome sequences of two novel strains of Streptomyces (NWU339 and NWU49) isolated from South African rhizosphere soils. Both strains were found to possess strong cellulolytic activity and contain numerous putative cellulase genes. Both genomes possess benzoate degradation pathways, while NWU49 contains the genomic potential for enediyne biosynthesis.

Hyperthermus butylicus is a peptide-fermenting hyperthermophilic archaeon producing 1-butanol as an end-product. The pathway for 1-butanol production in H. butylicus is unknown. Alcohol dehydrogenase (ADH) is a key enzyme responsible for alcohol production, which has to be present in H. butylicus, however, the gene encoding the enzyme is unknown. From the H. butylicus genome sequence, the gene Hbut_0414 is the only one annotated as an zinc-containing ADH, but it was unclear if it would be the enzyme catalyzing the production of 1-butanol. The gene was cloned and overexpressed in Escherichia coli. The recombinant enzyme encoded by Hbut_0414 was shown to be an NAD(H)-specific primary/secondary ADH capable of using a wide range of substrates including butyraldehyde and butanol. Optimal pH values for catalyzing aldehyde reduction and alcohol oxidation were of 5.0 and 8.5 respectively. Apparent Km values for acetaldehyde, propanal, acetone, butyraldehyde, ethanol, 1-propanol, 2-propanol and butanol were 0.61 ± 0.11, 0.41 ± 0.07, 1.41 ± 0.19, 1.75 ± 0.29, 1.79 ± 0.17, 2.30 ± 0.24, 4.85 ± 0.21 and 1.68 ± 0.15 mM, respectively. The specificity constant kcat/Km for propanal was the highest (15,975 s-1M-1) compared to that of butyraldehyde (1120 ± 345 s-1M-1). Its optimal temperature was found to be 60°C, and it lost 50% of its activity when incubated at 60°C for 3 hours, and 85% of its activity when incubated at 95°C for 5 min. It is unlikely that the ADH is functional for catalyzing the production of 1-butanol in H. butylicus growing optimally at 95°C. Further investigation is required for identifying the ADH responsible for catalyzing the 1-butanol production in this hypertehrmophilic archaeon.

An efficient method for the synthesis of the thermally stable and pharmaceutically important gem-difluoromethylene azo compounds is developed. This protocol achieved gem-difluoromethylenation of the nitrogen center of arenediazonium salts through in situ generated benzo-1,3-diazolic difluoromethylene radical addition to arenediazonium salts under mild Ag-initiated conditions.

HNH endonucleases in bacteriophages play a variety of roles in the phage lifecycle as key components of phage DNA packaging machines. The deep-sea thermophilic bacteriophage Geobacillus virus E2 (GVE2) encodes an HNH endonuclease (GVE2 HNHE). Here, the crystal structure of GVE2 HNHE is reported. This is the first structural study of a thermostable HNH endonuclease from a thermophilic bacteriophage. Structural comparison reveals that GVE2 HNHE possesses a typical ββα-metal fold and Zn-finger motif similar to those of HNH endonucleases from other bacteriophages, apart from containing an extra α-helix, suggesting conservation of these enzymes among bacteriophages. Biochemical analysis suggests that the alanine substitutions of the conserved residues (H93, N109 and H118) in the HNH motif of GVE2 HNHE abolished 94%, 60% and 83% of nicking activity, respectively. Compared to the wild type enzyme, the H93A mutant displayed almost the same conformation while the N108A and H118A mutants had different conformations. In addition, the wild type enzyme was more thermostable than the mutants. In the presence of Mn2+ or Zn2+, the wild type enzyme displayed distinct DNA nicking patterns. However, high Mn2+ concentrations were needed for the N109A and H118A mutants to nick DNA while Zn2+ inactivated their nicking activity.

The reversible redox reaction between coenzyme F420 and H2 to F420H2 is catalyzed by an F420-reducing [NiFe]-hydrogenase (FrhABG), which is an enzyme of the energy metabolism of methanogenic archaea. FrhABG is a group 3 [NiFe]-hydrogenase with a dodecameric quaternary structure of 1.25MDa as recently revealed by high-resolution cryo-electron microscopy. We report on the crystal structure of FrhABG from Methanothermobacter marburgensis at 1.7A resolution and compare it with the structures of group 1 [NiFe]-hydrogenases, the only group structurally characterized yet. FrhA is similar to the large subunit of group 1 [NiFe]-hydrogenases regarding its core structure and the embedded [NiFe]-center but is different because of the truncation of ca 160 residues that results in similar but modified H2 and proton transport pathways and in suitable interfaces for oligomerization. The small subunit FrhG is composed of an N-terminal domain related to group 1 enzymes and a new C-terminal ferredoxin-like domain carrying the distal and medial [4Fe-4S] clusters. FrhB adopts a novel fold, binds one [4Fe-4S] cluster as well as one FAD in a U-shaped conformation and provides the F420-binding site at the Si-face of the isoalloxazine ring. Similar electrochemical potentials of both catalytic reactions and the electron-transferring [4Fe-4S] clusters, determined to be E°'≈-400mV, are in full agreement with the equalized forward and backward rates of the FrhABG reaction. The protein might contribute to balanced redox potentials by the aspartate coordination of the proximal [4Fe-4S] cluster, the new ferredoxin module and a rather negatively charged FAD surrounding.

