摘要
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.
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.
