ABB  Vol.5 No.4 , March 2014
An Approach to the Identification and Characterisation of a Psychrotrophic Lipase Producing Pseudomonas sp ADT3 from Arctic Region
ABSTRACT

The aim of this research work was to explore psychrotrophic microbes from soil sample of NyAlesund, Svalbard, arctic region and to investigate their potential use as an effective tool for industrial application. A novel psychrotrophic bacterial strain showed good growth on minimal medium containing lipid as the only carbon source. Microbiological characterisation of the isolate showed that it was a gram negative rod. The strain was tested for the production of extracellular lipase enzyme. The enzymes were partially purified by 90% saturated ammonium sulfate and dialysis for desalting. The bacterium was identified as Pseudomonas sp ADT3 by 16S rRNA amplification and sequencing which had been deposited in the NCBI GenBank with accession number JX914667. Phylogenetic tree was also constructed with MEGA5 software and showed the highest level of sequence similarity with Pseudomonas sp. HC3-13 strain. The microorganism had a growth optimum at pH 8.0 and temperature 22°C. Optimization of different parameters e.g. temperature, pH, incubation time, cofactors etc. was performed for the extracellular lipase activity. The hydrolytic activity of the enzyme was enhanced 5 times by Pb2+ but strongly inhibited by heavy metals Hg2+ as well as EDTA and β-mercaptoethanol. For the molecular weight estimation of enzyme SDS-PAGE was done which showed an inducible band of approximately 13.9 KDa. Activity staining and mass spectrometry techniques were also performed.


Cite this paper
Dey, A. , Chattopadhyay, A. , Saha, P. , Mukhopadhyay, S. , Maiti, T. , Chatterjee, S. and Roy, P. (2014) An Approach to the Identification and Characterisation of a Psychrotrophic Lipase Producing Pseudomonas sp ADT3 from Arctic Region. Advances in Bioscience and Biotechnology, 5, 322-332. doi: 10.4236/abb.2014.54040.
References
[1]   Jaeger, K.-E. and Eggert, T. (2002) Lipases for Biotechnology. Current Opinion in Biotechnology, 13, 390-397.
http://dx.doi.org/10.1016/S0958-1669(02)00341-5

[2]   Rubin, B. and Dennis, E.A. (1997) Lipases: Part A. Biotechnology Methods in Enzymology. Vol. 284, Academic Press, New York, 1-408.

[3]   Kazlauskas, R.J. and Bornscheuer, U. (1998) Biotransformations with Lipases. In: Rehm, H.J. and Reeds, G., Eds., Biotechnology, Wiley-VCH, New York, 37-192. http://dx.doi.org/10.1002/9783527620906.ch3

[4]   Pandey, A., Benjamin, S., Soccol, C.R., Nigam, P., Krieger, N. and Soccol, V.T. (1999) The Realm of Microbial Lipases in Biotechnology. Biotechnology and Applied Biochemistry, 29, 119-131.

[5]   Saxena, R.K., Ghosh, P.K., Gupta, R., Davidson, W.S., Bradoo, S. and Gulati, R. (1999) Microbial Lipases: Potential Biocatalysts for the Future Industry. Current Science, 77, 101-115.

[6]   Sharma, R., Chisti, Y. and Banerjee, U. (2001) Production, Purification, Characterization and Applications of Lipases. Biotechnology Advances, 19, 627-662. http://dx.doi.org/10.1016/S0734-9750(01)00086-6

[7]   Iftikhar, T. and Hussain, A. (2002) Effect of Nutrients on the Extracellular Lipase Production by the Mutant Strain of R. oligosporous Tuv-31. Biotechnology, 1, 15-20. http://dx.doi.org/10.3923/biotech.2002.15.20

[8]   Iftikhar, T., Haq, I.U. and Javed, M.M. (2003) Optimization of Cultural Conditions for the Production of Lipase by Submerged Fermentation of Rhizopus oligosporous Tuv 31. Pakistan Journal of Botany, 35, 519-525.

