Recent advances in immobilized enzymes on nanocarriers

Recent progress in nanotechnology has provided high-performance nanomaterials for enzyme immobilization.Nanobiocatalysts combining enzymes and nanocarriers are drawing increasing attention because of their high catalytic performance,enhanced stabilities,improved enzyme-substrate affinities,and reusabilities.Many studies have been performed to investigate the efficient use of cellulose nanocrystals,polydopamine-based nanomaterials,and synthetic polymer nanogels for enzyme immobilization.Various nanobiocatalysts are highlighted in this review,with the emphasis on the design,preparation,properties,and potential applications of nanoscale enzyme carriers and nanobiocatalysts.

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.

RAPID DETERMINATION OF L-GLUTAMIC ACID WITH AN ENZYME REACTOR OF L-GLUTAMIC DECARBOXYLASE IMMOBILIZED ON ION EXCHANGE RESIN

The preparation and characterization of an immobilized L-glutamic decarboxylase (GDC) were studied. This work is to develop a sensitive method for the determination of L-glutamate using a new biosensor, which consists of an enzyme column reactor of GDC immobilized on a novel ion exchange resin (carboxymethyl-copolymer of allyl dextran and N.N?methylene-bisacrylamide CM-CADB) and ion analyzer coupled with a CO2 electrode. The conditions for the enzyme immobilization were optimized by the parameters: buffer composition and concentration, adsorption equilibration time, amount of enzyme, temperature, ionic strength and pH. The properties of the immobilized enzyme on CM-CADB were studied by investigating the initial rate of the enzyme reaction, the effect of various parameters on the immobilized GDC activity and its stability. An immobilized GDC enzyme column reactor matched with a flow injection system-ion analyzer coupled with CO2 electrode-data collection system made up the original form of the apparatus of biosensor for determining of L-glutamate acid. The limit of detection is 1.0×10-5 M. The linearity response is in the range of 5×10 -2-5×10 -5 M . The equation of linear regression of the calibration curve is y= 43.3x + 181.6 (y is the milli-volt of electrical potential response, x is the logarithm of the concentration of the substrate of L-glutamate acid). The correlation coefficient equals 0.99. The coefficient of variation equals 2.7%.

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.

Mathematical Simulation of Blood Purification for Leukemia by Immobilized L-asparaginase

Plasma was purified in an immobilized L-asparaginase column. The predicted results are in good agreement with experimental data. It is indicated that the mathematical model is suitable for the mass transfer and reaction of blood purification.

Activity and stability of bacterial cellulase immobilized on magnetic nanoparticles

Magnetic nanoparticles（Fe3O4） were synthesized by co-precipitating Fe^2＋ and Fe^3＋ ions in an ammonia solution and treating under hydrothermal conditions.Cellulase was immobilized onto Fe3O4 magnetic nanoparticles via glutaraldehyde activation.Using response surface methodology and Box-Behnken design,the variables such as magnetic nanoparticle concentration,glutaraldehyde concentration,enzyme concentration,and cross linking time were optimized.The Box-Behnken design analysis showed a reasonable adjustment of the quadratic model with the experimental data.Statistical contour plots were generated to evaluate the changes in the response surface and to understand the relationship between the nanoparticles and the enzyme activity.Scanning electron microscopy,X-ray diffraction analysis,and Fourier transform infrared spectroscopy were studied to characterize size,structure,morphology,and binding of enzyme onto the nanoparticles.The stability and activity of the bound cellulase was analyzed using various parameters including pH,temperature,reusability,and storage stability.The immobilized cellulase was compared with free cellulase and it shows enhanced stability and activity.

Fiber optic biosensor of immobilized firefly luciferase

Luciferase from firefly lantern extract was immobilized on CNBr activated Sepharose 4B. The kinetic properties of immobilized luciferase were extensively studied. The K m′ for D luciferin is 11.9 μmol/L, the optimum pH and temperature for Sepharose bound enzyme were 7.8 and 25℃ respectively. A luminescence fiber optic biosensor, making use of immobilized crude luciferase, was developed for assay of ATP. The peak light intensity was linear with respect to ATP concentration in range of 10 -9 -10 -5 mol/L. A biological application was also demonstrated with the determination of serum ATP from rats bred in low versus normal oxygen environments.

Characterization of cellulose acetate micropore membrane immobilized acylase Ⅰ

This paper describes an innovative method for the immobilization of acylase I, which was entrapped into the CA-CTA micropore membrane. The most suitable casting solutions proportion for immobilizing the enzyme was obtained through orthogonal experiment. Properties of the enzyme membrane were investigated and compared with those of free enzyme and blank membrane. The thermal stability and pH stability of the enzyme inside the membrane were changed by immobilization. The optimum pH was found to be 6.0, which changes 1.0 unit compared with that of free acylase I. The optimum temperature was found to be about 90℃, which is higher than that of free acylase I (60℃). Experimental results showed that immobilization had effects on the kinetic parameters of acylase I.

