多孔整体柱固定化酶研究进展
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摘要
高效固定化酶载体的构建对于提高酶催化的反应速率和效率以及延长酶的使用寿命至关重要。多孔整体柱具有大的贯穿孔道和良好的通透性,易于改性,化学稳定性高。作为固定化酶的载体,可以为底物和产物提供快速的对流传质性能,从而提高酶催化的速率和效率,在保持酶高效专一及温和的催化反应特性的同时,克服了游离酶的不足,具有贮存稳定性高、操作连续可控、易分离回收、可重复使用、工艺简便等一系列优点。本文中,笔者总结了多孔整体柱应用于固定化酶的研究进展,探讨了不同整体柱材料和固定化酶方法对酶催化反应性能的影响。
Exploration of effective solid supports for immobilization of enzymes is highly desirable to improve the enzymatic catalysis efficiency and increase the operational life of enzymes. Porous monoliths containing large through-pores feature the advantages of high permeability to flow,ease of surface functionalization,and high chemical stabilities. These properties can provide rapid convective mass transport of substrates and products to the active site of the enzymes,which will remarkably enhance the enzymatic catalysis efficiency. Moreover,monolithic bioreactors enable continuous operation with simple process and high reusability. In this review,we summarize the new development of monoliths as solid supports for enzyme immobilization. Effects of monolithic matrix and immobilization methods on the enzymatic catalysis efficiency have been discussed.
Exploration of effective solid supports for immobilization of enzymes is highly desirable to improve the enzymatic catalysis efficiency and increase the operational life of enzymes. Porous monoliths containing large through-pores feature the advantages of high permeability to flow,ease of surface functionalization,and high chemical stabilities. These properties can provide rapid convective mass transport of substrates and products to the active site of the enzymes,which will remarkably enhance the enzymatic catalysis efficiency. Moreover,monolithic bioreactors enable continuous operation with simple process and high reusability. In this review,we summarize the new development of monoliths as solid supports for enzyme immobilization. Effects of monolithic matrix and immobilization methods on the enzymatic catalysis efficiency have been discussed.
引文
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[16] YIN J,CHEN S,ZHANG N,et al.Multienzyme cascade bioreactor for a 10 min digestion of genomic DNA into single nucleosides and quantitative detection of structural DNA modifications in cellular genomic DNA[J]. ACS Appl Mater Interfaces,2018,10(26):21883-21890.
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[23] JONNADA M,EL RASSI Z. Poly(N-acryloxysuccinimide-coethylene glycol dimethacrylate)precursor monolith and its post polymerization modification with alkyl ligands,trypsin and lectins for reversed-phase chromatography,miniaturized enzyme reactors and lectin affinity chromatography, respectively[J].Electrophoresis,2017,38(22/23):2870-2879.
[24] WU S,ZHANG L,YANG K,et al.Preparing a metal-ion chelated immobilized enzyme reactor based on the polyacrylamide monolith grafted with polyethylenimine for a facile regeneration and high throughput tryptic digestion in proteomics[J]. Anal Bioanal Chem,2012,402(2):703-710.
[25] KRENKOVA J,LACHER N A,SVEC F. Highly Efficientenzyme reactors containing trypsin and endoproteinase Lys C immobilized on porous polymer monolith coupled to MS suitable for analysis of antibodies[J].Anal Chem,2009,81:2004-2012.
[26] SUN X,XIN Y,WANG X,et al. Functionalized acetoacetylated poly(vinyl alcohol)monoliths for enzyme immobilization:a phase separation method[J]. Colloid Polym Sci,2017,295(10):1827-1833.
[27] UYAMA H,WANG G.Reactive poly(ethylene-co-vinyl alcohol)monoliths with tunable pore morphology for enzyme immobilization[J].Colloid Polym Sci,2015,293(8):2429-2435.
[28] KOLB H C,FINN M G,SHARPLESS K B. Click chemistry:diverse chemical function from a few good reactions[J]. Angew Chem Int Ed,2001,40:2004-2021.
[29] CELEBI B,BAYRAKTAR A,TUNCEL A. Synthesis of a monolithic,micro-immobilised enzyme reactor via click-chemistry[J].Anal Bioanal Chem,2012,403(9):2655-2663.
[30] WU M,ZHANG H,WANG Z,et al."One-pot"fabrication of clickable monoliths for enzyme reactors[J]. Chem Commun(Camb),2013,49(14):1407-1409.
