| L-Asparaginase?EC 3.5.1.1,L-ASNase?is a hydrolase that deaminates L-asparagine to produce L-Aspartate and ammonia.L-ASNase has anti-tumor activity and has been widely used in the fields of food and medicine.In this study,a recombinant Bacillus subtilis/ASN?25/B2 which can secrete L-ASNase was used as the production strain,and its fermentation,extraction and stabilization process were optimized.The key research findings are as follows:?1?Optimization fermentation in shake flask to improve the yield of recombinant L-ASNaseEffect of culture conditions on cell growth and L-ASNase production were examined by single factor experiments.The initial pH had a great influence on cell growth and enzyme production.The optimum initial pH was 7.5.Then neural network algorithm was used to optimize its fermentation medium.First,the significant factors were screened through single factor experiment and Plackett-Burman experiment.Then the central composite experiment was used to establish the experimental data samples.Finally,a neural network model was established by JMP10.0 to optimize the composition of fermentation medium.After optimization,the best medium is obtained as follows:sucrose 65 g·L-1,yeast peptone 28 g·L-1,corn syrup 11 g·L-1,KH2PO4 11.5 g·L-1,NaCl 3.3 g·L-1,ammonium sulfate 4 g·L-1,K2HPO4·3H2O 22.5 g·L-1,MgSO4·7H2O 1 g·L-1,L-asparagine 2 g·L-1.L-ASNase activity reached 515.6 U·mL-1 under the condition of the optimal medium,which was 90.9%higher than that before optimization.?2?Effect of stirring speed on the fermentation of recombinant L-ASNase in 3 L fermenterThe results showed that different stirring speeds resulted in different levels of dissolved oxygen in the fermentation process,which led to big differences in cell growth and L-ASNase synthesis.Based on effect of the stirring speeds on the specific growth rate and specific enzyme production rate of the bacteria,a two-staged stirring speed control strategy was established.The stirring speed was set at 700 r·min-1 for the first 8 h and shifted to 900 r·min-1 till the end of the fermentation.Under the condition of two-staged stirring speed,the dissolved oxygen level can be maintained at more than30%,which can meet the demand of the bacteria for dissolved oxygen.The fermentation period was shortened from 40 h to 36 h,and the maximum L-ASNase activity was further increased to 850.6U·mL-1.?3?Effect of feeding conditions on the fermentation of recombinant L-ASNase in 3 L fermenter.Based on optimizing the stirring speed,fed-batch fermentation conditions were studied.Effect of feeding time,composition of feed medium,feeding quantity,and feeding mode on cell growth and L-ASNase production were investigated.After the optimization,conditions used for fed-batch fermentation were determined as follows:for fermentation at 1628 h,sucrose solution(800 g·L-1)and nitrogen sources(yeast peptone:200 g·L-1;corn syrup:80 g·L-1)were fed at 18.75 mL·h-1 and32 mL·h-1,respectively.Based on this fermentation condition,the L-ASNase yield reached 1413.6U·mL-1 at 48 h,which was 66.2%higher than that of batch fermentation.The final production intensity obtained here reached 29.45 U·mL-1·h-1,which was 24.6%higher than that of batch fermentation.?4?Study on extraction and stabilization of recombinant L-ASNaseBy optimizing the type of flocculant,the pH of the fermentation broth,and the amount of flocculant added,the L-ASNase activity recovery rate of flocculation filtration was 91.2%when 2%of calcium chloride was added at a pH of 8.0.When 40%anhydrous ethanol was added,the L-ASNase activity recovery rate of ethanol precipitation was 97.9%.With the addition of 5%(m·v-1)sucrose or10%(m·v-1)dextrin,the L-ASNase activity recovery rate in the freeze-drying process was 98.7%and98.6%,respectively.After 60 days of storage at room temperature,the survival rate of L-ASNase activity was 95.7%and 98.1%respectively.Adding compound protectants?6%glucose,6%sorbitol,1%soybean protein,0.5%potassium citrate?into liquid L-ASNase,the retention rate of enzyme activity was 96.6%after 2 h at 65?,which was 1.5 times as much as that of no protectant.After 80days of storage at room temperature,the retention rate of liquid L-ASNase activity reached 81.7%,which was 2.8 times as much as that without protectant. |
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