Directed evolution methods are increasingly needed to improve gene and protein properties. Error-prone PCR is the most efficient method to introduce random mutations by reducing the fidelity of the DNA polymerase. However, a highly efficient process is required for constructing and screening a diverse mutagenesis library since a large pool of transformants is needed to generate a desired mutant. We developed a method called in situ error-prone PCR (is-epPCR) to improve the efficiency of constructing a mutation library for directed evolution. This method offers the following advantages: (1) closed-circular PCR products can be directly transformed into competent E. coli cells and easily selected by using an alternative antibiotic; (2) a mutant library can be created and screened by one-step error-prone amplification of a variable DNA region in an expression plasmid; and (3) accumulation of desired mutations in one sequence can be obtained by multiple rounds of is-epPCR. Is-epPCR offers a novel, convenient, and efficient approach for improving genes and proteins through directed evolution.

Directed evolution methods are increasingly needed to improve gene and protein properties. Error-prone PCR is the most efficient method to introduce random mutations by reducing the fidelity of the DNA polymerase. However, a highly efficient process is required for constructing and screening a diverse mutagenesis library since a large pool of transformants is needed to generate a desired mutant. We developed a method called in situ error-prone PCR (is-epPCR) to improve the efficiency of constructing a mutation library for directed evolution. This method offers the following advantages: (1) closed-circular PCR products can be directly transformed into competent E. coli cells and easily selected by using an alternative antibiotic; (2) a mutant library can be created and screened by one-step error-prone amplification of a variable DNA region in an expression plasmid; and (3) accumulation of desired mutations in one sequence can be obtained by multiple rounds of is-epPCR. Is-epPCR offers a novel, convenient, and efficient approach for improving genes and proteins through directed evolution.

Biodiesel, a renewable fuel produced by transesterification of animal fats and vegetable oils, generates about 10% (v/v) of crude glycerol as a core byproduct. The high volume of this non bio-degradable glycerol is becoming of a great environmental and economical concern due to its worldwide ever-growing surplus. Herein we report a high production of 2,3-butanediol (2,3-BD) from pure and biodiesel derived crude glycerol using a mutant K. pneumoniae SRM2 obtained from a newly isolated strain Klebsiella pneumoniae SRP2. The mutant strain SRM2 with standing high glycerol concentration (220 g L-1 of medium) could rapidly convert glycerol aerobically to 2,3-BD, a versatile product extensively used in chemical, pharmaceutical and fuel industries Our study revealed that an increased GDH activity led to a substantially enhanced production of 2,3-BD. The mutant strain exhibited 1.3-fold higher activity of GDH than that of parent strain (500.08 vs. 638.6 µmol min -1 mg -1 protein), yielding of 32.3 g L-1 and 77.5 g L-1 2,3-BD with glycerol in batch and fed-batch process respectively. However, in batch culture with crude glycerol, cell growth and glycerol consumption were expressively boosted, and 2,3-BD production was 27.7 g L-1 from 75.0 g/L crude glycerol. In this report, the optimal conditions for high production of 2,3-BD were defined in a completely aerobic process, and 0.59 g g-1 product yield of 2,3-BD was attained by the mutated strain K. pneumoniae SRM2, which is the highest amount obtained from batch biotransformation process of glycerol metabolism till today. These results indicated that our newly developed mutant can tolerate high concentration of glycerol, have a high glycerol utilization rate, and high product yield of 2,3-BD. It is demonstrated that the mutant strain K. pneumoniae SRM2 has an ability to produce fewer co-products at trace concentrations at higher glycerol concentrations, and could be a potential candidate for 2,3-DB production in an industrial bioconversion process.

Active deep-sea hydrothermal vents harbor abundant thermophilic and hyperthermophilic microorganisms. However, microbial communities in inactive hydrothermal vents have not been well documented. Here, we investigated bacterial and archaeal communities in the two deep-sea sediments (named as TVG4 and TVG11) collected from inactive hydrothermal vents in the Southwest India Ridge using the high-throughput sequencing technology of Illumina MiSeq2500 platform. Based on the V4 region of 16S rRNA gene, sequence analysis showed that bacterial communities in the two samples were dominated by Proteobacteria, followed by Bacteroidetes, Actinobacteria and Firmicutes. Furthermore, archaeal communities in the two samples were dominated by Thaumarchaeota and Euryarchaeota. Comparative analysis showed that (i) TVG4 displayed the higher bacterial richness and lower archaeal richness than TVG11; (ii) the two samples had more divergence in archaeal communities than bacterial communities. Bacteria and archaea that are potentially associated with nitrogen, sulfur metal and methane cycling were detected in the two samples. Overall, we first provided a comparative picture of bacterial and archaeal communities and revealed their potentially ecological roles in the deep-sea environments of inactive hydrothermal vents in the Southwest Indian Ridge, augmenting microbial communities in inactive hydrothermal vents.