[9]   Iftikhar, T., Niaz, M., Zia, M.A., Qadeer, M.A., Abbas, S.Q., Rajoka, M.I. and Haq, I.U. (2007) Screening of Media and Kinetic Studies for the Production of Lipase from a Mutant Strain of Rhizopus oligosporus. Biotechnologies International Symposium “Which Biotechnologies for South Countries”. Proceedings of Biotech World 2007, Oran Algerie, 24-25 November 2007.

[10]   Ftikhar, T., Niaz, M., Hussain, Y., Abbas, S.Q., Ashraf, I. and Zia, M.A. (2010) Improvement of Selected Strains through Gamma Irradiation for Enhanced Lipolytic Potential. Pakistan Journal of Botany, 42, 2257-2267.

[11]   Wiseman, A. (1995) Introduction to Principles. In: Wiseman, A., Ed., Hand Book of Enzyme Biotechnology, 3rd Edition, Ellis Horwood Ltd. T.J. Press Ltd. Padstow, 3-8.

[12]   Vakhlu, J. and Kour, A. (2006) Yeast Lipases: Enzyme Purification, Biochemical Properties and Gene Cloning. Electronic Journal of Biotechnology, 9, 69-85. http://dx.doi.org/10.2225/vol9-issue1-fulltext-9

[13]   Abada, E.A.E. (2008) Production and Characterization of a Mesophilic Lipase Isolated from Bacillus stearothermophilus AB-1. Pakistan Journal of Biological Sciences, 11, 1100-1106. http://dx.doi.org/10.3923/pjbs.2008.1100.1106

[14]   Srinivas, T.N.R., Nageswara Rao, S.S.S., Vishnu Vardhan Reddy, P., et al. (2009) Bacterial Diversity and Bioprospecting for Cold-Active Lipases, Amylases and Proteases from Culturable Bacteria of Kongsfjorden and Ny-Ålesund, Svalbard, Arctic. Current Microbiology, 59, 537-547. http://dx.doi.org/10.1007/s00284-009-9473-0

[15]   Bornscheuer, U.T., Bessler, C., Srinivas, R., et al. (2002) Optimizing Lipases and Related Enzymes for Efficient Application. Trends in Biotechnology, 20, 433-437. http://dx.doi.org/10.1016/S0167-7799(02)02046-2

[16]   Menoncin, S., Domingues, N.M., Freire, D.M.G., et al. (2008) Study of the Extraction, Concentration, and Partial Characterization of Lipases Obtained from Penicillium verrucosum Using Solid-State Fermentation of Soybean Bran. Food and Bioprocess Technology, 3, 537-544. http://dx.doi.org/10.1007/s11947-008-0104-8.

[17]   Choo, D.W., Kurihara, T., Suzuki T., et al. (1998) A Cold-Adapted Lipase of an Alaskan Psychrotroph. Pseudomonas sp. Strain B11-1: Gene Cloning and Enzyme Purification and Characterization. Applied and Environmental Microbiology, 64, 486-491. http://dx.doi.org/10.1007/s11947-008-0104-8

[18]   Stackebrandt, E., Murray, R.G.E. and Truper, H.G. (1988) Proteobacteria Classis nov., a Name for the Phylogenetic Taxon Includes the “Purple Bacteria and Their Relatives”. International Journal of Systematic and Evolutionary Microbiology, 38, 321-325. http://dx.doi.org/10.1099/00207713-38-3-321

[19]   Laemmli, U.K. (1970) Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature, 277, 621-626.

[20]   Gregersen, T. (1978) Rapid Method for Distinction of Gram Negative from Gram Positive Bacteria. Applied Micro-Biology and Biotechnology, 5, 123-127. http://dx.doi.org/10.1007/BF00498806

[21]   Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., et al. (1997) Gapped BLAST and PSI-BLAST: A New Generation of Protein Database Search Programs. Nucleic Acids Research, 25, 3389-3402.
http://dx.doi.org/10.1093/nar/25.17.3389

 
 
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