[31] LAFLEUR J P,SENKBEIL S,NOVOTNY J,et al. Rapid and simple preparation of thiol-ene emulsion-templated monoliths and their application as enzymatic microreactors[J].Lab Chip,2015,15(10):2162-2172.
[32] JONSSON A,SVEJDAL R R,BOGELUND N,et al. Thiolenemonolithic pepsin microreactor with a 3D-printed interface for efficient UPLC-MS peptide mapping analyses[J]. Anal Chem,2017,89(8):4573-4580.
[33] LI N,ZHENG W,SHEN Y,et al.Preparation of a novel polymer monolith with functional polymer brushes by two-step atomtransfer radical polymerization for trypsin immobilization[J].J Sep Sci,2014,37(23):3411-3417.
[34] XIONG M,GU B,ZHANG J D,et al. Glucose microfluidic biosensors based on reversible enzyme immobilization on photopatterned stimuli-responsive polymer[J]. Biosens Bioelectron,2013,50:229-234.
[35] WU S,MA J,YANG K,et al. A novel organic-inorganic hybrid monolith for trypsin immobilization[J].Sci China Life Sci,2011,54(1):54-59.
[36] MA J,HOU C,LIANG Y,et al. Efficient proteolysis using a regenerable metal-ion chelate immobilized enzyme reactor supported on organic-inorganic hybrid silica monolith[J].Proteomics,2011,11(5):991-995.
[37] YUAN H,ZHANG L,ZHANG Y. Preparation of high efficiency and low carry-over immobilized enzymatic reactor with methacrylic acid-silica hybrid monolith as matrix for on-line protein digestion[J].J Chromatogr A,2014,1371:48-57.
[38] CHEN Y,WU M,WANG K,et al. Vinyl functionalized silica hybrid monolith-based trypsin microreactor for on line digestion and separation via thiol-ene"click"strategy[J].J Chromatogr A,2011,1218(44):7982-7988.
[39] ZHOU Y,WAN M M,GAO L,et al. One-pot synthesis of a hierarchical PMO monolith with superior performance in enzyme immobilization[J].J Mater Chem B,2013,1(12):1738-1748.
[40] ANSARI S A,HUSAIN Q. Potential applications of enzymes immobilized on/in nano materials:a review[J]. Biotechnol Adv,2012,30(3):512-523.
[41] LV Y,ALEJANDRO F M,FRECHET J M,et al. Preparation of porous polymer monoliths featuring enhanced surface coverage with gold nanoparticles[J]. J Chromatogr A,2012,1261:121-128.
[42] SHANGGUAN L,ZHANG L,XIONG Z,et al.Investigation of bienzymatic reactor based on hybrid monolith with nanoparticles embedded and its proteolytic characteristics[J].J Chromatogr A,2015,1388:158-166.
[43] XU S,MO R,JIN C,et al. Mesoporous silica nanoparticles incorporated hybrid monolithic stationary phase immobilized with pepsin for enantioseparation by capillary electrochromatography[J].J Pharm Biomed Anal,2017,140:190-198.
[44] HONG T,CHEN X,XU Y,et al.Preparation of graphene oxidemodified affinity capillary monoliths based on three types of amino donor for chiral separation and proteolysis[J]. J Chromatogr A,2016,1456:249-256.
[2]白云岫,曹逊,戈钧.高分子修饰无机晶体固定化酶研究进展[J].生物加工过程,2018,16(1):1672-3678.
[3] MOULD D L,SYNGE R L M.Electrokinetic ultrafiltration analysis of polysaccharides:a new approach to the chromatography of large molecules[J].Analyst,1952,77:964-969.
[4] KUBN M,PACˇEK P,CHROMECˇEK R. Gel permeation chromatography on porous poly(ethylene glycol methacrylate)[J].Chem Commun,1967,32(11):3881-3887.
[5] HJERTN S,LIAO J L,ZHANG R. High-performance liquid chromatography on continuous polymer beds[J].J Chromatogr A,1989,473(1):273-275.
[6] SVEC F,FRCHET J M J. Continuous rods of macroporous polymer as high performance liquid chromatography separation media[J].Anal Chem,1992,64(7):820-822.
[7] ISHIZUKA N,KOBAYASHI H,MINAKUCHI H.Monolithic silica columns for high-efficiency separations by high-performance liquid chromatography[J]. J Chromatogr A,2002,960(1/2):85-96.
[8] HE P,GREENWAY G,HASWELL S J. Development of a monolith based immobilized lipase micro-reactor for biocatalytic reactions in a biphasic mobile system[J]. Process Biochem,2010,45(4):593-597.
