Abstract: In order to improve the yield of fumonisin B1 (FB1) carboxylesterase produced by recombinant Pichia pastoris in shaking flask, the fermentation condition and medium were optimized with enzyme activity as index. The fermentation conditions were optimized through single factor experiment. The key components of fermentation medium were screened through Plackett-Burman experiment and then orthogonal experiment was carried out to establish the experimental data samples. Finally, the error Back Propagation (BP) neural network model was established to predict and search for the optimal composition of fermentation medium. After optimization, the optimal fermentation conditions were induction temperature 28 ℃, initial pH 6.0, 1% of methanol addition every 24 h, induction for 96 h. The optimal fermentation medium was peptone 23 g/L, KH2PO4 44 g/L, PTM4 1.5 mL/L, K2SO4 7.15 g/L, MgSO4·7H2O 5.85 g/L, yeast extract 5 g/L. FB1 carboxylesterase activity was over 402 U/mL, 1.19-fold higher than that of before optimization. Fermentation optimization in shaking flask can significantly improve the enzyme production capacity of recombinant strains. These results presented basic data for the optimization of FB1 carboxylesterase in fermenter.
赵一凡,常晓娇,杜稳,等. 毕赤酵母产伏马毒素B1羧酸酯酶发酵条件和培养基的优化[J]. 食品与发酵工业, 2021, 47(1): 43-49.
ZHAO Yifan,CHANG Xiaojiao,DU Wen,et al. Optimization of fermentation condition and medium for fumonisin B1 carboxylesterase production in Pichia pastoris[J]. Food and Fermentation Industries, 2021, 47(1): 43-49.
 YAMAZOE Y,KOYAMA N,KUMAGAI S.Possible role of phosphatidylcholine and sphingomyelin on fumonisin B1-mediated toxicity[J].Food Safety,2017,5(3):75-97.  ZHU Y,HASSAN Y I,WATTS C,et al.Innovative technologies for the mitigation of mycotoxins in animal feed and ingredients-A review of recent patents[J].Animal Feed Science and Technology,2016,216:19-29.  OMURTAG G Z.Fumonisins,trichothecenes and zearalenone in cereals[J].International Journal of Molecular Sciences,2008,9(11):2 062-2 090.  CAO C,ZHU X,LI X,et al.Assessment of ionic homeostasis imbalance and cytochrome P450 system disturbance in mice during fumonisin B1 (FB1) exposure[J].Chemosphere,2020.DOI:10.1016/j.chemosphere.2020.126393.  WEI T,ZHU W,PANG M,et al.Natural occurrence of fumonisins B1 and B2 in corn in four provinces of China[J].Food Additives & Contaminants:Part B,2013,6(4):270-274.  FALLAH B,ZAINI F,GHAZVINI R D,et al.The antagonistic effects of Candida parapsilosis on the growth of Fusarium species and fumonisin production[J].Current Medical Mycology,2016,2(1):1-6.  MINERVINI F,GARBETTA A,D’ANTUONO I,et al.Toxic mechanisms induced by fumonisin B1 mycotoxin on human intestinal cell line[J].Archives of Environmental Contamination and Toxicology,2014,67(1):115-123.  杨李梅, 苏建明,雷红宇,等.伏马毒素研究进展[J].动物医学进展,2014,35(3):97-100. YANG L M,SU J M,LEI H Y,et al.Progress on fumonisins[J].Progress in Veterinary Medicine, 2014,35(3):97-100.  BRYŁA M,WAŚKIEWICZ A,SZYMCZYK K,et al.Effects of pH and temperature on the stability of fumonisins in maize products[J].Toxins,2017,9(3):88-103.  ZHU Y,HASSAN Y I,LEPP D,et al.Strategies and methodologies for developing microbial detoxification systems to mitigate mycotoxins[J].Toxins,2017,9(4):130-155.  HEINL S,HARTINGER D,THAMHESL M,et al.Degradation of fumonisin B1 by the consecutive action of two bacterial enzymes[J].Journal of Biotechnology,2010,145(2):120-129.  