The Research On The Relationship Between Rhizopus Chinensis Morphology And The Production Of Its Membrane-bound Lipase In The Liquid State Fermentation

Lipase is a kind of enzyme to hydrolyze oil into glycerol and fatty acid and to catalyze the synthesis of esters. Lipase has region selectivity, stereo-selectivity and high substrate specificity, in which the lipase with ability of transesterification and esterification in nonaqueous phase has important signification in industrial production such as chiral compounds and fine chemicals. Rhizopus chinensis membrane-bound lipase has great applied potential in the industry such as bio-energy, flavors and so on due to its high ability of ester synthesis. The pre-study found that Rhizopus chinensis could form different mycelial morphology in liquid state fermentation. With the change of mycelial morphology, the Rhizopus chinensis synthesis activity changed greatly. In this thesis, regulation and control of Rhizopus chinensis mycelial morphology was studied and Western Blot analysis was used to study the relationship between mycelial morphology and the lipase synthesis activity. In the end, the preliminary research of Rhizopus chinensis fermentation regulation and control in fermentator and the catalytic application were carried out.(1) Morphology regulation of Rhizopus chinensis in flask. The influence of different fermentation parameters (addition amount of olive oil, initial pH, liquid medium volume, baffle condition and inoculums) on the Rhizopus chinensis (CCTCC M201021) morphology were studied and preliminary morphologic control of Rhiopus chinensis was achieved. The optimized fermentation conditions for the formation of clump thallus:liquid medium volume 20 mL/250 mL shake flask, rotation speed 200 rpm, initial pH 5.5, inoculum level 5×107 spores/mL,30℃. Under these conditions, clump thallus could form. Pellet thallus and diffused thallus could form when liquid medium volume was 40 mL/250 mL shake flask and baffled facility was used, respectively.(2) Study on the relationship between Rhizopus chinensis morphology and lipase synthesis activity. Polyclonal antibody against membrane-bound lipase of Rhizopus chinensis was produced and Western Blot analysis was used to study the production and distribution of lipases located in intracellular, membrane and extracellular domain, respectively. The result indicated that with the aggregation of Rhizopus chinensis mycelia, the total amount of lipase increased obviously. Meanwhile, lipase location changed with the change of Rhizopus chinensis macromorphology. The secretion of lipase from membrane to the culture medium was inhibited with the aggregation of mycelia.(3) Preliminary fermentation regulation of Rhizopus chinensis. As Rhizopus chinensis can't develop clump mycelia in common mechanical stirring fermentator, we designed a kind of sleeve on which Rhizopus chinensis could adhere. Under this condition, clump thallus could form and the lipase synthesis activity was improved 100%to 100 U/g contrast to common mechanical stirring fermentator.(4) Catalytic application research on Rhizopus chinensis lipase. This study based on the ability of synthesizing ethyl hexanoate and the result indicate that Rhizopus chinensis lipase could catalyze the synthesis of ethyl hexanoate effectively in external dehydrate-solvent free system.