Biocatalyst For Pharmaceutical Industry

Lipases (triacylglycerol hydrolase E.C.3.1.1.3 ) are enzymes widely distributed in nature, which catalyse the hydrolysis, synthesis and interesterification of lipoid, and also capable to catalyse the esterification, interesterification and transesterification in organic media, with control of the water activity. They show unique chemo-, regio- and enatioselectivities as well as temperature and pH stabilitity. This ancient enzyme has new and broad prospects for development because of its inhibition of the water participation side reaction, the relative ease of recyling, the relative ease of recovery of products in organic phase and separation and assymetrically synthesis of racemic compound which is difficult with the chemical process to carry on. The non-aqueous phase enzymatic catalytic reactions have received increased attention due to the enzyme in the non-aqueous medium has many advantages which is unable to compare in the water. Lipases in microorganism distribute very broad, but it is difficult to seek a suitable high production strain for the industrialization production. at present the domestic need lipase still relied on the import, in order to impel the non- aqueous phase enzymatic catalysis in our country's research and the application, it is necessary to develop the lipase suitable for non- aqueous phase function.In this thesis, the screening of Lipase-Producing Strain for Catalytic synthesis of ester in Organic Media was conducted. The Lipase-Producing Conditions and its application in synthesis of L-ascorbyl palmitate and biodiesel were also investigated consequently. The main results are as follows:(1)An organic solvent tolerant isolate A213 originating from soil samples were successfully isolated via direct plating method using 10 g/L of toluene as the sole carbon source and transparent cycle plate assay method. It was identified as Yarrowia based on its characteristics.(2)The results in shake flask cultivation showed that the suitable lipase producing media were (g/L): yeast extract 40, vegetable oil 10, MgSO₄·7H₂O1,KH₂PO₄5. Under optimal culture conditions (27℃and pH 6.5), the maximal lipase activity could reach 67.8 IU/mL. The optimal pH and temperature for the hydrolysis of p-nitrophenyl acetate by crude lipase were pH6.5 and 40℃. After 40 min, the enzyme had 50% activity at 40℃and was stable between pH 5.5～8.5. Then isolate A213 was found to produce the lipase which can synthesize L-ascorbyl palmitate in tert-amyl alcohol validated by the thin-layer chromatography.(3)A novel route for synthesis of L-ascorbyl fatty acid esters (AFAE) has been revealed in...