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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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Analysis of Cel01 production and purification by SDS PAGE (a) and Western blot analysis (b). His6-tagged Cel01 was purified from cell extract of E. coli BL21/pCel01 by nickel affinity chromatography. aLanes: M, marker proteins; 1, crude extract of E. coli BL21/pCel01; 2, flow through fraction; 3, wash fraction; 4, eluate (further purified by ultrafiltration); bLanes: M, marker proteins 5, crude extract; 6, purified Cel01
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Fig3: Analysis of Cel01 production and purification by SDS PAGE (a) and Western blot analysis (b). His6-tagged Cel01 was purified from cell extract of E. coli BL21/pCel01 by nickel affinity chromatography. aLanes: M, marker proteins; 1, crude extract of E. coli BL21/pCel01; 2, flow through fraction; 3, wash fraction; 4, eluate (further purified by ultrafiltration); bLanes: M, marker proteins 5, crude extract; 6, purified Cel01

Mentions: To facilitate purification of the cellulase Cel01 the corresponding gene was amplified by PCR without the signal peptide sequence. The latter necessitated the addition of a start codon to the 5′ end of the coding region. The resulting PCR product was cloned into the expression vector pET101/D, thereby placing the genes under control of the IPTG-inducible T7 promoter and adding sequences encoding a His6 tag and a V5 epitope. The resulting construct pCel01 (Table 1) was transformed into E. coli BL21 and production of Cel01 was induced by addition of 0.5 mM IPTG. After incubation for 12 h at 37°C, cells were harvested and crude cell-free extracts were prepared. The production of His6-tagged Cel01 in the cell extracts was confirmed by Western-Blot analysis using antibodies against the V5 epitope (Fig. 3) and detection of cellulase activity. Subsequently, His6-tagged Cel01 was purified from cell-free extracts by metal ion affinity chromatography and ultrafiltration. The specific cellulase activity of the final enzyme preparation was 780 ± 11.5 U mg−1 with barley glucan as substrate. SDS–PAGE of the purified enzyme revealed that Cel01 has a MW of approx. 90 kDa (Fig. 3). The observed molecular mass is in good agreement with the one deduced from the sequence of His6-tagged version of Cel01 (91.9 kDa).Fig. 3


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)

Analysis of Cel01 production and purification by SDS PAGE (a) and Western blot analysis (b). His6-tagged Cel01 was purified from cell extract of E. coli BL21/pCel01 by nickel affinity chromatography. aLanes: M, marker proteins; 1, crude extract of E. coli BL21/pCel01; 2, flow through fraction; 3, wash fraction; 4, eluate (further purified by ultrafiltration); bLanes: M, marker proteins 5, crude extract; 6, purified Cel01
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Related In: Results  -  Collection

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Fig3: Analysis of Cel01 production and purification by SDS PAGE (a) and Western blot analysis (b). His6-tagged Cel01 was purified from cell extract of E. coli BL21/pCel01 by nickel affinity chromatography. aLanes: M, marker proteins; 1, crude extract of E. coli BL21/pCel01; 2, flow through fraction; 3, wash fraction; 4, eluate (further purified by ultrafiltration); bLanes: M, marker proteins 5, crude extract; 6, purified Cel01
Mentions: To facilitate purification of the cellulase Cel01 the corresponding gene was amplified by PCR without the signal peptide sequence. The latter necessitated the addition of a start codon to the 5′ end of the coding region. The resulting PCR product was cloned into the expression vector pET101/D, thereby placing the genes under control of the IPTG-inducible T7 promoter and adding sequences encoding a His6 tag and a V5 epitope. The resulting construct pCel01 (Table 1) was transformed into E. coli BL21 and production of Cel01 was induced by addition of 0.5 mM IPTG. After incubation for 12 h at 37°C, cells were harvested and crude cell-free extracts were prepared. The production of His6-tagged Cel01 in the cell extracts was confirmed by Western-Blot analysis using antibodies against the V5 epitope (Fig. 3) and detection of cellulase activity. Subsequently, His6-tagged Cel01 was purified from cell-free extracts by metal ion affinity chromatography and ultrafiltration. The specific cellulase activity of the final enzyme preparation was 780 ± 11.5 U mg−1 with barley glucan as substrate. SDS–PAGE of the purified enzyme revealed that Cel01 has a MW of approx. 90 kDa (Fig. 3). The observed molecular mass is in good agreement with the one deduced from the sequence of His6-tagged version of Cel01 (91.9 kDa).Fig. 3

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