基于全细胞催化合成NADP重组细胞复合通透剂的筛选
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摘要
本文以重组大肠杆菌E. coli BL21(DE3) p ET30α(+)-Nmnat为研究对象,考察化学通透剂处理重组菌对其催化合成NADP的影响。实验显示,在单因素筛选实验中,四种通透剂曲拉通X-100、吐温80、二甲基亚砜和十六烷基三甲基溴化铵(CTAB)对细胞进行通透化处理均可提高全细胞催化产率,且提高幅度差距并不显著。进一步采用析因设计考察四种通透剂的复合效应,除了曲拉通X-100与吐温80和曲拉通X-100与CTAB交互作用的P大于0. 05为不显著,其余所有交互作用项P值均小于0. 05,呈现显著性,且复合通透剂中,仅单一增加CTAB量,复合体系的协同作用将减少,制约作用增加,不适宜用于复合配方。最后采用中心组合设计进一步对复配效果进行优化,通过拟合得到最佳通透剂配比为曲拉通X-100(1. 64%),吐温80(2. 53%)以及二甲基亚砜(1. 06%),此时NADP预测产率达到77. 46%,与实际验证产率均值77. 05%相差甚微,说明实验优化模型具有良好预测性,优化结果与未添加通透剂相比提升近47%,表明优化复合通透剂配方更有利于提高重组菌催化合成NADP。
The effects of chemical penetrators on NADP biosynthesis by recombinant E. coli BL21( DE3) pET30α( +)-Nmnat were investigated. The single factor screening showed that four kinds of permeable agents such as Triton X-100,Tween 80,dimethyl sulfoxide and cetyltrimethyl ammonium bromide( CTAB) all could increase the catalytic yield of the whole cell,however,the difference was not significant. Then,the factorial design was used to investigate the composite effects of the four penetrators on the whole cell catalysis. Except that the interactions of Triton X-100 with Tween 80 and Triton X-100 with CTAB( P > 0. 05),were not significant. All the other interaction items( P < 0. 05) showed significant difference,and the amount of CTAB was only increased in the composite penetrator. The synergy of the composite system would be reduced,and the restriction would be increased,so it was not suitable for compound formulation. Finally,the central composite design was applied to obtain the best ratio of the penetrators. The results showed that when 1. 64%Triton X-100,2. 53% Tween 80 and 1. 06% dimethyl sulfoxide were combined,the predicted NADP yield could reach the maximum 77. 46% in accordance with the verified yield,which revealed that the experimental optimization model had good predictability. The result of optimization was 47% higher than that of no penetrator. It indicated that optimized formula for composite penetrators was beneficial to improving the NADP biosynthesis by recombinant bacteria.
The effects of chemical penetrators on NADP biosynthesis by recombinant E. coli BL21( DE3) pET30α( +)-Nmnat were investigated. The single factor screening showed that four kinds of permeable agents such as Triton X-100,Tween 80,dimethyl sulfoxide and cetyltrimethyl ammonium bromide( CTAB) all could increase the catalytic yield of the whole cell,however,the difference was not significant. Then,the factorial design was used to investigate the composite effects of the four penetrators on the whole cell catalysis. Except that the interactions of Triton X-100 with Tween 80 and Triton X-100 with CTAB( P > 0. 05),were not significant. All the other interaction items( P < 0. 05) showed significant difference,and the amount of CTAB was only increased in the composite penetrator. The synergy of the composite system would be reduced,and the restriction would be increased,so it was not suitable for compound formulation. Finally,the central composite design was applied to obtain the best ratio of the penetrators. The results showed that when 1. 64%Triton X-100,2. 53% Tween 80 and 1. 06% dimethyl sulfoxide were combined,the predicted NADP yield could reach the maximum 77. 46% in accordance with the verified yield,which revealed that the experimental optimization model had good predictability. The result of optimization was 47% higher than that of no penetrator. It indicated that optimized formula for composite penetrators was beneficial to improving the NADP biosynthesis by recombinant bacteria.
引文
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[9]蔡海松,林晓栩,郭永华,等.信号肽及化学通透剂对环糊精葡萄糖基转移酶胞外分泌的影响[J].微生物学通报,2017,44(3):601-610.
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[12]侯堃,李红梅,侯立琪.嗜热脂肪地芽孢杆菌NAD激酶克隆表达及酶学性质研究[J].工业微生物,2014,44(6):39-44.
[13]袁飞飞,李红梅.保护剂对大肠杆菌重组NAD激酶热稳定性的影响[J].精细化工,2016,33(12):1344-1350.
[14]何璞,余龙江,周蓬蓬,等.响应面法对红法夫酵母合成虾青素主要影响因素的优化[J].工业微生物,2007,37(1):63-66.
[15] NI Y,CHEN R R. Accelerating whole-cell biocatalysis by reducing outer membrane permeability barrier[J]. Biotechnol Bioeng,2010,87(6):804-811.
[16] WERF M J V D,HARTMANS S,TWEEL W J J V D. Permeabilization and lysis of Pseudomonas pseudoalcaligenes cells by Triton X-100 for efficient production of d-malate[J]. Appl Microbiol Biot,1995,43(4):590-594.
[17] CHEN R R. Permeability issues in whole-cell bioprocesses and cellular membrane engineering[J]. Appl Microbiol Biot,2007,74(4):730-8.
[18] ZHOU J,CHEN X,LU L,et al. Enhanced production of N-acetyl-d-neuraminic acid by whole-cell bio-catalysis of Escherichia coli[J]. J Mol CataL B-enzym,2016,125(5):42-48.