Extremophiles are a group of organisms growing optimally in a wide range of extreme environments. They have the capability of using many different types of nutrients for their growth, but only those heterotrophic extremophilic microorganisms are well studied and included for reviewing in this chapter. The utilization of both carbon and nitrogen sources from environments are essential for their thriving. Their growth rates are relatively comparable to mesophilic/neutrophilic microorganisms except oligotrophs. They also use novel enzymes for metabolizing carbohydrates including polysaccharides and peptides and amino acids. Conventional and also modified central metabolic pathways including Embden–Meyerhof–Parnas, Entner-Doudoroff and Pentose Phosphate pathways and Tricarboxylic Acid cycle are operational. However, which one(s) would be used is dependent on the type of the extremophiles. In general, those that grow on carbohydrates can also on peptides and amino acids or both, but some of them can only grow on peptides and amino acids. Their growth, substrate specificity and connections between the carbohydrates metabolism and peptides and amino acids metabolism are discussed in detail. Understanding their growth and metabolic pathways will provide insight into the future development of applications of extremophilic microorganisms in industrial processes.

Acetohydroxyacid synthase (AHAS) catalyzes the production of acetolactate from pyruvate. The enzyme from the hyperthermophilic bacterium Thermotoga maritima has been purified and characterized (kcat ~100 s−1). It was found that the same enzyme also had the ability to catalyze the production of acetaldehyde and CO2 from pyruvate, an activity of pyruvate decarboxylase (PDC) at a rate approximately 10% of its AHAS activity. Compared to the catalytic subunit, reconstitution of the individually expressed and purified catalytic and regulatory subunits of the AHAS stimulated both activities of PDC and AHAS. Both activities had similar pH and temperature profiles with an optimal pH of 7.0 and temperature of 85 °C. The enzyme kinetic parameters were determined, however, it showed a non-Michaelis-Menten kinetics for pyruvate only. This is the first report on the PDC activity of an AHAS and the second bifunctional enzyme that might be involved in the production of ethanol from pyruvate in hyperthermophilic microorganisms., Graphical abstract fx1, Highlights • The acetohydroxyacid synthase of T. maritima has pyruvate decarboxylase activity • The AHAS and PDC activities share the same temperature and pH optima • Reconstitution of the catalytic and regulatory subunits increases both PDC and AHAS activities

An alcohol dehydrogenase (ADH) from hyperthermophilic archaeon Thermococcus guaymasensis was purified to homogeneity and was found to be a homotetramer with a subunit size of 40 ± 1 kDa. The gene encoding the enzyme was cloned and sequenced; this gene had 1,095 bp, corresponding to 365 amino acids, and showed high sequence homology to zinc-containing ADHs and l -threonine dehydrogenases with binding motifs of catalytic zinc and NADP + . Metal analyses revealed that this NADP + -dependent enzyme contained 0.9 ± 0.03 g-atoms of zinc per subunit. It was a primary-secondary ADH and exhibited a substrate preference for secondary alcohols and corresponding ketones. Particularly, the enzyme with unusual stereoselectivity catalyzed an anti-Prelog reduction of racemic ( R / S )-acetoin to (2 R ,3 R )-2,3-butanediol and meso -2,3-butanediol. The optimal pH values for the oxidation and formation of alcohols were 10.5 and 7.5, respectively. Besides being hyperthermostable, the enzyme activity increased as the temperature was elevated up to 95°C. The enzyme was active in the presence of methanol up to 40% (vol/vol) in the assay mixture. The reduction of ketones underwent high efficiency by coupling with excess isopropanol to regenerate NADPH. The kinetic parameters of the enzyme showed that the apparent K m values and catalytic efficiency for NADPH were 40 times lower and 5 times higher than those for NADP + , respectively. The physiological roles of the enzyme were proposed to be in the formation of alcohols such as ethanol or acetoin concomitant to the NADPH oxidation.

