Fermentation Control For Membrane-bound Lipase Produced By Rhizopus Chinensis Based On Morphological Engineering

With high synthetic activity, the membrane-bound lipase (mb-RCL) produced byRhizopus chinensis (CCTCC2010021) could catalyze the synthesis of esters in non-aqueousphase and had potential applications in the production of flavor and fragrance compounds,biodiesel, and so on. Firstly，the relationship between fungal morphology and RCL synthesisactivity in fermentor was investigated. Based on the biological characteristics andrequirements of mb-RCL fermentation by Rhizopus chinensis, the improvement of7Lmechanical stirring fermentor facilities and the cold form properties were carried out toincrease the lipase fermentation level using morphology engineering. And produced mb-RCLwas used as the catalyst in a―solvent-free‖reaction system. The main results were as follows:(1) Liquid fermentation of mb-RCL produced by Rhizopus chinensis was scaled up in a7L stirred-tank fermentor. The results showed that Rhizopus chinensis mycelium appearedtwo morphologies: aggregation closed to the baffle, electrode, impeller, etc. and freelydispersed mycelium in the fermentation broth. And mycelium of aggregation displayed highersynthesis activity of lipase. Based on SDS-PAGE and Western Blot analysis, it was found thatthe aggregation of mycelium helped mb-RCL produce and accumulate on the cell membrane.(2) The influence of starvation stress on mb-RCL liquid fermentation of Rhizopuschinensis was investigated. The fermentation results in shake flask showed that nutrientstarvation stress had no positive effect on growth and lipase expression of Rhizopus chinensis;anaerobic starvation stress had a positive effect on lipase activity, increased18.8%. However，the results in the fermentor showed that anaerobic starvation stress had impact on fungalmorphology changes, causing the decrease of lipase fermentation level.(3) In order to improve liquid fermentation level of the mb-RCL produced by Rhizopuschinensis, the fermentor facilities were modified and fermentation conditions were optimized.Based on the biological characteristics and fermentation requirements of Rhizopus chinensis,and the optimized fermentation processes obtained by the cold form experimental, ultimatelythe activity of Rhizopus chinensis lipase reached324.78U/g, cell dry weight reached19.08g/L, total activity of unit broth volume reached6196.88U/L, utilization rate of olive oilreached100%, the fermentation level was6.5times as the previous.(4) The produced mb-RCL, as the catalyst, was used in a―solvent-free‖reaction systemfor the synthesis of ethyl hexanoate. Using the product, ethyl hexanoate, as the solvent, thesubstrate (ethanol and hexanoic acid) concentration of3.56mol/L, reaction temperature37℃, shake speed of200r/min, the conversion of catalytic reaction can reach77%bysupplement with lipase on third day.