Culture conditions and magnetic immobilization of nitrilase-producing strain
SHANG Yuting1, GONG Jinsong2, WANG Shunzhi1, LU Zhenming1, LI Heng2, SHI Jinsong2, XU Zhenghong1*
1(School of Biotechnology, National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China) 2(School of Pharmaceutical Sciences, Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China)
Abstract: Previously, a recombinant Bacillus subtilis pMA5-NITR harboring nitrilasewas constructed in our laboratory, which could catalyze 3-cyanopyridine into nicotinic acid. In this study, the recombinant B. subtilis pMA5-NITR was cultured by different cultivation strategies including batch fermentation, constant-rate feeding, and pH-stat feeding, to achieve resting cells with high nitrilase activity. In order to enhance the stability of nitrilase in cells, the aminated core-shell magnetic Fe3O4 nanoparticles was used to immobilize the recombinant B. subtilis nitrilase with immobilization conditions optimized. In addition, the effects of temperature stability, substrate tolerance and initial substrate concentration on the transformation efficiency of magnetically immobilized cells were examined to determine the process conditions for further enhancing the nicotinic acid production capacity of recombinant nitrilase. The results showed that pH-stat feeding strategy produced the highest nitrilase activity. Under the pH-stat feeding strategy, the activity of nitrilase could reach 167.32 U/mL, which was 2.64 times that of the batch fermentation. The magnetically immobilized cells of recombinant nitrilase were more resistant to high concentrations of 3-cyanopyridine. Thirty batches of 3-cyanopyridine could be completely converted within 450 min, and the cumulative concentration of nicotinic acid reached 738.66 g/L, which was 2.5 times higher than that of free cells. And it was also the highest yield of nicotinic acid produced by nitrilase derived from B. subtilis. In summary, different culture conditions and the magnetic immobilization strategy could improve the ability of the recombinant nitrilase to produce nicotinic acid from 3-cyanopyridine, and the recombinant nitrilase had great potential in the industrial production of nicotinic acid.
 ZUORRO A, LAVECCHIA R.Protective effect of nicotinic acid on human albumin during UV-C irradiation[J].Korean Journal of Chemical Engineering, 2011, 28(10):1 965-1 968.  ADEBOWALE T O, LIU H, OSO A O, et al.Effect of dietary niacin supplementation on performance, total tract nutrient retention, carcass yield and meat lipid profile of growing turkeys[J].Animal Production Science, 2019, 59(6):1 098-1 107.  陈国忠, 郭秋翠, 谭其秀, 等.1株产烟酸羟基化酶海洋细菌H9的分离鉴定及培养条件优化[J].食品科学, 2017, 38(10):130-136. CHEN G Z, GUO Q C, TAN Q X, et al.Isolation, identification and culture optimization of Pseudomona sputida H9, a marine bacterium producing nicotinic acid hydroxylase[J].Food Science, 2017, 38(10):130-136.  CHANG Y H, WU F, LUO H.Conversion of 3-cyanopyridine to nicotinic acid by nitrilase in Rhodococcus rhodochrous tg1-A6[J].Journal of University of Science & Technology Beijing, 2007, 29(2):223-226.  FAN H Y, CHEN L F, SUN H H, et al.A novel nitrilase from Ralstonia eutropha H16 and its application to nicotinic acid production[J].Bioprocess & Biosystems Engineering, 2017, 40(8):1-11.  SUBRAMANIAM R, THIRUMAL V, CHISTOSERDOV A, et al.High-density cultivation in the production of microbial products[J].Chemical and Biochemical Engineering Quarterly, 2018, 32(4):451-464.  LEE S Y.High cell-density culture of Escherichia coli[J].Trends in Biotechnology, 1996, 14(3):98-105.  KWON E Y, KIM K M, KIM M K, et al.Production of nattokinase by high cell density fed-batch culture of Bacillus subtilis[J].Bioprocess and Biosystems Engineering, 2011, 34(7):789-793.  ZHOU Y L, LU Z H, WANG X, et al.Genetic engineering modification and fermentation optimization for extracellular production of recombinant proteins using Escherichia coli[J].Applied Microbiology and Biotechnology, 2018, 102(4):1 545-1 556.  顾炳琛, 龚劲松, 龙华, 等.产腈水解酶重组菌的发酵工艺及催化性质研究[J].分子催化, 2019, 33(2):174-180. GU B C, GONG J S, LONG H, et al.The fermentation process of a nitrilase-producing strain and its catalytic properties[J].Journal of Molecular Catalysis, 2019, 33(2):174-180.  VAGHARI H, JAFARIZADEHMALMIRI H, MOHAMMADLOU M, et al.Application of magnetic nanoparticles in smart enzyme immobilization[J].Biotechnology Letters, 2016, 38(2):223-233.  HUSAIN Q.Magnetic nanoparticles as a tool for the immobilization/stabilization of hydrolases and their applications:An overview[J].Biointerface Research in Applied Chemistry, 2016, 6(6):1 585-1 606.  邢朝晖, 苏跃龙, 张琦, 等.磁性纳米材料载体固定纤维素酶技术研究进展[J].生物技术通报, 2015, 31(8):59-65. XING Z H, SU Y L, ZHANG Q, et al.Research progress on cellulase immobilized by magnetic nanoparticles as carriers[J].Biotechnology Bulletin, 2015, 31(8):59-65.  DISHISHA T, ALVAREZ M T, HATTI-KAUL R.Batch-and continuous propionic acid production from glycerol using free and immobilized cells of Propionibacterium acidipropionici[J].Bioresource Technology, 2012, 118:553-562.  王顺治, 汪子凯, 龚劲松, 等.腈水解酶在枯草芽胞杆菌中表达及在烟酸合成中的应用[EB/OL].北京:中国科技论文在线, 2020-04-29. WANG S Z, WANG Z K, GONG J S, et al.Expression of nitrilase in Bacillus subtilis and its application in nicotinic synthesis[EB/OL].Beijing:Sciencepaper Online, 2020-04-29.  李刚. 氨基化Fe3O4纳米粒子的制备及在烟酸生物催化中的应用[D].无锡:江南大学, 2017. LI G.Preparation of aminated Fe3O4 nanoparticles and its application in nicotinic acid biocatalysis[D].Wuxi:Jiangnan University, 2017.  WU W, WU Z H, YU T, et al.Recent progress on magnetic iron oxide nanoparticles:Synthesis, surface functional strategies and biomedical applications[J].Science and Technology of Advanced Materials, 2015, 16(2):23 501-23 543.  TU G Y, LI M.Research progress of high density fermentation process of genetic engineering microorganisms[J].Industrial Microbiology, 2004, 34(3):48-52.  黄燕, 孙益荣, 吴敬, 等.重组Humicola insolens角质酶的高密度发酵优化[J].中国生物工程杂志, 2019, 39(1):63-70. HUANG Y, SUN Y R, WU J, et al.Optimization of high density fermentation of recombinant Humicola insolens cutinase[J].Chinese Biotechnology, 2019, 39(1):63-70.  PAI O, BANOTH L, GHOSH S, et al.Biotransformation of 3-cyanopyridine to nicotinic acid by free and immobilized cells of recombinant Escherichia coli[J].Process Biochemistry, 2014, 49(4):655-659.  DONG T T, GONG J S, GU B C, et al.Significantly enhanced substrate tolerance of Pseudomonas putida nitrilase via atmospheric and room temperature plasma and cell immobilization[J].Bioresource Technology, 2017, 244:1 104-1 110.