聚乙烯亚胺/多巴胺改性氧化硅固定碳酸酐酶
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摘要
利用聚乙烯亚胺(PEI)/多巴胺(DA)共沉积法改性氧化硅,并以此为载体固定化碳酸酐酶(CA)。考察了PEI/DA质量比、沉积时间对沉积率的影响,用傅里叶红外光谱(FTIR)和扫描电子显微镜(SEM)对改性前后的微球进行了表征;研究了沉积率、载体用量、酶浓度及戊二醛(GA)浓度对固定化酶活回收率的影响;考察了固定化酶的储存稳定性和重复利用性。结果表明,随PEI/DA质量比增加,沉积率先增加后降低,质量比为1∶1时最大;随沉积时间增加,沉积率线性增长,10 h后PEI/DA体系沉积率为单独DA沉积改性的2.66倍,但沉积时间对N元素含量和酶活回收率影响不大;酶固定化时载体用量存在饱和值,CA和GA浓度的最优值分别为0.8 mg/ml和0.1%(质量),此时酶活回收率可达78.8%。在25℃下储存30 d后,固定化酶的保留活性为77.2%,而游离酶只有12%;重复使用10次后,固定化酶仍能保持88.3%的相对活性。
Silica was modified by a polyethyleneimine(PEI)/dopamine(DA)co-deposition method,and then carbonic anhydrase(CA)was covalently immobilized via glutaraldehyde(GA).First,the effects of the mass ratio of PEI and DA,deposition time on the deposition rate were investigated,and silica microspheres before and after modification were characterized by Fourier transform infrared spectroscopy(FTIR)and scanning electron microscope(SEM).Then,the influences of the immobilization conditions including the deposition rate,the addition amount of PEI/PDA-SiO_2,CA and GA concentration on the activity recovery of immobilized enzyme were studied.At last,the storage stability and reusability of immobilized enzyme were also examined.The results show that the deposition rate increased firstly and then decreased with the mass ratio of PEI and DA increased,and reached the maximum at a mass ratio of 1∶1.The deposition rate increased linearly with the time prolonged,which respectively reached 1.33%and 0.5%after 10 h for PEI/DA co-deposited system and pure DA-deposited that.The deposition rate had no obvious effect on the content of N element and activity recovery of immobilized CA.For enzyme immobilization,there was a saturated addition amount of the carrier which was 0.25 g PEI/PDA-SiO_2/mg CA,and the optimal concentration of CA and GA was 0.8 mg/ml and 0.1%(mass),respectively.Under these conditions,the activity recovery of CA-PEI/PDA-SiO_2was about 78.8%.After 30 days of storage at 25℃,the remaining activity of immobilized CA was 77.2%while that of free CA was only 12%.The relative activity of immobilized CA was 88.3%after undergoing 10 cycles.
Silica was modified by a polyethyleneimine(PEI)/dopamine(DA)co-deposition method,and then carbonic anhydrase(CA)was covalently immobilized via glutaraldehyde(GA).First,the effects of the mass ratio of PEI and DA,deposition time on the deposition rate were investigated,and silica microspheres before and after modification were characterized by Fourier transform infrared spectroscopy(FTIR)and scanning electron microscope(SEM).Then,the influences of the immobilization conditions including the deposition rate,the addition amount of PEI/PDA-SiO_2,CA and GA concentration on the activity recovery of immobilized enzyme were studied.At last,the storage stability and reusability of immobilized enzyme were also examined.The results show that the deposition rate increased firstly and then decreased with the mass ratio of PEI and DA increased,and reached the maximum at a mass ratio of 1∶1.The deposition rate increased linearly with the time prolonged,which respectively reached 1.33%and 0.5%after 10 h for PEI/DA co-deposited system and pure DA-deposited that.The deposition rate had no obvious effect on the content of N element and activity recovery of immobilized CA.For enzyme immobilization,there was a saturated addition amount of the carrier which was 0.25 g PEI/PDA-SiO_2/mg CA,and the optimal concentration of CA and GA was 0.8 mg/ml and 0.1%(mass),respectively.Under these conditions,the activity recovery of CA-PEI/PDA-SiO_2was about 78.8%.After 30 days of storage at 25℃,the remaining activity of immobilized CA was 77.2%while that of free CA was only 12%.The relative activity of immobilized CA was 88.3%after undergoing 10 cycles.
引文
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[2]Vinoba M,Bhagiyalakshmi M,Jeong S K,et al.Carbonic anhydrase immobilized on encapsulated magnetic nanoparticles for CO2sequestration[J].Chemistry,2012,18(38):12028-12034.
[3]Zhang S H,Lu Y Q,Ye X H,et al.Catalytic behavior of carbonic anhydrase enzyme immobilized onto nonporous silica nanoparticles for enhancing CO2absorption into a carbonate solution[J].Greenhouse Gas Control,2013,13:17-25.
[4]Liu N,Bond G M,Abel A,et al.Biomimetic sequestration of CO2in carbonate form:role of produced waters and other brines[J].Fuel Processing Technology,2005,86:1615-1625.
[5]Perice S,Perfetto R,Russo M E,et al.Characterization of technical grade carbonic anhydrase as biocatalyst for CO2capture in potassium carbonate solutions[J].Original Research Article,2018,8(2):279-291.
[6]陈功,卢滇楠,吴建中,等.水化层影响碳酸酐酶内CO2扩散行为的分子动力学模拟[J].化工学报,2015,66(8):2903-2910.Chen G,Lu D N,Wu J Z,et al.Molecular dynamics simulation for hydration effect on CO2diffusion in carbonic anhydrase[J].CIESCJournal,2015,66(8):2903-2910.
