title: Cloning and characterization of a bifunctional glycosyl hydrolase from an antagonistic Pseudomonas putida strain P3(4).
abstract: 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;-D-glucosaminide and glycol chitosan, respectively. For hydrolysis of 4-nitrophenyl-N -acetyl-β;-D-glucosaminide (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. Fe(3+) and Cu(2+) 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.
authors.affiliation: Floriculture Pathology Laboratory, Institute of Himalayan Bioresource Technology, Council of Scientific and Industrial Research, Palampur, Himachal Pradesh, India.
authors.forename: Naosekpam Ajit
authors.initials: NA
authors.lastname: Singh
authors.forename: Veerubommu
authors.initials: V
authors.lastname: Shanmugam
mesh_terms: D000528:Alternaria / Q000254:growth & development; D000898:Antibiosis; D002686:Chitin / Q000378:metabolism*; D048271:Chitosan / Q000378:metabolism*; D003001:Cloning, Molecular; D003470:Culture Media / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004586:Electrophoresis; D020536:Enzyme Activators / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005670:Fusarium / Q000254:growth & development; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D003470:Culture Media; D004269:DNA, Bacterial; D020536:Enzyme Activators; D008670:Metals; D011994:Recombinant Proteins; D002686:Chitin; D048271:Chitosan; D006026:Glycoside Hydrolases