Microfluidic reactor with immobilized enzyme-from construction to applications:A review

Microfluidic,as the systems for using microchannel(micron-or sub-micron scale)to process or manipulate microflow,is being widely applied in enzyme biotechnology and biocatalysis.Microfluidic immobilized enzyme reactor(MIER)is a tool with great value for the study of catalytic property and optimal reaction parameter in a flourishing and highly producing manner.In view of its advantages in efficiency,economy,and addressable recognition especially,MIER occupies an important position in the investigation of life science,including molecular biology,bioanalysis and biosensing,biocatalysis etc.Immobilization of enzymes can generally improve their stability,and upon most occasions,the immobilized enzyme is endowed with recyclability.In this review,the enzyme immobilization techniques applied in MIER will be discussed,followed by summarizing the novel developments in the field of MIER for biocatalysis,bioconversion and bioanalysis.The preponderances and deficiencies of the current state-of-the-art preparation ways of MIER are peculiarly discussed.In addition,the prospects of its future study are outlined.

Immobilized enzyme reactors in HPLC and its application in inhibitor screening:A review

This paper sets out to summarize the literatures based on immobilized enzyme bio-chromatography and its application in inhibitors screening in the last decade.In order to screen enzyme inhibitors from a mass of compounds in preliminary screening,multi-pore materials with good biocompatibility are used for the supports of immobilizing enzymes,and then the immobilized enzyme reactor applied as the immobilized enzyme stationary phase in HPLC.Therefore,a technology platform of high throughput screening is gradually established to screen the enzyme inhibitors as new anti-tumor drugs.Here,we briefly summarize the selective methods of supports,immobilization techniques,co-immobilized enzymes system and the screening model.

Enhanced production of cytidine 5'-monophosphate using biocatalysis of di-enzymes immobilized on amino-functionalized sepharose

Cytidine 5'-monophosphate(5'-CMP)is an essential nucleotide for additives.In this study,enhanced production of 5'-CMP was realized by the transformation of cytidine using co-immobilized di-enzymes,uridine-cytidine kinase(UCK)and acetate kinase(AcK).The immobilization yield of the enzyme had a clear correlation with the surface charges as zeta potential(ξ).Among them,ε-polylysinefunctionalized sepharose(SA-EPL,ξ=9.31 m V)showed high immobilization yield(78.8%),which was4.9-fold than that of nitrilotriacetic acid functionalized sepharose(SA-NTA,ξ=-12.6 m V).The residual activity of affinity co-immobilized enzyme(EPL-Ni/EPL@Ac K-UCK)was higher than 70.6%after recycled 10 times.Thus,this study provides an effective approach for the production of 5'-CMP with the advantages of low adenosine 5'-triphosphate(ATP)consumption,reduced side reactions,and improved reusability by co-immobilized UCK and Ac K on the functionalized Sepharose.

Studies on Manganese Peroxidase Immobilized in Gelatin-containing Microemulsion-based Gels

The immobilized technique of manganese peroxidase(MnP) in gelatin-containing microemulsion-based gels and the effects of storage time and reuse times on its catalytic activity were studied. The results show that the MnP immobilized together with Mn 2+ and H_2O_2 could effectively oxidize syringaldazine in n-heptane. The immobilized MnP still had a high catalytic activity after one-month storage under a freezing condition. The reuse times have a relation to the amount of the immobilized H_2O_2. When the amount of the immobilized H_2O_2 is sufficient, the microemulsion-based gels containing MnP could be used many times.

Recent advances in screening of enzymes inhibitors based on capillary electrophoresis

Capillary electrophoresis with many advantages plays an important role in pharmaceutical analysis and drug screening. This review gives an overview on the recent advances in the developments and applications of capillary electrophoresis in the field of enzyme inhibitor screening. The period covers 2013 to 2017. Both the pre-capillary enzyme assays and in-capillary enzyme assays which include electrophoretically mediated microanalysis（EMMA） and immobilized enzyme microreactor（IMER） are summarized in this article.

Corrosion Inhibition of the Bi-enzyme Doped Epoxy Resin for Q235 Carbon Steel in Circulating Cooling Water

Carboxyl-functionalized SBA-15(COOH/SBA-15)was prepared by a one-pot synthesis method and characterized.COOH-SBA-15/LZM-LP,an immobilized bi-enzyme(lipase and lysozyme),was prepared using COOH/SBA-15 as a carrier.The orthogonal experiments were used to optimize the immobilization conditions with the index of corrosion inhibition.Electrochemical tests show that COOH-SBA-15/LZM-LP can significantly inhibit the corrosion of carbon steel in circulating cooling water.The corrosion inhibition rate was higher than 93%when the amount of COOHSBA-15/LZM-LP was 0.2 g/L.The inhibition mechanism was proposed and discussed from the perspective of carboxyl and enzymes.Finally,when COOH-SBA-15/LZM-LP was doped into epoxy resin,the corrosion resistance of epoxy coatings can be significantly improved,and the corrosion resistance only decreased by 0.23%after 720 h of soaking.