[19]吴敏,杨天锦,苗茂栋,等.改善细胞通透性促进1,3-丙二醇生物合成[J].化学学报,2009,67(18):2133-2138.
[20]刘俊梅,聂海彦,李琢伟,等.海藻糖合酶重组菌LB35透性化细胞产海藻糖特性研究[J].农业机械,2011(8):111-115.
[21] KAUR G,MEHTA S K. Developments of Polysorbate(Tween)based microemulsions:Preclinical drug delivery,Toxicity and antimicrobial applications.[J]. Int J Pharm, 2017, 529(1-2):134.
[22] STUTZENBERGER F J. Interference of the detergent Tween 80in protein assays[J]. Aanl Biochem,1992,207(2):249-254.
[23] CORTEZ D V,ROBERTO I C. CTAB,Triton X-100 and freezing-thawing treatments of Candida guilliermondii:effects on permeability and accessibility of the glucose-6-phosphate dehydrogenase,xylose reductase and xylitol dehydrogenase enzymes[J].New Biotechnol,2012,29(2):192.
[2]还晓静.三种棒杆菌NAD激酶的酶学性质及其对L-异亮氨酸合成的影响[D].无锡:江南大学,2012. 5-6
[3] MAILLOUX R J,LEMIRE J,APPANNA V D. Metabolic networks to combat oxidative stress in Pseudomonas fluorescens[J].Anton Leeuw Int J G,2011,99(3):433-42.
[4]刘根,周永达,庄季昌,等.一种氧化型辅酶ii的酶催化制备方法:,CN 102605027 A[P]. 2012.
[5] KRATZER R,WOODLEY J M,NIDETZKY B. Rules for biocatalyst and reaction engineering to implement effective,NAD(P)Hdependent,whole cell bioreductions.[J]. Biotechnol Adv,2015,33(8):1641-1652.
[6] RUNDBCK F,FIDANOSKA M,ADLERCREUTZ P. Coupling of permeabilized cells of Gluconobacter oxydans and Ralstonia eutropha for asymmetric ketone reduction using H2as reductant[J].J Biotechnol,2012,157(1):154-158.
[7]罗杰.细胞通透性的改变及其应用[J].微生物学报,2001,41(3):386-389.
[8]袁东超,王敏.提高氢化可的松发酵过程中的微生物转化效率的初探[J].药物生物技术,2004,11(6):399-400.
[9]蔡海松,林晓栩,郭永华,等.信号肽及化学通透剂对环糊精葡萄糖基转移酶胞外分泌的影响[J].微生物学通报,2017,44(3):601-610.
[10] CHENG S,WEI D,SONG Q,et al. Immobilization of permeabilized whole cell penicillin G acylase from Alcaligenes faecalis,using pore matrix crosslinked with glutaraldehyde[J]. Biotechnol Lett,2006,28(14):1129-1133.
[11]侯堃,李红梅.重组大肠杆菌产NAD激酶发酵条件的优化研究[J].工业微生物,2015(3):41-47.
[12]侯堃,李红梅,侯立琪.嗜热脂肪地芽孢杆菌NAD激酶克隆表达及酶学性质研究[J].工业微生物,2014,44(6):39-44.
[13]袁飞飞,李红梅.保护剂对大肠杆菌重组NAD激酶热稳定性的影响[J].精细化工,2016,33(12):1344-1350.
[14]何璞,余龙江,周蓬蓬,等.响应面法对红法夫酵母合成虾青素主要影响因素的优化[J].工业微生物,2007,37(1):63-66.
[15] NI Y,CHEN R R. Accelerating whole-cell biocatalysis by reducing outer membrane permeability barrier[J]. Biotechnol Bioeng,2010,87(6):804-811.
[16] WERF M J V D,HARTMANS S,TWEEL W J J V D. Permeabilization and lysis of Pseudomonas pseudoalcaligenes cells by Triton X-100 for efficient production of d-malate[J]. Appl Microbiol Biot,1995,43(4):590-594.
[17] CHEN R R. Permeability issues in whole-cell bioprocesses and cellular membrane engineering[J]. Appl Microbiol Biot,2007,74(4):730-8.
[18] ZHOU J,CHEN X,LU L,et al. Enhanced production of N-acetyl-d-neuraminic acid by whole-cell bio-catalysis of Escherichia coli[J]. J Mol CataL B-enzym,2016,125(5):42-48.
[19]吴敏,杨天锦,苗茂栋,等.改善细胞通透性促进1,3-丙二醇生物合成[J].化学学报,2009,67(18):2133-2138.
[20]刘俊梅,聂海彦,李琢伟,等.海藻糖合酶重组菌LB35透性化细胞产海藻糖特性研究[J].农业机械,2011(8):111-115.
[21] KAUR G,MEHTA S K. Developments of Polysorbate(Tween)based microemulsions:Preclinical drug delivery,Toxicity and antimicrobial applications.[J]. Int J Pharm, 2017, 529(1-2):134.
[22] STUTZENBERGER F J. Interference of the detergent Tween 80in protein assays[J]. Aanl Biochem,1992,207(2):249-254.
[23] CORTEZ D V,ROBERTO I C. CTAB,Triton X-100 and freezing-thawing treatments of Candida guilliermondii:effects on permeability and accessibility of the glucose-6-phosphate dehydrogenase,xylose reductase and xylitol dehydrogenase enzymes[J].New Biotechnol,2012,29(2):192.
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