A metagenome-derived thermostable ß-glucanase with an unusual module architecture which defines the new glycoside hydrolase family GH148.

The discovery of novel and robust enzymes for the breakdown of plant biomass bears tremendous potential for the development of sustainable production processes in the rapidly evolving new bioeconomy. By functional screening of a metagenomic library from a volcano soil sample a novel thermostable endo-ß-glucanase (EngU) which is unusual with regard to its module architecture and cleavage specificity was identified. Various recombinant EngU variants were characterized. Assignment of EngU to an existing glycoside hydrolase (GH) family was not possible. Two regions of EngU showed weak sequence similarity to proteins of the GH clan GH-A, and acidic residues crucial for catalytic activity of EngU were identified by mutation. Unusual, a carbohydrate-binding module (CBM4) which displayed binding affinity for ß-glucan, lichenin and carboxymethyl-cellulose was found as an insertion between these two regions. EngU hydrolyzed ß-1,4 linkages in carboxymethyl-cellulose, but displayed its highest activity with mixed linkage (ß-1,3-/ß-1,4-) glucans such as barley ß-glucan and lichenin, where in contrast to characterized lichenases cleavage occurred predominantly at the ß-1,3 linkages of C4-substituted glucose residues. EngU and numerous related enzymes with previously unknown function represent a new GH family of biomass-degrading enzymes within the GH-A clan. The name assigned to the new GH family is GH148.

A novel, versatile family IV carboxylesterase exhibits high stability and activity in a broad pH spectrum.

OBJECTIVES: To investigate the properties of a novel metagenome-derived member of the hormone-sensitive lipase family of lipolytic enzymes. RESULTS: A forest soil metagenome-derived gene encoding an esterase (Est06) belonging to the hormone-sensitive lipase family of lipolytic enzymes was subcloned, heterologously expressed and characterized. Est06 is a polypeptide of 295 amino acids with a molecular mass of 31 kDa. The deduced protein sequence shares 61% similarity with a hypothetical protein from the marine symbiont Candidatus Entotheonella sp. TSY1. Purified Est06 exhibited high affinity for acyl esters with short-chain fatty acids, and showed optimum activity with p-nitrophenyl valerate (C5). Maximum enzymatic activity was at 50 °C and pH 7. Est06 exhibited high stability at moderate temperatures by retaining all of its catalytic activity below 30 °C over 13 days. Additionally, Est06 displayed high stability between pH 5 and 9. Esterase activity was not inhibited by metal ions or detergents, although organic solvents decreased activity. CONCLUSIONS: The combination of Est06 properties place it among novel biocatalysts that have potential for industrial use including low temperature applications.

VOC emission of various Serratia species and isolates and genome analysis of Serratia plymuthica 4Rx13.

Bacteria emit a wealth of volatile organic compounds. Gas chromatography coupled to mass spectrometry analysis of five Serratia strains revealed ketones, dimethyl di- and trisulfide and 2-phenylethanol commonly released in this genus. The polymethylated bicyclic hydrocarbon sodorifen was uniquely released by the rhizobacterium Serratia plymuthica 4Rx13. Of 10 Serratia strains, only S. plymuthica isolates originating from plants grown on fields near Rostock (Germany) released this new and unusual compound. Since the biosynthetic pathway of sodorifen was unknown, the genome sequence of S. plymuthica 4Rx13 was determined and annotated. Genome comparison of S. plymuthica 4Rx13 with sodorifen non-producing Serratia species highlighted 246 unique candidate open reading frames.

Identification and characterization of novel cellulolytic and hemicellulolytic genes and enzymes derived from German grassland soil metagenomes.

Soil metagenomes represent an unlimited resource for the discovery of novel biocatalysts from soil microorganisms. Three large-inserts metagenomic DNA libraries were constructed from different grassland soil samples and screened for genes conferring cellulase or xylanase activity. Function-driven screening identified a novel cellulase-encoding gene (cel01) and two xylanase-encoding genes (xyn01 and xyn02). From sequence and protein domain analyses, Cel01 (831 amino acids) belongs to glycoside hydrolase family 9 whereas Xyn01 (170 amino acids) and Xyn02 (255 amino acids) are members of glycoside hydrolase family 11. Cel01 harbors a family 9 carbohydrate-binding module, previously found only in xylanases. Both Xyn01 and Xyn02 were most active at 60°C with high activities from 4 to 10 and optimal at pH 7 (Xyn01) and pH 6 (Xyn02). The cellulase gene, cel01, was expressed in E. coli BL21 and the recombinant enzyme (91.9 kDa) was purified. Cel01 exhibited high activity with soluble cellulose substrates containing ß-1,4-linkages. Activity with microcrystalline cellulose was not detected. These data, together with the analysis of the degradation profiles of carboxymethyl cellulose and barley glucan indicated that Cel01 is an endo 1,4-ß-glucanase. Cel01 showed optimal activity at 50°C and pH 7 being highly active from pH range 5 to 9 and possesses remarkable halotolerance.