Non-canonical amino acids as a useful synthetic biological tool for lipase-catalysed reactions in hostile environments

The incorporation of several non-canonical amino acids into the Thermoanaerobacter thermohydrosulfuricus lipase confers not only activity enhancement upon treatment with organic solvents (by up to 450%) and surfactants (resp. 1630%), but also protective effects against protein reducing (resp. 140%), alkylating (resp. 160%), and denaturing (resp.190%) agents as well as inhibitors (resp. 40%). This approach offers novel chemically diversified biocatalysts for hostile environments.DFG, EXC 314, Unifying Concepts in Catalysi

Isolation and biochemical characterization of a glucose dehydrogenase from a hay infusion metagenome

Glucose hydrolyzing enzymes are essential to determine blood glucose level. A high-throughput screening approach was established to identify NAD(P)-dependent glucose dehydrogenases for the application in test stripes and the respective blood glucose meters. In the current report a glucose hydrolyzing enzyme, derived from a metagenomic library by expressing recombinant DNA fragments isolated from hay infusion, was characterized. The recombinant clone showing activity on glucose as substrate exhibited an open reading frame of 987 bp encoding for a peptide of 328 amino acids. The isolated enzyme showed typical sequence motifs of short-chain-dehydrogenases using NAD(P) as a co-factor and had a sequence similarity between 33 and 35% to characterized glucose dehydrogenases from different Bacillus species. The identified glucose dehydrogenase gene was expressed in E. coli, purified and subsequently characterized. The enzyme, belonging to the superfamily of short-chain dehydrogenases, shows a broad substrate range with a high affinity to glucose, xylose and glucose-6-phosphate. Due to its ability to be strongly associated with its cofactor NAD(P), the enzyme is able to directly transfer electrons from glucose oxidation to external electron acceptors by regenerating the cofactor while being still associated to the protein. © 2014 Basner, Antranikian

Microorganisms for a circular bioeconomy to remove CO2 from the atmosphere and reduce pollution

Microorganisms for a circular bioeconomy to remove CO2 from the atmosphere and reduce pollution: The greatest global and human-induced challenges of our time are climate change caused by fossil fuels, global pollution, and rapidly evolving infections in plants, humans, and animals. To address the necessary global changes, a dramatic transformation in science and society must take place. This change will require very intensive and forward-looking industrial and basic research, strongly focused on the development of (bio)technologies and industrial bioprocesses towards a low carbon sustainable bioeconomy. In this transition, efficient microorganisms will play a significant and global role as technology drivers. They harbor in their genomes the keys and blueprints for sustainable biotechnology. In this article, we outline urgent and important areas of microbial research and technological development that will ultimately contribute significantly to the transition from a linear to a circular bioeconomy

pH profile of GDH 1E5.

<p>The enzyme activity was assayed at various pH values in Britton & Robinson universal buffer in the range of pH 2–11 using DCPIP as electron acceptor. The enzymatic activity was assayed for 5 min at 35°C and the according pH using DCPIP as electron acceptor. All data represent the average of triplicate determinations ± standard derivation.</p

Isolation and Biochemical Characterization of a Glucose Dehydrogenase from a Hay Infusion Metagenome

<div><p>Glucose hydrolyzing enzymes are essential to determine blood glucose level. A high-throughput screening approach was established to identify NAD(P)-dependent glucose dehydrogenases for the application in test stripes and the respective blood glucose meters. In the current report a glucose hydrolyzing enzyme, derived from a metagenomic library by expressing recombinant DNA fragments isolated from hay infusion, was characterized. The recombinant clone showing activity on glucose as substrate exhibited an open reading frame of 987 bp encoding for a peptide of 328 amino acids. The isolated enzyme showed typical sequence motifs of short-chain-dehydrogenases using NAD(P) as a co-factor and had a sequence similarity between 33 and 35% to characterized glucose dehydrogenases from different <i>Bacillus</i> species. The identified glucose dehydrogenase gene was expressed in <i>E. coli</i>, purified and subsequently characterized. The enzyme, belonging to the superfamily of short-chain dehydrogenases, shows a broad substrate range with a high affinity to glucose, xylose and glucose-6-phosphate. Due to its ability to be strongly associated with its cofactor NAD(P), the enzyme is able to directly transfer electrons from glucose oxidation to external electron acceptors by regenerating the cofactor while being still associated to the protein.</p></div

What we learn from extremophiles

Extremophiles are microorganisms that love extreme conditions, such as high temperatures up to the boiling point of water or low temperatures down to below the freezing point. Moreover, some extreme microbes prefer to live in acidic or alkaline environments, under high pressure or high salinity. Three extremophilic species are presented in this article: Lacinutrix algicola, a psychrophilic bacterium that grows at temperatures between 0 and 25 °C, Anaerobranca gottschalkii, a thermophilic and alkaliphilic bacterium growing optimally at 50–55 °C under alkaline conditions, and Pyrococcus furiosus, a famous hyperthermophilic archaeon that prefers 100 °C for growth. These extraordinary microorganisms are examples of extremophiles that possess remarkable adaptation mechanisms and additionally produce unique enzymes called extremozymes. These robust biocatalysts can be applied in various biotechnologic processes to enable substrate conversions under extreme process conditions. Due to their unusual properties, extremophiles and extremozymes will play a pivotal role in the development of modern circular bioeconomy

Purification table of the GDH1E5.

<p>^a Total protein was determined after cell disruption in the soluble fraction by the method of Bradford.</p><p>^b Total activity was determined using <i>p</i>-nitrosoaniline BM53.0861 as electron acceptor at 35°C and pH 6.0.</p

First Glycoside Hydrolase Family 2 Enzymes from Thermus antranikianii and Thermus brockianus with β-Glucosidase Activity

Two glycoside hydrolase encoding genes (tagh2 and tbgh2) were identified from different Thermus species using functional screening. Based on amino acid similarities, the enzymes were predicted to belong to glycoside hydrolase (GH) family 2. Surprisingly, both enzymes (TaGH2 and TbGH2) showed twofold higher activities for the hydrolysis of nitrophenol-linked β-D-glucopyranoside than of -galactopyranoside. Specific activities of 3,966 U/mg for TaGH2 and 660 U/mg for TbGH2 were observed. In accordance, K m values for both enzymes were significantly lower when β-D-glucopyranoside was used as substrate. Furthermore, TaGH2 was able to hydrolyze cellobiose. TaGH2 and TbGH2 exhibited highest activity at 95 and 90°C at pH 6.5. Both enzymes were extremely thermostable and showed thermal activation up to 250% relative activity at temperatures of 50 and 60°C. Especially, TaGH2 displayed high tolerance toward numerous metal ions (Cu(2+), Co(2+), Zn(2+)), which are known as glycoside hydrolase inhibitors. In this study, the first thermoactive GH family 2 enzymes with β-glucosidase activity have been identified and characterized. The hydrolysis of cellobiose is a unique property of TaGH2 when compared to other enzymes of GH family 2. Our work contributes to a broader knowledge of substrate specificities in GH family 2.German Federal Ministry of Education and Research (BMBF), German Research Foundation (DFG