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

Nacke H, Engelhaupt M, Brady S, Fischer C, Tautzt J, Daniel R - Biotechnol. Lett. (2011)

Bottom Line: Cel01 harbors a family 9 carbohydrate-binding module, previously found only in xylanases.Activity with microcrystalline cellulose was not detected.Cel01 showed optimal activity at 50°C and pH 7 being highly active from pH range 5 to 9 and possesses remarkable halotolerance.

View Article: PubMed Central - PubMed

Affiliation: Department of Genomic and Applied Microbiology, Göttingen Genomics Laboratory, Institute of Microbiology and Genetics, Georg-August University Göttingen, Grisebachstr. 8, 37077, Göttingen, Germany.

ABSTRACT
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.

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Multiple sequence alignment of conserved regions in glycoside hydrolases belonging to family 9 (a) and family 11 (b). Identical amino acid residues are shown as white letters on a dark background. Triangles indicate catalytic residues and asterisks represent residues that are involved in substrate binding. References: Cel01, Xyn01, and Xyn02 (this study); ZP_07606292, GH9 from Streptomyces violaceusniger Tu 4113; YP_004081973, GH9 from Micromonospora sp. L5; YP_001612873, cellulase from Sorangium cellulosum ‘So ce 56’; ZP_05497266, GH9 from Clostridium papyrosolvens DSM 2782; AAK24198, GH9 from Caulobacter crescentus CB15; BAA06837, xylanase I precursor from Aeromonas punctata; AAB72117, endo-beta-1,4-xylanase from Geobacillus stearothermophilus; AAQ14588, xylanase from Bacillus firmus; CAJ87325, endo-1,4-beta-xylanase B from Thermobacillus xylanilyticus; AAZ17386, endo-1,4-beta-xylanase from Paenibacillus macerans; YP_001310136, endo-1,4-beta-xylanase from Clostridium beijerinckii NCIMB 8052; AAD54767, endo-1,4-beta-xylanase from Xylanimicrobium pachnodae; ABL11222, xylanase from uncultured bacterium
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Fig2: Multiple sequence alignment of conserved regions in glycoside hydrolases belonging to family 9 (a) and family 11 (b). Identical amino acid residues are shown as white letters on a dark background. Triangles indicate catalytic residues and asterisks represent residues that are involved in substrate binding. References: Cel01, Xyn01, and Xyn02 (this study); ZP_07606292, GH9 from Streptomyces violaceusniger Tu 4113; YP_004081973, GH9 from Micromonospora sp. L5; YP_001612873, cellulase from Sorangium cellulosum ‘So ce 56’; ZP_05497266, GH9 from Clostridium papyrosolvens DSM 2782; AAK24198, GH9 from Caulobacter crescentus CB15; BAA06837, xylanase I precursor from Aeromonas punctata; AAB72117, endo-beta-1,4-xylanase from Geobacillus stearothermophilus; AAQ14588, xylanase from Bacillus firmus; CAJ87325, endo-1,4-beta-xylanase B from Thermobacillus xylanilyticus; AAZ17386, endo-1,4-beta-xylanase from Paenibacillus macerans; YP_001310136, endo-1,4-beta-xylanase from Clostridium beijerinckii NCIMB 8052; AAD54767, endo-1,4-beta-xylanase from Xylanimicrobium pachnodae; ABL11222, xylanase from uncultured bacterium

Mentions: Cellulases and xylanases are subdivided in different glycoside hydrolase families based on amino acid sequence similarity (Cantarel et al. 2009; Henrissat 1991). The amino acid sequence deduced from the putative cellulase gene cel01 of pLC01 comprised 831 amino acids with a calculated molecular mass of 90.4 kDa. A potential signal peptide of 21 amino acids was predicted at the N-terminus of Cel01. The amino acid sequence of the putative signal peptide showed the typical orientation of signal peptides with three distinct parts (N, H, and C domains) (Pugsley 1993). Protein domain analyses suggested that Cel01 belongs to the glycoside hydrolase family 9, as it contains a catalytic domain (amino acids 350–814) that is typical for family 9 members (Fig. 1). The multiple sequence alignment revealed that Cel01 contained putative catalytic aspartate and glutamate residues (D418, D421, and E808), which act as nucleophile (aspartate) or proton donor (glutamate) during substrate hydrolysis (Parsiegla et al. 2002) (Fig. 2a). In addition to the catalytic domain of family 9 glycoside hydrolases, Cel01 harbored a family 9 carbohydrate-binding module (CBM9, amino acids 46–231) and a N-terminal cellulase domain (Cel-N-term, amino acids 255–338) (Fig. 1). To our knowledge, family 9 carbohydrate-binding modules have previously been detected only in xylanases. The protein sequence of Cel01 was most similar (50% identity) to a cellulase from Sorangium cellulosum ‘So ce 56’ (Schneiker et al. 2007), but not over the entire length. The cellulase from Sorangium cellulosum possesses a similar modular structure as Cel01, but lacks a family 9 carbohydrate-binding module. Most of the metagenome-derived cellulase genes that have been identified by activity-based screening belonged to glycoside hydrolase families 5 and 9 (Duan and Feng 2010). Metagenome-derived family 9 cellulases were isolated from an aquatic community (Pottkämper et al. 2009), elephant dung (Wang et al. 2009), and an enrichment culture of an alkaline lake (Grant et al. 2004) but not from other soils with the exception of compost soil (Pang et al. 2009). In summary, molecular analysis indicated that Cel01 is an extracellular cellulase belonging to glycoside hydrolase family 9. In addition, the enzyme exhibited a modular structure, which is not known from other cellulases.Fig. 1