STUDY ON IMMOBILIZED PORCINE PANCREATIC LIPASE CATALYZING TRANSESTERIFICATION BETWEEN METHYL－BUTYRATE AND 1－BUTANOL IN NONAQUEOUS SYSTEMS

Transesterification between methyl-butyrate and 1-butanol in nonaqueous systems was catalyzed by porcine pancreatic lipase which was immobilized on cross- linked polystyrene. Organic solvents, substrate concentration, contents of water and other parameters which affect the immobilized enzyme activity were studied. Lipase immobilized on hydrophobic crosslinked polystyrene can reduce its diffusion limit in the reaction. It was found that the activity of immobilized lipase in organic systems was two times as high as that of free lipase.

Covalent Assembly of Penicillin Acylase in Mesoporous Silica Based on Macromolecular Crowding Theory

To improve the covalent immobilization of penicillin acylase （PA）, macromolecular crowding theory was applied to its immobilization. Influence of mass ratio of enzyme to the silica, as well as, activation time with glutaraldehyde on the activity of assembled PA, was studied. In the mesopores, the effect of fl-cyclodextrin （β-CD） on the immobilization of the enzyme was also investigated. It was remarkable that the coupled yield and relative activity reached 99.5% and 92.3%, respectively, when penicillin acylase assembled covalently in the mesopores. The results here indicate that mimicked macromolecule crowding could significantly ameliorate the performance of covalently immobilized PA.

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.

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.

Immobilization of Penicillin G Acylase on Magnetic Nanoparticles Modified by Ionic Liquids

Functionalized ionic liquids containing ethyoxyl groups were synthesized and immobilized on magnetic silica nanoparticles （MSNP） prepared by two steps, i.e., Fe304 synthesis and silica shell growth on the surface. This magnetic nanoparticle supported ionic liquid （MNP-IL） were applied in the immobilization of penicillin G acylase （PGA）. The MSNPs and MNP-ILs were characterized by themeans of Fourier transform infrared spectroscopy （FTIR）, scanning electron microscopy （SEM）, transmission electron microscopy （TEM）, and vibrating sample magnetometer （VSM）. The results showed that the average size of magnetic Fe304 nanoparticles and MSNPs were -10 and -90 nm, respectively. The saturation magnetizations of magnetic Fe304 nanoparticles and MNP-ILs were 63.7 and 26.9 A＇m2·kg^-1, respectively. The MNP-IL was successfully applied in the immobilization of PGA. The maximum amount of loaded enzyme-was about 209 mg·g^-1 （based on carder）, and the highest enzyme activity of immobilized PGA （based on ImPGA） was 261 U·g^-1. Both the amount of loaded enzyme and the activity of ImPGA are at the same leyel of or higher than that in previous reports. After 10 consecutive operat!ons, ImPGA still mainrained 62% of its initial activity, indicating the＇good recovery property of ImPGA activity. The ionic liquid modified magnetic particles integrate the magnetic properties of Fe304 and the structure-tunable properties of ionic liquids, and have extensive potential uses in protein immobilization and magnetic bioseparation. This work may open up a novel strategy to immobilize proteins by ionic liquids.

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.

Preparation and evaluation of enzyme encapsulated hydrogels(single gels and double network gels) and enzyme immobilized magnetic beads

In the present research,enzyme encapsulated hydrogels（single gels and double network gels）and enzyme immobilized magnetic beads,which allow high-throughput screening,were fabricated and evaluated in terms of the preservation,precision, and repeatability of enzyme activity.The fabricated gels and magnetic beads were analyzed in a 96-well microassay plate.Trypsin was successfully encapsulated in both types of gels and immobilized to the magnetic beads.However,pepsin,either encapsulated in the gels or immobilized to the magnetic beads,could not react with its substrates.The adaptability to various enzymes （e.g.,trypsin,β-glucuronidase,and CYP1A1）in the single gels and magnetic beads was superior to that in double network gels.However,the soak out of the enzymes was observed in the single gels.The double network gels could encapsulate trypsin,whereas the fabrication of the other enzymes（e.g.β-glucuronidase,CYP1A1,and pepsin）failed because of the inactivation of the enzymes by acryl amide and ammonium peroxodisulfate,which are the components of the gel formulation. The enzyme reaction in the magnetic beads exhibited the highest efficiency among the three fabrication methods.Furthermore, the stability of the enzymes immobilized to the magnetic beads was better than that fabricated by the other methods,and the activities of trypsin andβ-glucuronidase did not decline for up to one week.In addition,in the magnetic beads,the activities of trypsin andβ-glucuronidase can be well repeated.Hence,although the adaptability of the double network gels to various enzymes is currently limited,the efficiency of the enzyme encapsulation can be improved by optimizing the formulation of acryl amide gels.