Electrospun Poly(acrylonitrile-co-acrylic acid) Nanofibrous Membranes for Catalase Immobilization:Effect of Porphyrin Filling on the Enzyme Activity

Porphyrin-filled nanofibrous membranes were facilely prepared by electrospinning of the mixtures of poly（acrylonitrile-co-acrylic acid）（PANCAA） and porphyrins. 5,10,15,20-Tetraphenylporphyrin（TPP） and its metal-loderivatives（ZnTPP and CuTPP） were studied as filling mediators for the immobilization of redox enzyme. Results indicate that the introduction of TPP, ZnTPP and CuTPP improves the retention activity of the immobilized catalase. Among these three porphyrins, the ZnTPP-filled PANCAA nanofibrous membrane exhibits an activity retention of 93%, which is an exciting improvement. This improvement is attributed to both the strong catalase-porphyrin affinity and the possible facilitated electron transfer induced by the porphyrin as evidenced by quartz crystal microbalance （QCM） and fluorescence spectroscopy studies.

Enzymes immobilized in wood-derived cellulose scaffold for constructing a novel modular bioreactor

Modular bioreactors can provide a flexible platform for constructing complex multi-step pathways,which may be a solution for maximizing reactions and overcoming the complexity of multi-enzyme systems.Here,we selected wood-derived cellulose scaffold as a support for enzyme immobilization and constructed the modular bioreactor.Cellulose scaffold was prepared after removing lignin from wood,followed by citric acid functionalization and the addition of glutaraldehyde finally allowed the cross-linking of enzymes.Three enzymes,horseradish peroxidase(HRP),glucose oxidase(GOD),and catalase(CAT),were separately immobilized,resulting in the immobilized enzyme amount to over 40 mg/g.The introduction of carboxyl groups from citric acid facilitated the rapid enzyme adsorption on the support surface and immobilized enzymes possess~65%expressed activity.Modular bioreactors were constructed by using the immobilized enzymes.With the immobilized HRP module,reactor showed desired catalytic performance with the phenol degradation rate of>90%.Also,a pH regulation can occur in the bioreactors for preserving enzyme activities and neutralizing acid products.In the GOD/CAT modular bioreactor,the cascade reaction with adjusting pH values can achieve a 95%yield of sodium gluconate and exhibit a favorable reusability of 5 operation cycles.

CATase-immobilized hydrogel platform molded by photo-enzyme coupling polymerization for effectively preventing postoperative abdominal adhesion

Redox homeostasis,which is regulated by enzymes acting as regulatory valves,is crucial for maintaining the proper functioning of biomolecules and a stable microenvironment for physiological processes by modulating the homeostasis of reactive oxygen species(ROS).Antioxidant enzymes in biocatalysis are used in the prevention or treatment of oxidative stress-related disease by counteracting the harmful effects of ROS.However,designing a system that can efficiently immobilize antioxidant enzymes with high catalytic activity and stability is still challenging.Bioinspired by photo-biocatalysis,a novel and effective catalase(CATase)-immobilized hydrogel platform has been developed by the proposed photo-enzymatic coupled radical polymerization strategy of the visible light coupling with the porphyrin-centered CATase.The higher catalytic stability and activity can therefore be achieved due to the preferential polymerization of CATase-immobilized hydrogel platform with a favorable three-dimensional network of enhanced coupling efficacy between light and enzymes.The mechanisms of free radical-initiated polymerization as well as the antioxidant cycle in the photo-CATase coupling system have been explored.Intriguingly,the CATase-immobilized hydrogel platform affords an unprecedented antioxidant ability to scavenge ROS and provide an effective cellular protection mechanism against external oxidative stress.Additionally,the CATase-immobilized hydrogel platform can effectively prevent peritoneal adhesion by reducing the expression of inflammatory cytokines.Therefore,the novel CATase-immobilized hydrogel platform is a potential candidate for physical barriers that effectively prevent postoperative adhesion formation,offering a new anti-adhesion strategy for clinical applications.

