Lipases, essential enzymes for hydrolyzing triglycerides, are widely produced by various microorganisms, including bacteria, fungi, and yeast. Among microbial lipases, those from Pseudomonas species are particularly notable for their thermal resistance and alkaline activity, making them valuable for industrial applications. This study focuses on a novel recombinant lipase gene from Pseudomonas fluorescens AFPF19, aiming to express this gene in Escherichia coli (E.coli) and evaluate its potential for biodiesel production. The lipase gene was successfully isolated, cloned into the pET28a expression vector, and expressed in E. coli BL21 CodonPlusTM (DE3). The recombinant lipase was purified using Ni-NTA affinity chromatography. To enhance the enzyme’s stability and reusability, immobilization techniques were employed. The immobilized lipase demonstrated robust activity in the transesterification of Millettia pinnata oil to produce biodiesel. Various reaction conditions, including oil-to-methanol molar ratios and enzyme concentrations, were optimized to maximize biodiesel yield. Gas chromatography-mass spectrometry (GC-MS) analysis confirmed the high purity of the produced biodiesel, while its properties, such as density, kinematic viscosity, and calorific value, met international standards. This study highlights the potential of using immobilized recombinant lipases for sustainable and efficient biodiesel production, addressing challenges associated with free enzyme use and contributing to the advancement of renewable energy sources.
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