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

Nacke H, Engelhaupt M, Brady S, Fischer C, Tautzt J, Daniel R - Biotechnol. Lett. (2011)

Multiple sequence alignment of conserved regions in glycoside hydrolases belonging to family 9 (a) and family 11 (b). Identical amino acid residues are shown as white letters on a dark background. Triangles indicate catalytic residues and asterisks represent residues that are involved in substrate binding. References: Cel01, Xyn01, and Xyn02 (this study); ZP_07606292, GH9 from Streptomyces violaceusniger Tu 4113; YP_004081973, GH9 from Micromonospora sp. L5; YP_001612873, cellulase from Sorangium cellulosum ‘So ce 56’; ZP_05497266, GH9 from Clostridium papyrosolvens DSM 2782; AAK24198, GH9 from Caulobacter crescentus CB15; BAA06837, xylanase I precursor from Aeromonas punctata; AAB72117, endo-beta-1,4-xylanase from Geobacillus stearothermophilus; AAQ14588, xylanase from Bacillus firmus; CAJ87325, endo-1,4-beta-xylanase B from Thermobacillus xylanilyticus; AAZ17386, endo-1,4-beta-xylanase from Paenibacillus macerans; YP_001310136, endo-1,4-beta-xylanase from Clostridium beijerinckii NCIMB 8052; AAD54767, endo-1,4-beta-xylanase from Xylanimicrobium pachnodae; ABL11222, xylanase from uncultured bacterium
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Related In: Results  -  Collection

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Fig2: Multiple sequence alignment of conserved regions in glycoside hydrolases belonging to family 9 (a) and family 11 (b). Identical amino acid residues are shown as white letters on a dark background. Triangles indicate catalytic residues and asterisks represent residues that are involved in substrate binding. References: Cel01, Xyn01, and Xyn02 (this study); ZP_07606292, GH9 from Streptomyces violaceusniger Tu 4113; YP_004081973, GH9 from Micromonospora sp. L5; YP_001612873, cellulase from Sorangium cellulosum ‘So ce 56’; ZP_05497266, GH9 from Clostridium papyrosolvens DSM 2782; AAK24198, GH9 from Caulobacter crescentus CB15; BAA06837, xylanase I precursor from Aeromonas punctata; AAB72117, endo-beta-1,4-xylanase from Geobacillus stearothermophilus; AAQ14588, xylanase from Bacillus firmus; CAJ87325, endo-1,4-beta-xylanase B from Thermobacillus xylanilyticus; AAZ17386, endo-1,4-beta-xylanase from Paenibacillus macerans; YP_001310136, endo-1,4-beta-xylanase from Clostridium beijerinckii NCIMB 8052; AAD54767, endo-1,4-beta-xylanase from Xylanimicrobium pachnodae; ABL11222, xylanase from uncultured bacterium
Mentions: Cellulases and xylanases are subdivided in different glycoside hydrolase families based on amino acid sequence similarity (Cantarel et al. 2009; Henrissat 1991). The amino acid sequence deduced from the putative cellulase gene cel01 of pLC01 comprised 831 amino acids with a calculated molecular mass of 90.4 kDa. A potential signal peptide of 21 amino acids was predicted at the N-terminus of Cel01. The amino acid sequence of the putative signal peptide showed the typical orientation of signal peptides with three distinct parts (N, H, and C domains) (Pugsley 1993). Protein domain analyses suggested that Cel01 belongs to the glycoside hydrolase family 9, as it contains a catalytic domain (amino acids 350–814) that is typical for family 9 members (Fig. 1). The multiple sequence alignment revealed that Cel01 contained putative catalytic aspartate and glutamate residues (D418, D421, and E808), which act as nucleophile (aspartate) or proton donor (glutamate) during substrate hydrolysis (Parsiegla et al. 2002) (Fig. 2a). In addition to the catalytic domain of family 9 glycoside hydrolases, Cel01 harbored a family 9 carbohydrate-binding module (CBM9, amino acids 46–231) and a N-terminal cellulase domain (Cel-N-term, amino acids 255–338) (Fig. 1). To our knowledge, family 9 carbohydrate-binding modules have previously been detected only in xylanases. The protein sequence of Cel01 was most similar (50% identity) to a cellulase from Sorangium cellulosum ‘So ce 56’ (Schneiker et al. 2007), but not over the entire length. The cellulase from Sorangium cellulosum possesses a similar modular structure as Cel01, but lacks a family 9 carbohydrate-binding module. Most of the metagenome-derived cellulase genes that have been identified by activity-based screening belonged to glycoside hydrolase families 5 and 9 (Duan and Feng 2010). Metagenome-derived family 9 cellulases were isolated from an aquatic community (Pottkämper et al. 2009), elephant dung (Wang et al. 2009), and an enrichment culture of an alkaline lake (Grant et al. 2004) but not from other soils with the exception of compost soil (Pang et al. 2009). In summary, molecular analysis indicated that Cel01 is an extracellular cellulase belonging to glycoside hydrolase family 9. In addition, the enzyme exhibited a modular structure, which is not known from other cellulases.Fig. 1

Bottom Line: Cel01 harbors a family 9 carbohydrate-binding module, previously found only in xylanases.Activity with microcrystalline cellulose was not detected.Cel01 showed optimal activity at 50°C and pH 7 being highly active from pH range 5 to 9 and possesses remarkable halotolerance.

View Article: PubMed Central - PubMed

Affiliation: Department of Genomic and Applied Microbiology, Göttingen Genomics Laboratory, Institute of Microbiology and Genetics, Georg-August University Göttingen, Grisebachstr. 8, 37077, Göttingen, Germany.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus