海洋源低温微生物产苹果酸脱氢酶发酵条件优化
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摘要
苹果酸脱氢酶(MDH)是参与生物三羧酸循环(TCA)的关键酶,广泛用于临床肝脏相关疾病的诊断,并在生物制药、化工检测等领域具有极大的市场需求。该研究以墓画大洋芽孢杆菌(Oceanobacillus picturae)XJH-11为苹果酸脱氢酶(MDH)产生菌,基于单因素试验,通过Plackett-Burman响应面试验并结合Box-Behnken试验设计对发酵条件进行响应面优化。结果表明,菌株XJH-11产MDH的发酵培养基为葡萄糖10 g/L、酵母膏20 g/L、硫酸镁0.1 g/L、初始pH值8.0,最佳发酵条件为发酵温度27℃、摇床转速150 r/min、接种量5%、装液量125 mL/500 mL。在此优化条件下,MDH酶活为43.67 U/m L,比优化前提高了197.87%。
Malic dehydrogenase(MDH) is a key enzyme involved in the biological tricarboxylic acid cycle(TCA), which is widely used in the diagnosis of clinical liver-related diseases, and has great market demand in the fields of biopharmaceuticals, chemical industry detection and so on. In this study,using the Oceanobacillus picturae XJH-11 as a MDH producing strain, the fermentation conditions were optimized by Plackett-Burman response surface method combined with Box-Behnken experiments design based on the single factor experiments. The results showed that the compositions of fermentation medium for MDH production by strain XJH-11 were glucose 10 g/L, yeast extract 20 g/L, magnesium sulfate 0.1 g/L and initial pH 8.0. The optimum fermentation conditions were fermentation temperature 27 ℃, shaking speed 150 r/min, inoculum 5% and liquid loading volume125 ml/500 ml. Under these conditions, the activity of MDH was 43.67 U/ml, which was 197.87% higher than that of before optimization.
Malic dehydrogenase(MDH) is a key enzyme involved in the biological tricarboxylic acid cycle(TCA), which is widely used in the diagnosis of clinical liver-related diseases, and has great market demand in the fields of biopharmaceuticals, chemical industry detection and so on. In this study,using the Oceanobacillus picturae XJH-11 as a MDH producing strain, the fermentation conditions were optimized by Plackett-Burman response surface method combined with Box-Behnken experiments design based on the single factor experiments. The results showed that the compositions of fermentation medium for MDH production by strain XJH-11 were glucose 10 g/L, yeast extract 20 g/L, magnesium sulfate 0.1 g/L and initial pH 8.0. The optimum fermentation conditions were fermentation temperature 27 ℃, shaking speed 150 r/min, inoculum 5% and liquid loading volume125 ml/500 ml. Under these conditions, the activity of MDH was 43.67 U/ml, which was 197.87% higher than that of before optimization.
引文
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[21]孟利强,赵晓宇,陈静宇,等.响应面法优化枯草芽孢杆菌B91发酵培养基[J].安徽农业科学,2015,43(25):26-29.
[22]林秀芳,尹文哲,张智,等.基于design-expert软件设计优化动物双歧杆菌培养条件工艺[J].食品研究与开发,2017,38(7):144-148.
[23]孙丽娜,范英兵,胡冬慧,等.响应面优化纤维素酶提取蓝莓多糖工艺研究[J].食品研究与开发,2017,38(23):57-60.
[24]刘水英,李新生,党娅,等.响应面法优化紫山药花青苷提取工艺及其抗氧化活性[J].食品科学,2014,35(22):84-91.
[2]TAKAHASHIINIGUEZ T,ABURTO R,NVILCHIS G A,et al.Function,kinetic properties,crystallization,and regulation of microbial malate dehydrogenase[J].J Zhejiang Univ-Sci B,2016,17(4):247-261.
[3]AN Y,CAO Y,XU Y.Purification and characterization of the plastid-localized NAD-dependent malate dehydrogenase from Arabidopsis thaliana[J].Biotechnol Appl Biochem,2016,63(4):490-496.