[9] ANUAR S T,MUGO S M,CURTIS J M.A flow-through enzymatic microreactor for the rapid conversion of triacylglycerols into fatty acid ethyl ester and fatty acid methyl ester derivatives for GC analysis[J].Anal Methods,2015,7(14):5898-5906.
[10] KAWAKAMI K,UENO M,TAKEI T,et al. Application of a Burkholderia cepacia lipase-immobilized silica monolith microbioreactor to continuous-flow kinetic resolution for transesterification of(R,S)-1-phenylethanol[J]. Process Biochem,2012,47(1):147-150.
[11] SZYMAN'SKA K,PUDO W,MROWIEC-BIAON'J,et al.Immobilization of invertase on silica monoliths with hierarchical pore structure to obtain continuous flow enzymatic microreactors of high performance[J].Micropor Mesopor Mater,2013,170:75-82.
[12] HE P,DAVIES J,GREENWAY G,et al. Measurement of acetylcholinesterase inhibition using bienzymes immobilized monolith micro-reactor with integrated electrochemical detection[J].Anal Chim Acta,2010,659(1/2):9-14.
[13] YESIL-CELIKTAS O,CUMANA S,SMIRNOVA I. Silica-based monoliths for enzyme catalyzed reactions in microfluidic systems with an emphasis on glucose 6-phosphate dehydrogenase and cellulase[J].Chem Eng J,2013,234:166-172.
[14] PIROZZI D,ABAGNALE M,MINIERI L,et al. In-situ sol-gel modification strategies to develop a monolith continuous microreactor for enzymatic green reactions[J].Chem Eng J,2016,306:1010-1016.
[15] CHEN S,WEN L,SVEC F,et al. Magnetic metal-organic frameworks as scaffolds for spatial co-location and positional assembly of multi-enzyme systems enabling enhanced cascade biocatalysis[J].RSC Adv,2017,7(34):21205-21213.
[16] YIN J,CHEN S,ZHANG N,et al.Multienzyme cascade bioreactor for a 10 min digestion of genomic DNA into single nucleosides and quantitative detection of structural DNA modifications in cellular genomic DNA[J]. ACS Appl Mater Interfaces,2018,10(26):21883-21890.
[17] RIVERA J G,MESSERSMITH P B. Polydopamine-assisted immobilization of trypsin onto monolithic structures for protein digestion[J].J Sep Sci,2012,35(12):1514-1520.
[18] FOO H C,SMITH N W,STANLEY S M.Fabrication of an on-line enzyme micro-reactor coupled to liquid chromatography-tandem mass spectrometry for the digestion of recombinant human erythropoietin[J].Talanta,2015,135:18-22.
[19]吕永琴.新型功能化整体柱的制备、应用和分离识别机理的研究[D].北京:北京化工大学,2010.
[20] XIE S,SVEC F,FRECHET J M J. Design of reactive porous polymer supports for high throughput bioreactors:poly(2-vinyl-4,4-dimethylazlactone-co-acrylamide-co-ethyl dimethacrylate[J].Biotechnol Bioeng,1999,62(1):30-35.
[21] YAO C,QI L,HU W,et al. Immobilization of trypsin on submicron skeletal polymer monolith[J].Anal Chim Acta,2011,692(1/2):131-137.
[22] OZONER S K,KESKINLER B,ERHAN E. HRP immobilized microporous poly(styrene-divinylbenzene-polyglutaraldehyde)monolith for forced flow injected phenol biosensing[J].Mater Sci Eng C,2011,31(3):663-668.
[23] JONNADA M,EL RASSI Z. Poly(N-acryloxysuccinimide-coethylene glycol dimethacrylate)precursor monolith and its post polymerization modification with alkyl ligands,trypsin and lectins for reversed-phase chromatography,miniaturized enzyme reactors and lectin affinity chromatography, respectively[J].Electrophoresis,2017,38(22/23):2870-2879.
[24] WU S,ZHANG L,YANG K,et al.Preparing a metal-ion chelated immobilized enzyme reactor based on the polyacrylamide monolith grafted with polyethylenimine for a facile regeneration and high throughput tryptic digestion in proteomics[J]. Anal Bioanal Chem,2012,402(2):703-710.
[25] KRENKOVA J,LACHER N A,SVEC F. Highly Efficientenzyme reactors containing trypsin and endoproteinase Lys C immobilized on porous polymer monolith coupled to MS suitable for analysis of antibodies[J].Anal Chem,2009,81:2004-2012.
