Study On The Improving Enzymatic Catalysis Synthetic Activity Of Lipase From Rhizopus Chinensis

Rhizopus chinensis lipase (RCL) has a good potential in industrial application, with high esterification conversion. This dissertation studied on enzymatic catalysis synthetic activity of RCL in order to improve industrial application of Rhizopus chinensis lipase. It included established determine method of lipase synthetic activity in organic media, improved RCL synthetic activity by mutation and optimized media based synthetic activity and improved RCL-mediated esterification substrate concentration.It was established determine method of lipase synthetic activity in organic media. Lipase mediates esterification between caproic acid (0.6mol/L) and ethanol (0.6mol/L) in n-heptane at 40℃. The decrease in caproic acid is measured by titration and 1 unit of lipase synthetic activity is defined as 1 μmol caproic acid consumed per min. Measured synthetic activities of lipases from seven microorganisms, the relative errors of results were small and it showed this method had well veracity.Comparison of synthetic activities in organic media and hydrolytic activities in aqueous media of lipases from seven microorganisms proved an absence of general correlation between synthetic activity and hydrolytic activity. When lipases from seven microorganisms were used to catalyze synthesis of ethyl hexanoate, synthetic activity was more adapt to indicate lipase esterification ability compared hydrolytic activity. So synthetic activity can direct lipase research and application in esterification in non-aqueous phase.Rhizopus chinensis was mutated and optimized media based synthetic activity. Mutant 2113 lipase synthetic activity was improved 29.6% after mutation and increased 534% after optimized media compared to contrast. The synthetic activity improvement of Mutant 2113 lipase resulted in esterification ability increase. Ethyl hexanoate synthesis mediated by Mutant 2113 lipase had 90% conversion at 15h reaction time that was 15.6% of contrast. The optimized media were: maltose 1%, peptone 4%, bean powder 4%, olive oil 2%, MgSO4·7H2O 0.05%, K2HPO4 0.2%, initial pH 5.5. The cultivation conditions were: 2.2～4.3×105spores/30mL inoculum size, 30mL medium volume, 150 r/min or 200 r/min agitation speed. Fed maltose or olive oil fermentation of mutant 2113 lipase was not improved lipase synthetic activity but improved biomass resulted in total cell synthetic activity yield increase.Rhizopus chinensis whole-cell lipases of different culture time were used to catalyze synthesis of ethyl hexanoate and whole-cell lipase with high synthetic activity catalyzed esterification quickly. So cell used to catalyze esterification in organic media with high synthetic activity had good esterification ability and cell quality.It was increased ethyl hexanoate synthesis substrate concentration mediated by Rhizopus chinensis 2113 whole-cell lipase after improved synthetic activity and optimized reaction condition. The reaction condition were: caproic acid concentration 2.4mol/L, substrate molar concentration ratio 1:1.1, initial water content ≤0.2%, lipase water activity 0.3~0.66, enzyme amount 16%(g/v), reaction temperature 30℃, agitation speed 150 r/min. The esterification conversion was about 90%. It was increased conversion to 92.5% by added desiccant.