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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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Effect of metal ions and chemical agents on activity of Cel01. Activity without addition of metal ions or chemical agents was defined as 100%. The average of triplicate experiments is presented
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Fig6: Effect of metal ions and chemical agents on activity of Cel01. Activity without addition of metal ions or chemical agents was defined as 100%. The average of triplicate experiments is presented

Mentions: Purified Cel01 had significant activity between 30 and 50°C and exhibited optimal activity at 45 and 50°C (Fig. 5a). Cel01 was stable for 96 h below 50°C with more than 55% remaining activity (Supplementary Fig. 2). Incubation of Cel01 at 60°C caused complete inactivation of the enzyme. Interestingly, the optimal activities of other cellulases derived from soil metagenomic libraries ranged from 45 to 50°C and incubation at 60°C also resulted in rapid inactivation (Kim et al. 2008; Liu et al. 2011; Voget et al. 2006) (Table S3). Cel01 was highly active from pH 5 to pH 9 with optimal activity at pH 7 (Fig. 5b, Table S3). The cellulase activity was reduced by addition of MnCl2, ethanol, EDTA, and SDS to 62 ± 3.8%, 84 ± 5.5%, 12 ± 1.1%, and 18 ± 2.4%, respectively (Fig. 6). CoCl2 weakly stimulated the enzyme activity. When the enzyme was incubated in 3 M KCl or 4 M NaCl, it showed high halotolerance and retained more than 70% of its activity after 12 h incubation (Fig. 7).Fig. 5


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)

Effect of metal ions and chemical agents on activity of Cel01. Activity without addition of metal ions or chemical agents was defined as 100%. The average of triplicate experiments is presented
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3298741&req=5

Fig6: Effect of metal ions and chemical agents on activity of Cel01. Activity without addition of metal ions or chemical agents was defined as 100%. The average of triplicate experiments is presented
Mentions: Purified Cel01 had significant activity between 30 and 50°C and exhibited optimal activity at 45 and 50°C (Fig. 5a). Cel01 was stable for 96 h below 50°C with more than 55% remaining activity (Supplementary Fig. 2). Incubation of Cel01 at 60°C caused complete inactivation of the enzyme. Interestingly, the optimal activities of other cellulases derived from soil metagenomic libraries ranged from 45 to 50°C and incubation at 60°C also resulted in rapid inactivation (Kim et al. 2008; Liu et al. 2011; Voget et al. 2006) (Table S3). Cel01 was highly active from pH 5 to pH 9 with optimal activity at pH 7 (Fig. 5b, Table S3). The cellulase activity was reduced by addition of MnCl2, ethanol, EDTA, and SDS to 62 ± 3.8%, 84 ± 5.5%, 12 ± 1.1%, and 18 ± 2.4%, respectively (Fig. 6). CoCl2 weakly stimulated the enzyme activity. When the enzyme was incubated in 3 M KCl or 4 M NaCl, it showed high halotolerance and retained more than 70% of its activity after 12 h incubation (Fig. 7).Fig. 5

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