Secondary Alcohol Dehydrogenases from Thermoanaerobacter pseudoethanolicus and Thermoanaerobacter brockii as Robust Catalysts.

Alcohol dehydrogenases (ADHs) are an important type of enzyme that have significant applications as biocatalysts. Secondary ADHs from Thermoanaerobacter pseudoethanolicus (TeSADH) and Thermoanaerobacter brockii (TbSADH) are well-known as robust catalysts. However, like most other ADHs, these enzymes suffer from their high substrate specificities (i.

Novel Bioengineered Cassava Expressing an Archaeal Starch Degradation System and a Bacterial ADP-Glucose Pyrophosphorylase for Starch Self-Digestibility and Yield Increase.

To address national and global low-carbon fuel targets, there is great interest in alternative plant species such as cassava (), which are high-yielding, resilient, and are easily converted to fuels using the existing technology. In this study the genes encoding hyperthermophilic archaeal starch-hydrolyzing enzymes, α-amylase and amylopullulanase from and glucoamylase from , together with the gene encoding a modified ADP-glucose pyrophosphorylase () from , were simultaneously expressed in cassava roots to enhance starch accumulation and its subsequent hydrolysis to sugar. A total of 13 multigene expressing transgenic lines were generated and characterized phenotypically and genotypically.

Activity of select dehydrogenases with sepharose-immobilized N(6)-carboxymethyl-NAD.

N(6)-carboxymethyl-NAD (N(6)-CM-NAD) can be used to immobilize NAD onto a substrate containing terminal primary amines. We previously immobilized N(6)-CM-NAD onto sepharose beads and showed that Thermotoga maritima glycerol dehydrogenase could use the immobilized cofactor with cofactor recycling. We now show that Saccharomyces cerevisiae alcohol dehydrogenase, rabbit muscle L-lactate dehydrogenase (type XI), bovine liver L-glutamic dehydrogenase (type III), Leuconostoc mesenteroides glucose-6-phosphate dehydro-genase, and Thermotoga maritima mannitol dehydrogenase are active with soluble N(6)-CM-NAD.

Respiratory glycerol metabolism of Actinobacillus succinogenes 130Z for succinate production.

Actinobacillus succinogenes 130Z naturally produces among the highest levels of succinate from a variety of inexpensive carbon substrates. A few studies have demonstrated that A. succinogenes can anaerobically metabolize glycerol, a waste product of biodiesel manufacture and an inexpensive feedstock, to produce high yields of succinate.

Characterization of Thermotoga maritima glycerol dehydrogenase for the enzymatic production of dihydroxyacetone.

NAD-dependent Thermotoga maritima glycerol dehydrogenase (TmGlyDH) converts glycerol into dihydroxyacetone (DHA), a valuable synthetic precursor and sunless tanning agent. In this work, recombinant TmGlyDH was characterized to determine if it can be used to catalyze DHA production. The pH optima for glycerol oxidation and DHA reduction at 50 °C were 7.

Development of a markerless knockout method for Actinobacillus succinogenes.

Actinobacillus succinogenes is one of the best natural succinate-producing organisms, but it still needs engineering to further increase succinate yield and productivity. In this study, we developed a markerless knockout method for A. succinogenes using natural transformation or electroporation.

Cell-specific gene expression in Anabaena variabilis grown phototrophically, mixotrophically, and heterotrophically.

Background: When the filamentous cyanobacterium Anabaena variabilis grows aerobically without combined nitrogen, some vegetative cells differentiate into N2-fixing heterocysts, while the other vegetative cells perform photosynthesis. Microarrays of sequences within protein-encoding genes were probed with RNA purified from extracts of vegetative cells, from isolated heterocysts, and from whole filaments to investigate transcript levels, and carbon and energy metabolism, in vegetative cells and heterocysts in phototrophic, mixotrophic, and heterotrophic cultures.

Results: Heterocysts represent only 5% to 10% of cells in the filaments.

Racemization of enantiopure secondary alcohols by Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase.

Controlled racemization of enantiopure phenyl-ring-containing secondary alcohols is achieved in this study using W110A secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus (W110A TeSADH) and in the presence of the reduced and oxidized forms of its cofactor nicotinamide-adenine dinucleotide. Racemization of both enantiomers of alcohols accepted by W110A TeSADH, not only with low, but also with reasonably high, enantiomeric discrimination is achieved by this method. Furthermore, the high tolerance of TeSADH to organic solvents allows TeSADH-catalyzed racemization to be conducted in media containing up to 50% (v/v) of organic solvents.

A genomic perspective on the potential of Actinobacillus succinogenes for industrial succinate production.

Background: Succinate is produced petrochemically from maleic anhydride to satisfy a small specialty chemical market. If succinate could be produced fermentatively at a price competitive with that of maleic anhydride, though, it could replace maleic anhydride as the precursor of many bulk chemicals, transforming a multi-billion dollar petrochemical market into one based on renewable resources. Actinobacillus succinogenes naturally converts sugars and CO2 into high concentrations of succinic acid as part of a mixed-acid fermentation.

Crystallization and preliminary X-ray diffraction analysis of the Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase I86A mutant.

The Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase I86A mutant is stereospecific for (R)-alcohols instead of (S)-alcohols. Pyramidal crystals grown in the presence of (R)-phenylethanol via the hanging-drop vapour-diffusion method diffracted to 3.2 A resolution at the Canadian Light Source.

Recent advances in the biological production of mannitol.

Mannitol is a fructose-derived, 6-carbon sugar alcohol that is widely found in bacteria, yeasts, fungi, and plants. Because of its desirable properties, mannitol has many applications in pharmaceutical products, in the food industry, and in medicine. The current mannitol chemical manufacturing process yields crystalline mannitol in yields below 20 mol% from 50% glucose/50% fructose syrups.

Dynamics in Thermotoga neapolitana adenylate kinase: 15N relaxation and hydrogen-deuterium exchange studies of a hyperthermophilic enzyme highly active at 30 degrees C.

Backbone conformational dynamics of Thermotoga neapolitana adenylate kinase in the free form (TNAK) and inhibitor-bound form (TNAK*Ap5A) were investigated at 30 degrees C using (15)N NMR relaxation measurements and NMR monitored hydrogen-deuterium exchange. With kinetic parameters identical to those of Escherichia coli AK (ECAK) at 30 degrees C, TNAK is a unique hyperthermophilic enzyme. These catalytic properties make TNAK an interesting and novel model to study the interplay between protein rigidity, stability, and activity.

Thermotoga maritima TM0298 is a highly thermostable mannitol dehydrogenase.

Thermotoga maritima TM0298 is annotated as an alcohol dehydrogenase, yet it shows high identity and similarity to mesophilic mannitol dehydrogenases. To investigate this enzyme further, its gene was cloned and expressed in Escherichia coli. The purified recombinant enzyme was most active on fructose and mannitol, making it the first known hyperthermophilic mannitol dehydrogenase.

Activity and selectivity of W110A secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus in organic solvents and ionic liquids: mono- and biphasic media.

The asymmetric reduction of hydrophobic phenyl-ring-containing ketones and the enantiospecific kinetic resolution of the corresponding racemic alcohols catalyzed by Thermoanaerobacter ethanolicus W110A secondary alcohol dehydrogenase were performed in mono- and biphasic systems containing either organic solvents or ionic liquids. Both yield and enantioselectivity for these transformations can be controlled by changing the reaction medium. The enzyme showed high tolerance to both water-miscible and -immiscible solvents, which allows biotransformations to be conducted at high substrate concentrations.

13C-metabolic flux analysis of Actinobacillus succinogenes fermentative metabolism at different NaHCO3 and H2 concentrations.

Actinobacillus succinogenes naturally produces high concentrations of succinate, a potential intermediary feedstock for bulk chemical productions. A. succinogenes responds to high CO(2) and H(2) concentrations by producing more succinate and by producing less formate, acetate, and ethanol.

Crystallization, preliminary X-ray diffraction and structure analysis of Thermotoga maritima mannitol dehydrogenase.

