Cloning and characterization of a bifunctional glycosyl hydrolase from an antagonistic Pseudomonas putida strain P3(4)
IR@IHBT: CSIR-Institute of Himalayan Bioresource Technology, Palampur
View Archive Info| Field | Value | |
| Title |
Cloning and characterization of a bifunctional glycosyl
hydrolase from an antagonistic Pseudomonas putida
strain P3(4)
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| Creator |
Singh , N A
Shanmugam, V |
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| Subject |
Plant sciences
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| Description |
A fluorescent pseudomonad strain P3(4) showing chitinolysis on chitinase detection agar and
antagonism against Fusarium oxysporum f.sp dianthi causing vascular wilt of carnation was
isolated from pea rhizosphere soil. PCR primers specific for glycosyl hydrolase family 5 (GH5)
of Pseudomonas putida isolate KT2440 amplified a 947 bp fragment of the GH5 gene from P3(4).
Cloning of this gene into Escherichia coli M15 using an expression vector pQE-30UA and
screening on chitin and chitosan detection agar identified one positive clone (Pchi+). Sequence
analysis of the cloned insert revealed an open reading frame of 947 nucleotides corresponding
to a protein of 315 amino acids with a predicted molecular mass of 38.0 kDa. The deduced
amino acid sequence of the open reading frame (gene product/GH) showed 83–84% homology
to the GH5 of P. putida strains F1 and KT2440, respectively. The purified enzyme was homogenous,
as examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was
visualized as single fluorescent band in native gel assay with 4-methylumbelliferyl-N-acetyl-β-Dglucosaminide
and glycol chitosan, respectively. For hydrolysis of 4-nitrophenyl-N-acetyl-β-Dglucosaminide
(pNP-(GlcNAc) and colloidal chitosan, the enzyme had an optimal temperature
of 40 °C, and was stable within the temperature range of 10 °C to 40 °C. The enzyme showed an
optimal pH of 3.5, with maximum stabilities at 5.0 and 5.5 for hydrolysis of pNP-(GlcNAc) and
colloidal chitosan, respectively. Fe3+ and Cu2+ stimulated chitinase and chitosanase activities by
74.2 and 51.4%, respectively. The purified GH displayed 70 and 45% inhibition of spore
germination of the pathogenic fungi, Fusarium oxysporum f.sp. dianthi and Alternaria solani,
respectively
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| Date |
2011
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| Type |
Article
PeerReviewed |
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| Format |
application/pdf
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| Identifier |
http://ihbt.csircentral.net/1049/1/58_Cloning_and_Charatrization_2011.pdf
Singh , N A and Shanmugam, V (2011) Cloning and characterization of a bifunctional glycosyl hydrolase from an antagonistic Pseudomonas putida strain P3(4). Journal of Basic Microbiology , 51. pp. 1-10. |
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| Relation |
http://ihbt.csircentral.net/1049/
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