Abstract
Fifty fluorescent pseudomonads were isolated from rhizospheric soil of green gram from nearby area of Kaziranga, Assam, India and assayed for their extracellular proteinase production. Out of these isolates, 20 were found to be prominent in proteinase production. Genetic diversity of the 20 isolates were analyzed through BOX-PCR fingerprinting and 16S rDNA-RFLP along with three reference strains, viz., Pseudomonas fluorescens (NCIM2099T), Pseudomonas aureofaciens (NCIM2026T), and Pseudomonas aeruginosa (MTCC2582T). BOX-PCR produced two distinct clusters at 56% similarity coefficient and seven distinct BOX profiles. 16S rDNA-RFLP with three tetra-cutters restriction enzymes (HaeIII, AluI, and MspI) revealed two major clusters A and B; cluster A contained only single isolate FPS9 while the rest of 22 isolates belonged to the cluster B. Based on phenotypic characters and 16S rDNA sequence similarity, all the eight highly proteinase-producing strains were affiliated with P. aeruginosa. The proteinase was extracted from two most prominent strains (KFP1 and KFP2), purified by a three-step process involving (NH4)2SO4 precipitation, gel filtration, and ion exchange chromatography. The enzyme had an optimal pH of 8.0 and exhibit highest activity at 60°C and 37°C by KFP1 and KFP2 respectively. The specific activities were recorded as 75,050 (for KFP1) and 81,320 U/mg (for KFP2). The purified enzyme was migrated as a single band on native and SDS-PAGE with a molecular mass of 32 kDa. Zn2+, Cu2+, and Ni2+ ion inhibited the enzyme activity. Enzyme activity was also inhibited by EDTA established as their metallo-proteinase nature.
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Abbreviations
- EDTA:
-
Ethylenediaminetetraacetic acid
- PMSF:
-
Phenylmethylsulfonylfluoride
- TAE:
-
Tris-acetate ethylenediaminetetraacetic acid
- DEAE:
-
Diethylaminoethyl
- SDS-PAGE:
-
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
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Acknowledgments
The work is supported by a Network Project (AMAAS), sponsored by Indian Council of Agricultural Research (ICAR), Government of India, New Delhi. The authors are thankful to the funding agency. The authors are also thankful to P.G. Rao, Director, North-East Institute of Science and Technology (CSIR), Jorhat, India for providing necessary facilities to carry out the work.
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Supplementary material 1
Phenotypic and molecular traits of fluorescent pseudomonads (Pseudomonas aeruginosa) (DOC 39 kb)
Supplementary material 2
Fig. a, b, c Restriction patterns of PCR amplified fragment of 16S rDNA digested with HaeIII, AluI, and MspI, respectively. Lane name with respective isolates: M, 100 bp molecular marker; 1, FPS 26; 2, KFP 5; 3, FPS 9; 4, FPS 324; 5, FPS 14; 6, KFP 7; 7, KFP8; 8, FPS 37; 9, NAM 7; 10, FPS 22; 11, FPS 211; 12, KFP1; 13, FPS 5; 14, FPS 24; 15, KFP3; 16, KFP 9; 17 KFP 2; 18, KFP 357; 19, FPS 18; 20, KFP4; 21, P. fluorescens NCIM2099T; 22, P. aeruginosa MTCC2582T; and 23, P. aureofaciens NCIM 2026T (DOC 180 kb)
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Sarma, R.K., Debnath, R., Saikia, R. et al. Phylogenetic analysis of alkaline proteinase producing fluorescent pseudomonads associated with green gram (Vigna radiata L.) rhizosphere. Folia Microbiol 57, 129–137 (2012). https://doi.org/10.1007/s12223-012-0097-6
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DOI: https://doi.org/10.1007/s12223-012-0097-6