Application,Immobilization And Mutagenesis Of Recombinant Serratia Marcescens Lipase

The extracellular lipases from Serratia marcescens ECU1010are a kind of α/β hydrolase fold protein; they are widely used in the food, medicine and bio-energy industries. In this paper, we investigated and optimized the production conditions of biodiesel using S. marcescens LipA as the catalyst. Through the single-factor experiment and response surface analysis, the optimal production conditions of biodiesel were obtained and the results were as follows:Based on the weight of soybean oil, enzyme dosage10.8%(w/w), reaction temperature40℃, petroleum ether2.73mL/g, substrate molar ratio1:4, adding methanol for3times, time interval10.6h. Under the optimal conditions, the conversion rate of FAME reached84.2%, which increased10.7%than prior to optimization.The immobilization conditions of lipase present in active inclusion bodies were studied by response surface methodology. Plackett-Burman design and Box-Behnken design were used to optimize the immobilization conditions, respectively. Through the experiment, we obtained the optimal immobilized conditions. The details were as follows:glutaraldehyde0.13%(w/v), enzyme16.5mg/mL, ratio of glutaraldehyde and enzyme5.5:1. Under this condition, the enzymatic activity was increased45%than prior to optimization.Comparative studies on the enzymatic properties of the free and immobilized enzymes were performed. The main properties studied in this work included the stability of pH and temperature, as well as in the presence of various metal ions and organic solvents. In addition, the operation stability and the storage stability were investigated. The immobilized lipase showed high stability in a broad pH range of5.0-10.0and remained over48%of its activity at60℃for1h. The tolerance to metal ions and water-miscible organic solvents was raised. The operation stability and the storage stability were also significantly improved.The mutants (G33D, A187V and T270A) of S. marcescens LipB were obtained through the site-directed mutagenesis technology and the tolerance of LipB mutants to various organic solvents were investigated. Based on homology modeling and structural analysis, the33rd amino acid (Gly) of LipB played an important role in the tolerance against organic solvents.