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| Screen and Analysis of Enzymic Property of β-glucosidase from Metagenome of Goat (Capra hircus) Rumen |
| Fa-Zhi Xu 2, 2, 2 |
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Abstract The microbial DNA of goat (Capra hircus) rumen was extracted and a metagenomic library of uncultured microorganism was constructed, which contained 12 100 clones. Functional screening of the library led to the isolation of ten clones expressing β-glycosidase activity and a clone designated pET-6 was subcloned. Sequencing analysis showed that there was a β-glucosidase gene umcel 6X with an ORF of 1 836 bp. The encoded product shared highest homology with a Dictyoglomus thermophilumH-6-12 (GenBank No.: YP_002251384) at 55% identity and 65% similarity. The umcel 6X was expressed in Escherichia coli though IPTG and the size of the translated product Umcel 6X on SDS-PAGE was in agreement with the predicated molecular mass. Zymogram analysis revealed that Umcel 6X exhibited β-glucosidase activity, confirming that the ORF encoded a β-glucosidase. The Umcel 6X, purifed with Ni-NTA column, showed optimal activity at pH7.5 and at 50℃. Ca2+, Na+ and Mg2+ had significant positive effect on the activity of Umcel 6X, while Cu2+, Fe2+ and Hg+ gave significant inhibitory effect on the enzyme. The Ni-NTA purified recombinant β-glucosidase Umcel 6X had a specific activity of 33.6 IU/mg at pH 5.5, 35℃. Therefore, the cellulase gene from rumen would provide materials for research of degradation crude fiber in vitro.
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Received: 14 March 2013
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Corresponding Authors:
Fa-Zhi Xu
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