Characterization of a thermoactive endoglucanase isolated from a biogas plant metagenome.

A metagenomic library from DNA isolated from a biogas plant was constructed and screened for thermoactive endoglucanases to gain insight into the enzymatic diversity involved in plant biomass breakdown at elevated temperatures. Two cellulase-encoding genes were identified and the corresponding proteins showed sequence similarities of 59% for Cel5A to a putative cellulase from Anaerolinea thermolimosa and 99% for Cel5B to a characterized endoglucanase isolated from a biogas plant reactor. The cellulase Cel5A consists of one catalytical domain showing sequence similarities to glycoside hydrolase family 5 and comprises 358 amino acids with a predicted molecular mass of 41.2 kDa. The gene coding for cel5A was successfully cloned and expressed in Escherichia coli C43(DE3). The recombinant protein was purified to homogeneity using affinity chromatography with a specific activity of 182 U/mg, and a yield of 74%. Enzymatic activity was detectable towards cellulose and mannan containing substrates and over a broad temperature range from 40 °C to 70 °C and a pH range from 4.0 to 7.0 with maximal activity at 55 °C and pH 5.0. Cel5A showed high thermostability at 60 °C without loss of activity after 24 h. Due to the enzymatic characteristics, Cel5A is an attractive candidate for the degradation of lignocellulosic material.

Complete genome sequence of Thermus brockianus GE-1 reveals key enzymes of xylan/xylose metabolism.

Thermus brockianus strain GE-1 is a thermophilic, Gram-negative, rod-shaped and non-motile bacterium that was isolated from the Geysir geothermal area, Iceland. Like other thermophiles, Thermus species are often used as model organisms to understand the mechanism of action of extremozymes, especially focusing on their heat-activity and thermostability. Genome-specific features of T. brockianus GE-1 and their properties further help to explain processes of the adaption of extremophiles at elevated temperatures. Here we analyze the first whole genome sequence of T. brockianus strain GE-1. Insights of the genome sequence and the methodologies that were applied during de novo assembly and annotation are given in detail. The finished genome shows a phred quality value of QV50. The complete genome size is 2.38 Mb, comprising the chromosome (2,035,182 bp), the megaplasmid pTB1 (342,792 bp) and the smaller plasmid pTB2 (10,299 bp). Gene prediction revealed 2,511 genes in total, including 2,458 protein-encoding genes, 53 RNA and 66 pseudo genes. A unique genomic region on megaplasmid pTB1 was identified encoding key enzymes for xylan depolymerization and xylose metabolism. This is in agreement with the growth experiments in which xylan is utilized as sole source of carbon. Accordingly, we identified sequences encoding the xylanase Xyn10, an endoglucanase, the membrane ABC sugar transporter XylH, the xylose-binding protein XylF, the xylose isomerase XylA catalyzing the first step of xylose metabolism and the xylulokinase XylB, responsible for the second step of xylose metabolism. Our data indicate that an ancestor of T. brockianus obtained the ability to use xylose as alternative carbon source by horizontal gene transfer.

First Glycoside Hydrolase Family 2 Enzymes from Thermus antranikianii and Thermus brockianus with ß-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 ß-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 ß-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 ß-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.

Exploration of extremophiles for high temperature biotechnological processes.

Industrial processes often take place under harsh conditions that are hostile to microorganisms and their biocatalysts. Microorganisms surviving at temperatures above 60°C represent a chest of biotechnological treasures for high-temperature bioprocesses by producing a large portfolio of biocatalysts (thermozymes). Due to the unique requirements to cultivate thermophilic (60-80°C) and hyperthermophilic (80-110°C) Bacteria and Archaea, less than 5% are cultivable in the laboratory. Therefore, other approaches including sequence-based screenings and metagenomics have been successful in providing novel thermozymes. In particular, polysaccharide-degrading enzymes (amylolytic enzymes, hemicellulases, cellulases, pectinases and chitinases), lipolytic enzymes and proteases from thermophiles have attracted interest due to their potential for versatile applications in pharmaceutical, chemical, food, textile, paper, leather and feed industries as well as in biorefineries.

Characterization of a heat-active archaeal ß-glucosidase from a hydrothermal spring metagenome.

Thermostable enzymes are required for application in a wide range of harsh industrial processes. High stability and activity at elevated temperatures, as well as high tolerances toward various reagents and solvents, are needed. In this work, a glycoside hydrolase family 1 ß-glucosidase (Bgl1) of archaeal origin was isolated from a hydrothermal spring metagenome. The enzyme showed a broad substrate spectrum with activity toward cellobiose, cellotriose and lactose. Compared to most enzymes, extremely high specific activity with 3195U/mg was observed at 90°C and pH 6.5. Bgl1 was completely stable at pH 4.5-9.5 for 48 h at 4 °C. More than 40% of activity was measured at 105 °C. A thermal activation was observed at 90 °C after 30 min. Enzyme stability was enhanced (5- and 7-fold) after applying pressure of 100 and 200 bar at 90 °C for 2h, respectively. The affinity of the ß-glucosidase to its substrate was significantly increased in the presence of AlCl3. The K(i) value for glucose was 150 mM. These distinctive characteristics distinguish Bgl1 from other enzymes described so far and make this enzyme suitable for application in numerous processes that run at high temperatures.