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-β-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 β-glucan, lichenin and carboxymethyl-cellulose was found as an insertion between these two regions. EngU hydrolyzed β-1,4 linkages in carboxymethyl-cellulose, but displayed its highest activity with mixed linkage (β-1,3-/β-1,4-) glucans such as barley β-glucan and lichenin, where in contrast to characterized lichenases cleavage occurred predominantly at the β-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.

To investigate the properties of a novel metagenome-derived member of the hormone-sensitive lipase family of lipolytic enzymes.

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.

A fosmid library constructed with DNA from a naturally heated (67 °C), alkaline (pH 9.3) sample from the crater of the Avachinsky volcano in the Kamchatka region was screened functionally for cellulase, xylanase and β‐glucosidase activities. Sequence analyses of the fosmids conferring xylanase and cellulase activity led to the identification of an ORF coding for a putative xylanase and an ORF coding for a putative cellulase, Xyn10K and Cel5K. Both enzymes were produced in E. coli and characterized. Purified recombinant Xyn10K (50 kDa) was active against various xylans, with beech wood xylan being the preferred substrate. Cel5K (38 kDa) showed activity against CM cellulose (CMC), lichenan and barley beta-glucan. Hydrolysis product analyses indicated that Xyn10K acts as an endoxylanase whereas Cel5K exhibits endocellulase activity. Xyn10K was active over a pH range of 6-8 and the pH optimum of Cel5K was at pH 7. The maximum hydrolytic activity of Cel5K and Xyn10K under the assay conditions employed was found at 96 °C and 95 °C respectively. Cel5K is the first characterized endoglucanase from a metagenomic library with a temperature optimum above 90 °C. Also, its exceptional resistance against thermoinactivation at temperatures above 80 °C, with a half life of 8h at 86 °C, sets this enzyme apart from previously reported metagenomic cellulases. The xylanase Xyn10K, to our knowledge the first extreme thermostable xylanase from a metagenome study, also displayed a remarkable long term-stability against thermal inactivation, with a half life of 22 h at 85 °C. Both enzymes displayed, partially, a high hydrolytic activity and stability in different ionic liquids. In conclusion, the properties of the metagenome-derived (hemi)cellulolytic enzymes reported here indicate robustness and suitability for applications in biotechnology. Keywords: Cellulase, metagenomic, thermophilic, xylanase.

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 β-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-β-glucanase. Cel01 showed optimal activity at 50°C and pH 7 being highly active from pH range 5 to 9 and possesses remarkable halotolerance.