Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Purification, Characterization and Immobilization of Lipase from Proteus vulgaris OR34 for Synthesis of Methyl Oleate

  • Misbah, Asmae (Microbial Biotechnology Laboratory, Faculty of Sciences and Technologies, Sidi Mohamed Ben Abdellah University) ;
  • Koraichi, Saad Ibnsouda (Microbial Biotechnology Laboratory, Faculty of Sciences and Technologies, Sidi Mohamed Ben Abdellah University) ;
  • Jouti, Mohamed Ali Tahri (Microbial Biotechnology Laboratory, Faculty of Sciences and Technologies, Sidi Mohamed Ben Abdellah University)
  • Received : 2020.03.27
  • Accepted : 2020.09.07
  • Published : 2020.12.28
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Abstract

A newly isolated strain, Proteus vulgaris OR34, from olive mill waste was found to secrete an alkaline extracellular lipase at 11 U·ml-1 when cultivated on an optimized liquid medium. This lipase was purified 94.64-fold with a total yield of 9.11% and its maximal specific activity was shown to be 3232.58 and 1777.92 U·mg-1 when evaluated using the pH-stat technique at 55℃ and pH 9 and Tributyrin TC4 or olive oil as the substrate. The molecular mass of the pure OR34 lipase was estimated to be around 31 kDa, as revealed by SDS-PAGE and its substrate specificity was investigated using a variety of triglycerides. This assay revealed that OR34 lipase preferred short and medium chain fatty acids. In addition, this lipase was stable in the presence of high concentrations of bile salt (NaDC) and calcium ions appear not to be necessary for its activity. This lipase was inhibited by THL (Orlistat) which confirmed its identity as a serine enzyme. In addition, the immobilization of OR34 lipase by adsorption onto calcium carbonate increased its stability at higher temperatures and within a larger pH range. The immobilized lipase exhibited a high tolerance to organic solvents and retained 60% of its activity after 10 months of storage at 4℃. Finally, the OR34 lipase was applied in biodiesel synthesis via oleic acid mediated esterification of methanol when using hexane as solvent. The best conversion yield (67%) was obtained at 12 h and 40℃ using the immobilized enzyme and this enzyme could be reused for six cycles with the same efficiency.

Keywords

Acknowledgement

This study was supported by "Ministry of Higher Education and Scientific research of Morocco".

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