Dendrimer-induced synthesis of porous organosilica capsules for enzyme encapsulation

Organic matter-induced mineralization is a green and versatile method for synthesizing hybrid nanostructured materials,where the material properties are mainly influenced by the species of natural biomolecules,linear synthetic polymer,or small molecules,limiting their diversity.Herein,we adopted dendrimer poly(amidoamine)(PAMAM)as the inducer to synthesize organosilica-PAMAM network(OSPN)capsules for mannose isomerase(MIase)encapsulation based on a hard-templating method.The structure of OSPN capsules can be precisely regulated by adjusting the molecular weight and concentration of PAMAM,thereby demonstrating a substantial impact on the kinetic behavior of the MIase@OSPN system.The MIase@OSPN system was used for catalytic production of mannose from Dfructose.A mannose yield of 22.24% was obtained,which is higher than that of MIase in organosilica network capsules and similar to that of the free enzyme.The overall catalytic efficiency(kcat/Km)of the MIase@OSPN system for the substrate D-fructose was up to 0.556 s^(-1)·mmol^(-1)·L.Meanwhile,the MIase@OSPN system showed excellent stability and recyclability,maintaining more than 50% of the yield even after 12 cycles.

Dendritic mesoporous silica nanoparticles for enzyme immobilization

Dendritic mesoporous silica nanoparticles(DMSNs)are a new class of solid porous materials used for enzyme immobilization support due to their intrinsic characteristics,including their unique open central-radial structures with large pore channels and their excellent biocompatibility.In this review,we review the recent progress in research on enzyme immobilization using DMSNs with different structures,namely,flower-like DMSNs and tree-branch-like DMSNs.Three DMSN synthesis methods are briefly compared,and the distinct characteristics of the two DMSN types and their effects on the catalytic performance of immobilized enzymes are comprehensively discussed.Possible directions for future research on enzyme immobilization using DMSNs are also proposed.

Synthesis and Characterization of Amphiphific Block Copolymer Containing PVP and Poly（5-benzyloxytrimethylene carbonate）

Amphiphilic copolymer of 5-benzyloxytrimethylene carbonate （BTMC） with poly （vinyl pyrrolidone） （PVP） was successfully synthesized using immobilized porcine pancreas lipase （IPPL） or SnOct2 as catalyst. Hydroxyl terminated PVP, synthesized with 2-mercaptoethanol as a chain transfer reagent, was employed as a rnacroinitiator. The resulting copolymers were characterized by GPC, ^1H NMR and IR. Increasing the BTMC/PVP-OH feed ratio （[B]/[P]） resulted in the increase of Mn of corresponding copolymers and the decrease of Mw/Mn. Immobilized enzyme has comparable catalytic activity to SnOct2 for the copolymerization.

SYNTHESIS OF POLY(5,5-DIMETHYL-1,3-DIOXAN-2-ONE)BY LIPASE-CATALYZED RING-OPENING POLYMERIZATION

Porcine pancreas lipase (PPL) and PPL immobilized on narrow distributed micron-sized glass beads wereemployed successfully for the ring-opening polymerization of 5, 5-dimethyl-1, 3-dioxan-2-one (DTC) for the first time.Different polymerization conditions such as enzyme concentration and reaction temperature were studied. Immobilized PPLexhibits higher activity than native PPL. Along wth the increasing enzyme concentration, the molecular weigh of resultingPDTC decreases. PPL immobilized on narrow distributed micron-sized glass beads has outstanding recyclability. For thethird recycle time, immobilized PPL exhibits the highest catalytic activity and with high activity even after the fifth recyletime for the synthesis of PDTC. The ~1H-NMR spectra indicate that decarboxylation does not occur during the ring-openingpolymerization.

Research and Application Progress of Silk Fibroin Membranes

This paper mainly introduced the preparation of silk fibroin membranes and their structural change characteristics.Silk fibroin membranes can be used as tissue engineering materials,enzyme-immobilizing membranes,biosensors and drug controlled-release membranes and other different materials.They have excellent characteristics such as non-toxic,non-polluting and degradable,and thus have broad application prospects.

Visible light assisted enzyme-photocatalytic cascade degradation of organophosphorus pesticides

The worldwide application of organophosphorus pesticides(OPs)has promoted agricultural development,but their gradual accumulation in soil and water can seriously affect the central nervous system of humans and other mammals.Organophosphorus hydrolase(OPH)is an effective enzyme that can catalyze the degradation of the residual OPs.However,the degradation products such as p-nitrophenol(p-NP)is still toxic.Thus,it is of great significance to develop a multi-functional support that can be simultaneously used for the immobilization of OPH and the further degradation of p-NP.Herein,a visible light assisted enzyme-photocatalytic integrated catalyst was constructed by immobilizing OPH on hollow structured Au-TiO_(2)(named OPH@H-Au-TiO_(2))for the degradation of OPs.The obtained OPH@H-Au-TiO_(2)can degrade methyl parathion to p-NP by OPH and then degrade p-NP to hydroquinone with low toxicity by using H-Au-TiO_(2)under visible light.OPH molecules were immobilized on HAu-TiO_(2)through adsorption method to prepare OPH@H-Au-TiO_(2).After 2.5 h of reaction,methyl parathion is completely degraded,and about 82.64%of the generated p-NP is further degraded into hydroquinone.After reused for 4 times,the OPH@H-Au-TiO_(2)retains more than 80%of the initial degradation activity.This research presents a new insight in designing and constructing multi-functional biocatalyst,which greatly expands the application scenarios and industrial value of enzyme catalysis.