[26] SUN X,XIN Y,WANG X,et al. Functionalized acetoacetylated poly(vinyl alcohol)monoliths for enzyme immobilization:a phase separation method[J]. Colloid Polym Sci,2017,295(10):1827-1833.
[27] UYAMA H,WANG G.Reactive poly(ethylene-co-vinyl alcohol)monoliths with tunable pore morphology for enzyme immobilization[J].Colloid Polym Sci,2015,293(8):2429-2435.
[28] KOLB H C,FINN M G,SHARPLESS K B. Click chemistry:diverse chemical function from a few good reactions[J]. Angew Chem Int Ed,2001,40:2004-2021.
[29] CELEBI B,BAYRAKTAR A,TUNCEL A. Synthesis of a monolithic,micro-immobilised enzyme reactor via click-chemistry[J].Anal Bioanal Chem,2012,403(9):2655-2663.
[30] WU M,ZHANG H,WANG Z,et al."One-pot"fabrication of clickable monoliths for enzyme reactors[J]. Chem Commun(Camb),2013,49(14):1407-1409.
[31] LAFLEUR J P,SENKBEIL S,NOVOTNY J,et al. Rapid and simple preparation of thiol-ene emulsion-templated monoliths and their application as enzymatic microreactors[J].Lab Chip,2015,15(10):2162-2172.
[32] JONSSON A,SVEJDAL R R,BOGELUND N,et al. Thiolenemonolithic pepsin microreactor with a 3D-printed interface for efficient UPLC-MS peptide mapping analyses[J]. Anal Chem,2017,89(8):4573-4580.
[33] LI N,ZHENG W,SHEN Y,et al.Preparation of a novel polymer monolith with functional polymer brushes by two-step atomtransfer radical polymerization for trypsin immobilization[J].J Sep Sci,2014,37(23):3411-3417.
[34] XIONG M,GU B,ZHANG J D,et al. Glucose microfluidic biosensors based on reversible enzyme immobilization on photopatterned stimuli-responsive polymer[J]. Biosens Bioelectron,2013,50:229-234.
[35] WU S,MA J,YANG K,et al. A novel organic-inorganic hybrid monolith for trypsin immobilization[J].Sci China Life Sci,2011,54(1):54-59.
[36] MA J,HOU C,LIANG Y,et al. Efficient proteolysis using a regenerable metal-ion chelate immobilized enzyme reactor supported on organic-inorganic hybrid silica monolith[J].Proteomics,2011,11(5):991-995.
[37] YUAN H,ZHANG L,ZHANG Y. Preparation of high efficiency and low carry-over immobilized enzymatic reactor with methacrylic acid-silica hybrid monolith as matrix for on-line protein digestion[J].J Chromatogr A,2014,1371:48-57.
[38] CHEN Y,WU M,WANG K,et al. Vinyl functionalized silica hybrid monolith-based trypsin microreactor for on line digestion and separation via thiol-ene"click"strategy[J].J Chromatogr A,2011,1218(44):7982-7988.
[39] ZHOU Y,WAN M M,GAO L,et al. One-pot synthesis of a hierarchical PMO monolith with superior performance in enzyme immobilization[J].J Mater Chem B,2013,1(12):1738-1748.
[40] ANSARI S A,HUSAIN Q. Potential applications of enzymes immobilized on/in nano materials:a review[J]. Biotechnol Adv,2012,30(3):512-523.
[41] LV Y,ALEJANDRO F M,FRECHET J M,et al. Preparation of porous polymer monoliths featuring enhanced surface coverage with gold nanoparticles[J]. J Chromatogr A,2012,1261:121-128.
[42] SHANGGUAN L,ZHANG L,XIONG Z,et al.Investigation of bienzymatic reactor based on hybrid monolith with nanoparticles embedded and its proteolytic characteristics[J].J Chromatogr A,2015,1388:158-166.
[43] XU S,MO R,JIN C,et al. Mesoporous silica nanoparticles incorporated hybrid monolithic stationary phase immobilized with pepsin for enantioseparation by capillary electrochromatography[J].J Pharm Biomed Anal,2017,140:190-198.
[44] HONG T,CHEN X,XU Y,et al.Preparation of graphene oxidemodified affinity capillary monoliths based on three types of amino donor for chiral separation and proteolysis[J]. J Chromatogr A,2016,1456:249-256.