An NADH oxidase from the anaerobic hyperthermophilic bacterium Thermotoga maritima was purified. The enzyme was very active in catalyzing the reduction of oxygen to hydrogen peroxide with an optimal pH value of 7 at 80°C. The V max was 230 ± 14 μmol/min/mg ( k cat / K m = 548,000 min −1 mM −1 ), and the K m values for NADH and oxygen were 42 ± 3 and 43 ± 4 μM, respectively. The NADH oxidase was a heterodimeric flavoprotein with two subunits with molecular masses of 54 kDa and 46 kDa. Its gene sequences were identified, and the enzyme might represent a new type of NADH oxidase in anaerobes. An NADH-dependent peroxidase with a specific activity of 0.1 U/mg was also present in the cell extract of T. maritima .

Acetohydroxyacid synthase (AHAS) is the key enzyme in branched chain amino acid biosynthesis pathway. The enzyme activity and properties of a highly thermostable AHAS from the hyperthermophilic bacterium Thermotoga maritima is being reported. The catalytic and regulatory subunits of AHAS from T. maritima were over-expressed in Escherichia coli. The recombinant subunits were purified using a simplified procedure including a heat-treatment step followed by chromatography. A discontinuous colorimetric assay method was optimized and used to determine the kinetic parameters. AHAS activity was determined to be present in several Thermotogales including T. maritima. The catalytic subunit of T. maritima AHAS was purified approximately 30-fold, with an AHAS activity of approximately 160±27 U/mg and native molecular mass of 156±6 kDa. The regulatory subunit was purified to homogeneity and showed no catalytic activity as expected. The optimum pH and temperature for AHAS activity were 7.0 and 85 °C, respectively. The apparent Km and Vmax for pyruvate were 16.4±2 mM and 246±7 U/mg, respectively. Reconstitution of the catalytic and regulatory subunits led to increased AHAS activity. This is the first report on characterization of an isoleucine, leucine, and valine operon (ilv operon) enzyme from a hyperthermophilic microorganism and may contribute to our understanding of the physiological pathways in Thermotogales. The enzyme represents the most active and thermostable AHAS reported so far., Graphical abstract, Highlights • First report of AHAS from a hyperthermophilic bacterium. • Catalytic and regulatory subunits of AHAS of T. maritima was expressed in E. coli. • Recombinant proteins were purified using a simplified procedure. • Enzyme represents the most active and thermostable AHAS reported so far. • Kinetic parameters were determined for the purified recombinant enzyme

The manufacture of biodiesel generates about 10% crude glycerol as a core by- product. This research is to identify novel bacterial strains which are capable of efficiently converting glycerol aerobically, and improve the strains for large scale production of value-added products. The production of a major product 2,3-butanediol (2,3- BD) from glycerol as a sole carbon source by the newly isolated Klebsiella variicola SRP3 are reported in a series of batch processes under aerobic condition. This study also compares the bacterial cell biomass, bio-products and glycerol dehydrogenase (GDH) enzyme production of K. variicola SRP3 isolated from paper mill waste when grown in aerobic condition. The incubation temperature, pH, glycerol concentration and nitrogen sources are the most important factors ruling the glycerol dehydrogenase. Acidic initial pH (pH 5.0) led to enhanced GDH activity (558.2 μmol/min/mg protein), yielding 25.33 g/L 2,3-BD, 7.6 g/L 1,3-PDO and 2.2 g/L acetoin from 50.0 g/L glycerol. In our aerobic batch culture the mutated strain K. variicola SRM3 exhibited 1.3 fold increased GDH activity of 721.5 units/mg protein from 558.2 units/mg protein, yielding 29.87 g/L 2,3-BD, 7.08 g/L 1,3-PDO and 2.02 g/L acetoin from 50.0 g/L glycerol. In our report, the optimal conditions for maximal GDH enzyme activity were defined, and 0.79 g/g product yield was achieved by the muted strain K. variicola SRM3, which is the highest amount obtained from glycerol as a sole carbon source until now. The research has for the first time proved that this K. variicola species can efficiently convert glycerol.

Hydrogen peroxide can be conveniently determined using horseradish peroxidase (HRP) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). However, interference occurs among assay components in the presence of reduced nicotinamide adenine dinucleotide (NADH) that is also a substrate of NADH oxidase. So, depletion of NADH is required before using the HRP method. Here, we report simple and rapid procedures to accurately determine hydrogen peroxide generated by NADH oxidase. All procedures developed were based on the extreme acid lability of NADH and the stability of hydrogen peroxide, because NADH was decomposed at pH 2.0 or 3.0 for 10 min, while hydrogen peroxide was stable at pH 2.0 or 3.0 for at least 60 min. Acidification and neutralization were carried out by adjusting sample containing NADH up to 30 microM to pH 2.0 for 10 min before neutralizing it back to pH 7.0. Then, hydrogen peroxide in the sample was measured using the HRP method and its determination limit was found to be about 0.3 microM. Alternatively, hydrogen peroxide in samples containing NADH up to 100 microM could be quantitated using a modified HRP method that required an acidification step only, which was found to have a determination limit of about 3 microM hydrogen peroxide in original samples.