IMMOBILIZATION OF GLUCOSE OXIDASE AND CELLULASE BY CHITOSAN— POLYACRYLIC ACID COMPLEX

This study is concerned with chitosan-polyacrylic acid complex as a carrier to immobilize glucose oxidase (GOD)and cellulase. The optimum emperature of the immobilized GOD (IG) was determined to be 60℃ which is higher than that of the native GOD about 40℃. The optimum temperature of the immobilized cellulase (IC) was determined to be about 30℃ higher than that of native cellulase. Both of the optimum pH of IG and IC shifted one pH unit to acid. Immobilized enzyme may be used in more wide pH range. Their storage life are much longer compared with their native states. Both of them can be reused at least 12 times.

A New Application of Carbon Nanotubes Constructing Biosensor

Carbon nanotubes used for constructing biosensor was described for the first time. Single-wall carbon nanotubes (SWNTs) functionalized with carboxylic acid groups were used to immobilize glucose oxidase forming a glucose biosensor. The biosensor response can be determined by amperometric method at a low applied potential (0.40 V).

Immobilization of Penicillin G Acylase on Calcined Layered Double Hydroxides

A hydrotalcite-like Mg 2+ /Al 3+ layered double hydroxide(LDH) material was prepared by means of a modified coprecipitation method involving a rapid mixing step followed by a separate aging process. LDH calcined at 500 ℃, denoted as CLDH, was characterized by XRD, IR and BET surface area measurements. CLDH has a poor crystalline MgO-like structure with a high surface area and porosity. CLDH was used as a support for the immobilization of penicillin G acylase(PGA). The effect of varying the immobilization conditions, such as pH, contact time and the ratio of enzyme to support, on the activity of the immobilized enzyme in the hydrolysis of penicillin G has been studied. It was found that the activity of the immobilized enzyme decreased slightly with decreasing pH and reached a maximum after a contact time of 24 h. The activity of the immobilized enzyme increased with increasing the ratio of enzyme to support. It was found that the adsorption of PGA inhibited the expected reaction of CLDH with an aqueous medium to regenerate a LDH phase. Its original activity(36%) after 15 cycles of reuse of the immobilized enzyme was retained, but no further loss in the activity was observed.

Immobilization and Properties of Polyphenol Oxidase

[Objectives]This study was conducted to investigate the effects of embedding conditions on activity and catalytic properties of immobilized polyphenol oxidase.[Methods]Polyphenol oxidase was immobilized in a polymer material by the embedding method,and the optimal immobilization conditions were obtained by single factor tests:CaCl_(2) concentration 2.0%,sodium alginate concentration 2.0%,immobilization time 2 h and mass ratio of enzyme to carrier 10 mg/100 g,under which the immobilized enzyme activity was 93.33 U/g.Under the above conditions,the properties of polyphenol oxidase immobilized by sodium alginate(A-PPO)and free polyphenol oxidase were studied.[Results]The thermostability of A-PPO was better than that of the free enzyme,but the pH stability of A-PPO was inhibited.The Michaelis constant K_(m) values of free polyphenol oxidase and A-PPO were 0.37 and 0.48 mmol/L,respectively,and the maximum reaction rate V_(max) values were 0.38 and 0.51 mmol/(L·g),respectively.[Conclusions]This study provides a theoretical basis for the study of the properties of polyphenol oxidase.

Immobilization of carbonic anhydrase for facilitated CO2 capture and separation

Carbonic anhydrase(CA)as a typical metalloenzyme in biological system can accelerate the hydration/dehydration of carbon dioxide(CO2,the major components of greenhouse gases),which performer with high selectivity,environmental friendliness and superior efficiency.However,the free form of CA is quite expensive(~RMB 3000/100 mg),unstable,and non-reusable as the free form of CA is not easy for recovery from the reaction environment,which severely limits its large-scale industrial applications.The immobilization may solve these problems at the same time.In this context,many efforts have been devoted to improving the chemical and thermal stabilities of CA through immobilization strategy.Very recently,a wide range of available inorganic,organic and hybrid compounds have been explored as carrier materials for CA immobilization,which could not only improve the tolerance of CA in hazardous environments,but also improve the efficiency and recovery to reduce the cost of large-scale application of CA.Several excellent reviews about immobilization methods and application potential of CA have been published.By contrast,in our review,we stressed on the way to better retain the biocatalytic activity of immobilized CA system based on different carrier materials and to solve the problems facing in practical operations well.The concluding remarks are presented with a perspective on constructing efficient CO2 conversion systems through rational combining CA and advanced carrier materials.