ADDITIVES E P O,FEED P O S U I A.Scientific opinion on the safety and efficacy of fumonisin esterase (FUMzyme®) as a technological feed additive for pigs[J].EFSA Journal,2014,12(5):3 667-3 685.  YANG Y,GAO M,YU X,et al.Optimization of medium composition for two-step fermentation of vitamin C based on artificial neural network-genetic algorithm techniques[J].Biotechnology & Biotechnological Equipment,2015,29(6):1 128-1 134.  SHI Y,GAI G,ZHAO X,et al.Back propagation neural network (BPNN) simulation model and influence of operational parameters on hydrogen bio-production through integrative biological reactor (IBR) treating wastewater[C].2010 4th International Conference on Bioinformatics and Biomedical Engineering,2010:1-4.  坚乃丹. 伏马毒素B1降解酶YD的异源表达及其酶学性质研究 [D].天津:天津科技大学,2018. JIAN N D.Heterologous expression and characterization of fumonisin B1 degradation enzyme YD [D].Tianjin:Tianjin University of Science and Technology,2018.  李飞, 喻晓蔚,沙冲,等.基因拷贝数和甲醇浓度对重组毕赤酵母产华根霉脂肪酶的影响[J].微生物学通报,2011,38(3):301-309. LI F,YU X W,SHA C,et al.Impact of gene dosage and methanol concentration on Rhizopus chinensis recombinant lipase production in Pichia pastoris[J].Microbiology China,2011,38(3):301-309.  陈明祥, 谢万勇,廖锡豪,等.表面展示南极假丝酵母脂肪酶B的毕赤酵母FM22发酵培养基响应面优化[J].中国酿造,2012,31(10):52-56. CHEN M X,XIE W Y,LIAO X H,et al.The response surface optimization on FM22 induction medium of CALB displayed on Pichia pastoris cell surface production[J].China Brewing,2012,31(10):52-56.  CHAROENRAT T,KHUMRUAENGSRI N,PROMDONKOY P,et al.Improvement of recombinant endoglucanase produced in Pichia pastoris KM71 through the use of synthetic medium for inoculum and pH control of proteolysis[J].Journal of Bioscience and Bioengineering,2013,116(2):193-198.  WANDERLEY M S,OLIVEIRA C,BRUNESKA D,et al.Influence of trace elements supplementation on the production of recombinant frutalin by Pichia pastoris KM71H in fed-batch process[J].Chemical Papers,2013,67(7):682-687.  陈柳, 马广大.大气中SO2浓度的小波分析及神经网络预测[J].环境科学学报,2006,26(9):1 553-1 558. CHEN L,MA G D.Study on wavelet analysis and neural network prediction of SO2 concentration in air[J].Acta Scientiae Circumstantiae, 2006,26(9):1 553-1 558.  刘瑞江, 张业旺,闻崇炜,等.正交试验设计和分析方法研究[J].实验技术与管理,2010,27(9):52-55. LIU R J,ZHANG Y W,WEN C W,et al.Study on the design and analysis methods of orthogonal experiment[J].Experimental Technology and Management,2010,27(9):52-55.  MA H,LI H,GANG J,et al.Optimization of ethanol fermentation from fruit and vegetable waste by plackett-burman and orthogonal experimental design[J].BioResources,2019,14(1):1 210-1 218.  吴娜, 赵重博,胡美变,等.BP-ANN 结合正交试验法优化白附子多糖提取工艺[J].中成药,2016,38(6):1 248-1 253. WU N,ZHAO C B,HU M B,et al.Optimizing the extraction of polysaccharides from Typhonii rhizoma by BP-ANN combined with orthogonal test[J].Chinese Traditional Patent Medicine,2016,38(6):1 248-1 253.  王云龙, 刘松,堵国成,等.基于人工神经网络的L-天冬酰胺酶发酵培养基优化[J].食品与发酵工业,2018,44(8):27-33. WANG Y L,LIU S,DU G C,et al.Optimization of L-asparaginase fermentation medium based on artificial neural network[J].Food and Fermentation Industries,2018,44(8):27-33.  PENG W,ZHONG J,YANG J,et al.The artificial neural network approach based on uniform design to optimize the fed-batch fermentation condition:Application to the production of iturin A[J].Microbial Cell Factories,2014,13(1).DOI:10.1186/1475-2859-13-54.  DASARI V R R K,DONTHIREDDY S R R,NIKKU M Y,et al.Optimization of medium constituents for Cephalosporin C production using response surface methodology and artificial neural networks[J].Journal of Biochemical Technology,2009,1(3):69-74.