High Throughput Screening And Fermentation System Optimization Of Rhizomucor Miehei Lipase Producing Strain By Aspergillus Oryzae

Rhizomucor miehei lipase(RML)is a kind of serine hydrolase,which has broad application prospect in biodiesel,oil chemical,food processing,medicine,fine chemical and other industries.However,natural RML is difficult to be used in industrial production due to its low yield and unstable composition.In this study,the exogenous expression of RML by A.oryzae was used as the research object,starting from two aspects of both strain production performance improvement and fermentation system optimization,using high-throughput screening of high-yielding strains,response surface optimization of fermentation medium,and exogenous addition of surfactants to achieve the goal of efficient production of RML by A.oryzae,thus laying the foundation for the industrial production and application.Firstly,a high-throughput screening system for RML production by A.oryzae was established,and a high-yield strain was obtained.A high-throughput screening system for RML production by A.oryzae was successfully constructed by optimizing the double-indicator method for high-throughput detection of RML enzyme activity and developing the 24-well deep well plate high-throughput culture system.On this basis,ARTP was used for mutagenesis breeding.After 6 rounds of preliminary screening and re-screening,4 RML high-yielding mutant strains were successfully screened out from more than 36,000 spores,among which the enzyme activity of the mutant V7-6C-3 reached 293.5U/mL,which was 66.2%higher than that of the original strain(176.5U/mL).Secondly,response surface was used to optimize the fermentation medium for RML production by A.oryzae,which significantly increased RML enzyme activity.The optimal C/N ratio was determined to be corn dextrin:soybean peptone:yeast powder?75:45:2 by full factor optimization design;Two kinds of inorganic ions,ZnSO4·7H2O and CuSO4·5H2O,which can increase the yield of RML,were determined by single factor experiment.Plackett-Burman experiment showed that the three most important factors affecting the yield of RML were zein dextrin,pH value and magnesium sulfate heptahydrate.The results of steepest climb and CCD response surface experiment showed that the RML enzyme activity was the highest under the conditions of 75 g/L zein dexin,magnesium sulfate heptahydrate 0.1 g/L and pH 6.5,the theoretical value was 326.4 U/mL,and the real measured enzyme activity was 312.1 U/mL after shaking flask validation.Compared with the original medium(256.7 U/mL),it was increased by 21.5%.Finally,the addition strategy of non-ionic surfactants was optimized to realize the efficient production of RML by A.oryzae.The addition effect of seven kinds of non-ionic surfactants was investigated,and the most suitable surfactant S2 was selected.By optimizing the optimal addition time and amount,the results showed that after 24 h fermentation,adding 0.5%surfactant S2,the RML enzyme activity reached the its highest of 339.1 U/mL,which was increased by 42.7%compared with the group without addition surfactants(237.5 U/mL).The analysis of intracellular metabolites showed that the addition of surfactants was beneficial to the synthesis of amino acids,especially the important precursor amino acids in RML composition,so as to effectively promote the synthesis of RML.