[4]王庆杰.苹果细胞质苹果酸脱氢酶基因的抗逆功能鉴定及机制研究[D].泰安:山东农业大学,2016.
[5]CHEN K H,LEE S Y,SHOW P L,et al.Direct recovery of malate dehydrogenase from highly turbid yeast cell homogenate using dye-ligand affinity chromatography in stirred fluidized bed[J].J Chromatogr B,2018,1100-1101:65-75.
[6]曾英杰.荔枝酒发酵过程中酿酒酵母Ydcs101代谢醋酸的调控研究[D].海口:海南大学,2015.
[7]常平平,郝艳红,周遵武,等.食品中柠檬酸快速检测试剂盒的研制[J].中国酿造,2015,34(3):111-114.
[8]肖景惠,张庆芳,于爽,等.微生物中苹果酸脱氢酶研究现状及展望[J].中国酿造,2018,37(8):14-18.
[9]王俊,杨晓霞,魏云林,等.深黄被孢霉苹果酸脱氢酶基因的克隆和表达[J].中国微生态学杂志,2017,29(9):993-997.
[10]崔锦锦,季秀玲,林连兵,等.一个深黄被孢霉苹果酸脱氢酶基因的克隆与表达[J].生命科学研究,2017,21(3):208-212.
[11]刘俊.Thermus Thermophilus HB27苹果酸脱氢酶基因的克隆表达、酶抑制动力学及蛋白折叠研究[D].杭州:浙江理工大学,2010.
[12]龚韧,孙艳,王静,等.猪心肌苹果酸脱氢酶的制备及应用[J].食品与生物技术学报,2008,27(5):57-61.
[13]张翔,张彦昊,刘孝永,等.产壳聚糖酶菌株ncps116发酵条件优化及其酶学性质[J].化工进展,2018,37(6):2354-2363.
[14]周新尚,逄飞,窦少华,等.海洋生淀粉酶菌株发酵条件响应面优化[J].中国酿造,2017,36(7):80-84.
[15]吴优,周卫,李尧益,等.Arthrobacter ureafaciens CZ31丙氨酸脱氢酶可溶性表达及产酶条件优化[J].微生物学报,2017,57(12):1778-1787.
[16]肖冬来,张迪,林衍铨,等.金属离子对香菇纤维素酶、漆酶和木质素过氧化物酶活性的影响[J].中国食用菌,2018,37(3):56-58.
[17]王茜茜.基于iTRAQ技术对耐盐植物乳杆菌FS5-5的蛋白质组学分析[D].沈阳:沈阳农业大学,2016.
[18]宋爱荣,田雪梅.不同装瓶量对樟芝液体深层培养的影响[C].中国海峡两岸菌物学学术研讨会论文集.北京:中国菌物学会,2004:3-6.
[19]陈志杰,韩永斌,沈昌,等.Plackett-Burman设计在灵芝生长及产胞外多糖主要影响因子筛选中的应用[J].食品科学,2005,26(12):115-118.
[20]郭琪,杨晖.Plackett-Burman设计法筛选影响桃儿七内生真菌Penicillium sp.XJ产鬼臼类物质发酵培养的主要因子[J].内蒙古农业大学学报(自然科学版),2015,36(4):83-87.
[21]孟利强,赵晓宇,陈静宇,等.响应面法优化枯草芽孢杆菌B91发酵培养基[J].安徽农业科学,2015,43(25):26-29.
[22]林秀芳,尹文哲,张智,等.基于design-expert软件设计优化动物双歧杆菌培养条件工艺[J].食品研究与开发,2017,38(7):144-148.
[23]孙丽娜,范英兵,胡冬慧,等.响应面优化纤维素酶提取蓝莓多糖工艺研究[J].食品研究与开发,2017,38(23):57-60.
[24]刘水英,李新生,党娅,等.响应面法优化紫山药花青苷提取工艺及其抗氧化活性[J].食品科学,2014,35(22):84-91.