Abstract
The bacterial strain Paenibacillus xylanilyticus KJ-03 was isolated from a sample of soil used for cultivating Amorphophallus konjac. The cellulase gene, cel5A was cloned using fosmid library and expressed in Escherichia coli BL21 (trxB). The cel5A gene consists of a 1,743 bp open reading frame and encodes 581 amino acids of a protein. Cel5A contains N-terminal signal peptide, a catalytic domain of glycosyl hydrolase family 5, and DUF291 domain with unknown function. The recombinant cellulase was purified by Ni-affinity chromatography. The cellulase activity of Cel5A was detected in clear band with a molecular weight of 64 kDa by zymogram active staining. The maximum activity of the purified enzyme was displayed at a temperature of 40 °C and pH 6.0 when carboxymethyl cellulose was used as a substrate. It has 44% of its maximum activity at 70 °C and retained 66% of its original activity at 45 °C for 1 h. The purified cellulase hydrolyzed avicel, CMC, filter paper, xylan, and 4-methylumbelliferyl β-d-cellobiose, but no activity was detected against p-nitrophenyl β-d-glucoside. The end products of the hydrolysis of cellotetraose and cellopentaose by Cel5A were detected by thin layer chromatography, while enzyme did not hydrolyze cellobiose and cellotriose.
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Abbreviations
- Cel5A:
-
Cellulase from Paenibacillus xylanilyticus KJ-03
- LB:
-
Luria–Bertani
- TSA:
-
Tryptic soy agar
- CMC:
-
Carboxymethyl cellulose
- IPTG:
-
Isopropyl-β-d-thiogalactopyranoside
- SDS-PAGE:
-
Sodium dodecyl sulphate polyacrylamide gel electrophoresis
- DNS:
-
Dinitrosalicylic acid
- DTT:
-
Dithiothreitol
- EDTA:
-
Ethylenediaminetetraacetic acid
- TLC:
-
Thin layer chromatography
- ORF:
-
Open reading frame
- GH 5 family:
-
Glycosyl hydrolase 5 family
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Acknowledgment
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0008619).
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Park, IH., Chang, J., Lee, YS. et al. Gene Cloning of Endoglucanase Cel5A from Cellulose-Degrading Paenibacillus xylanilyticus KJ-03 and Purification and Characterization of the Recombinant Enzyme. Protein J 31, 238–245 (2012). https://doi.org/10.1007/s10930-012-9396-7
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DOI: https://doi.org/10.1007/s10930-012-9396-7