Diffraction data have been collected from a crystal of Thermotoga maritima mannitol dehydrogenase at the Canadian Light Source. The crystal diffracted to 3.3 A resolution and belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 83.

A Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase mutant derivative highly active and stereoselective on phenylacetone and benzylacetone.

The secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus 39E (TeSADH) is highly thermostable and solvent-stable, and it is active on a broad range of substrates. These properties make TeSADH an excellent template to engineer an industrial catalyst for chiral chemical synthesis. (S)-1-Phenyl-2-propanol was our target product because it is a precursor to major pharmaceuticals containing secondary alcohol groups.

Determining Actinobacillus succinogenes metabolic pathways and fluxes by NMR and GC-MS analyses of 13C-labeled metabolic product isotopomers.

Actinobacillus succinogenes is a promising candidate for industrial succinate production. However, in addition to producing succinate, it also produces formate and acetate. To understand carbon flux distribution to succinate and alternative products we fed A.

Asymmetric reduction and oxidation of aromatic ketones and alcohols using W110A secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus.

An enantioselective asymmetric reduction of phenyl ring-containing prochiral ketones to yield the corresponding optically active secondary alcohols was achieved with W110A secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus (W110A TESADH) in Tris buffer using 2-propanol (30%, v/v) as cosolvent and cosubstrate. This concentration of 2-propanol was crucial not only to enhance the solubility of hydrophobic phenyl ring-containing substrates in the aqueous reaction medium, but also to shift the equilibrium in the reduction direction. The resulting alcohols have S-configuration, in agreement with Prelog's rule, in which the nicotinamide-adenine dinucleotide phosphate (NADPH) cofactor transfers its pro-R hydride to the re face of the ketone.

Insights into Actinobacillus succinogenes fermentative metabolism in a chemically defined growth medium.

Chemically defined media allow for a variety of metabolic studies that are not possible with undefined media. A defined medium, AM3, was created to expand the experimental opportunities for investigating the fermentative metabolism of succinate-producing Actinobacillus succinogenes. AM3 is a phosphate-buffered medium containing vitamins, minerals, NH4Cl as the main nitrogen source, and glutamate, cysteine, and methionine as required amino acids.

Influence of divalent cations on the structural thermostability and thermal inactivation kinetics of class II xylose isomerases.

The effects of divalent metal cations on structural thermostability and the inactivation kinetics of homologous class II d-xylose isomerases (XI; EC 5.3.1.

Associative mechanism for phosphoryl transfer: a molecular dynamics simulation of Escherichia coli adenylate kinase complexed with its substrates.

The ternary complex of Escherichia coli adenylate kinase (ECAK) with its substrates adenosine monophosphate (AMP) and Mg-ATP, which catalyzes the reversible transfer of a phosphoryl group between adenosine triphosphate (ATP) and AMP, was studied using molecular dynamics. The starting structure for the simulation was assembled from the crystal structures of ECAK complexed with the bisubstrate analog diadenosine pentaphosphate (AP(5)A) and of Bacillus stearothermophilus adenylate kinase complexed with AP(5)A, Mg(2+), and 4 coordinated water molecules, and by deleting 1 phosphate group from AP(5)A. The interactions of ECAK residues with the various moieties of ATP and AMP were compared to those inferred from NMR, X-ray crystallography, site-directed mutagenesis, and enzyme kinetic studies.

Construction of a shuttle vector for the overexpression of recombinant proteins in Actinobacillus succinogenes.

To express foreign proteins in Actinobacillus succinogenes, a shuttle vector was constructed based on the Actinobacillus pleuropneumoniae-Escherichia coli shuttle vector, pGZRS-19. We demonstrated that A. succinogenes is transformed by electroporation at reasonably high efficiency, that pGZRS-19 is stable in A.

Effect of overexpression of Actinobacillus succinogenes phosphoenolpyruvate carboxykinase on succinate production in Escherichia coli.

Succinate fermentation was investigated in Escherichia coli strains overexpressing Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PEPCK). In E. coli K-12, PEPCK overexpression had no effect on succinate fermentation.