[7]Chang H L,Eui K J,Young J Y,et al.Stabilization of bovine carbonic anhydrase II through rational site-specific immobilization[J].Biochemical Engineering Journal,2018,138:29-36.
[8]Ren S Z,Feng Y X,Wen H,et al.Immobilized carbonic anhydrase on mesoporous cruciate flowerlike metal organic framework for promoting CO2sequestration[J].2018,117:189-198.
[9]刘文芳,魏利娜.碳酸酐酶固定化研究进展[J].分子催化,2016,30(2):1001-3555.Liu W F,Wei L N.Research progress on carbonic anhydrase immobilization[J].Journal of Molecular Catalysis(China),2016,30(2):1001-3555.
[10]袁定重,张秋禹,侯振宇.磁性高分子微球研究进展及其在生化分离中的应用[J].材料导报,2006,20:69-72.Yuan D Z,Zhang Q Y,Hou Z Y.Research advance of magnetic microsphere and its application in biochemical separation[J].Materials Reports,2006,20:69-72.
[11]Fei X Y,Chen S Y,Liu D,et al.Comparison of amino and epoxy functionalized SBA-15 used for carbonic anhydrase immobilization[J].Bioscience and Bioengineering,2016,122(3):314-321.
[12]张朝晖,杨玉莹.碳酸酐酶在多孔玻璃上的固定化及性质研究[J].材料导报,2015,29(25):237-241.Zhang Z H,Yang Y Y.Evaluation and characterization of immobilized bovine carbonic anhydrase on controlled pore glass[J].Materials Reports,2015,29(25):237-241.
[13]Al-Dhrub A H A,Sahin S,Ozmen I,et al.Immobilization and characterization of human carbonic anhydrase I on amine functionalized magnetic nanoparticles[J].Process Biochemistry,2017,57:95-104.
[14]Chew N G P,Zhao S S,Malde C,et al.Superoleophobic surface modification for robust membrane distillation performance[J].Journal of Membrane Science,2017,541:162-173.
[15]Zhou L Y,Jiang Y J,Ma L,et al.Immobilization of glucose oxidase on polydopamine-functionalized graphene oxide[J].Applied Biochemistry Biotechnology,2015,175(2):1007-1017.
[16]Qiu W Z,Yang H C,Xu Z K.Dopamine-assisted co-deposition:an emerging and promising strategy for surface modification[J].Advances in Colloid and Interface Science,2018,256:111-125.
[17]Wang T Y,Qiblawey H,Judd S,et al.Fabrication of high flux nanofiltration membrane via hydrogen bonding based codeposition of polydopamine with poly(vinyl alcohol)[J].Journal of Membrane Science,2018,552:222-233.
[18]李创,王彩云,江婷婷,等.PDA/RGO复合材料对水中Fe(Ⅲ)的吸附行为[J].化工学报,2017,68(6):2577-2584.Li C,Wang C Y,Jiang T T,et al.Adsorption behavior of Fe(III)on PDA/RGO composites[J].CIESC Journal,2017,68(6):2577-2584.
[19]张培斌,唐安琪,路景驭,等.基于贻贝仿生化学的分离功能材料[J].功能高分子学报,2017,(10):1008-9357.Zhang P B,Tang A Q,Lu J Y,et al.Separation materials based on mussel-bioinspired surface chemistry[J].Journal of Functional Polymers,2017,(10):1008-9357.
[20]Yang H C,Luo J Q,Lv Y,et al.Surface engineering of polymer membranes via mussel-inspired chemistry[J].Journal of Membrane Science,2015,48:42-59.
[21]Yang H C,Wu M B,Li Y J,et al.Effects of polyethyleneimine molecular weight and proportion on the membrane hydrophilization by codepositing with dopamine[J].Journal of Applied Polymer Science,2016,133(32):43792(1-9).
[22]Yang H C,Liao K J,Huang H,et al.Mussel-inspired modification of a polymer membrane for ultra-high water permeability and oilin-water emulsion separation[J].Materials Chemistry A,2014,2(26):10225-10230.
[23]Cao X T,Luo J Q,Woodley J M,et al.Mussel-inspired codeposition to enhance bisphenol A removal in a bifacial enzymatic membrane reactor[J].Chemical Engineering Journal,2018,336:315-324.
[24]Ozdemir E.Biomimetic CO2sequestration(1):Immobilization of carbonic anhydrase within polyurethane foam[J].American Chemical Society,2009,23(11):5725-5730.
[25]Cui J Y,Zhou Z P,Xie A,et al.Bio-inspired fabrication of superhydrophilic nanocomposite membrane based on surface modification of SiO2anchored by polydopamine towards effective oil-water emulsions separation[J].Separation and Purification Technology,2019,209:434-422.
[26]Xu Y,Li Z H,Su K M,et al.Mussel-inspired modification of PPSmembrane to separate and remove the dyes from the wastewater[J].Chemical Engineering Journal.2018,341:371-382.
[27]Sun J,Wei L,Wang Y Z,et al.Immobilization of carbonic anhydrase on polyvinylidene fluoride membranes[J].Biotechnology and Applied Biochemistry,2018,65:362-371.
[28]Sahoo P C,Sambudi N S,Park S B,et al.Immobilization of carbonic anhydrase on modified electrospun poly(lactic acid)membranes:quest for optimum biocatalytic performance[J].Catalysis Letters,2015,145:519-526.