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.

Integration of enzyme immobilization and biomimetic catalysis in hierarchically porous metal-organic frameworks for multi-enzymatic cascade reactions

Multiple enzymes-induced biological cascade catalysis is indispensable in biotechnology and industrial processes. Nevertheless,the drawbacks of most natural enzymes, including poor stability and recyclability and sensitivity to the environment, have hindered their broader application. Here, we report a facile strategy to prepare a biomimetic cascade reaction system by combining the advantages of enzyme immobilization and biomimetic catalysis in a one-pot reaction system based on the hierarchically porous metal-organic frameworks(HP-MOFs). The hierarchically porous zirconium-porphyrin-based MOF(HPPCN-222(Fe)) synthesized by modulator-induced strategy possessed tunable hierarchical porous and peroxidase-like activity,permitting them to act as not only an efficient immobilization matrix for glucose oxidase(GOx) but also peroxidase mimics to catalyze the cascade for glucose detection. A stable, anti-interference and reusable colorimetric biosensor for glucose detection was successfully established through GOx@HP-PCN-222(Fe) on the basis of the artificial tandem catalysis. Moreover, the GOx@HP-PCN-222(Fe)-fabricated electrode was available for glucose detection by electrochemical method. This work provides a potentially universal method to design functional multi-enzymatic cascade reaction systems by integrating the merits of enzyme encapsulation and biomimetic catalysis in HP-MOFs.

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.

POROUS SILICA NANO-TUBE AS HOST FOR ENZYME IMMOBILIZATION

With their hollow morphology and large openings, the as-synthesized porous silica nano-tubes (NTPS), prepared through a sol-gel routine by using nano-sized needle-shaped CaCO3 particles as templates, were used as host for enzyme immobilization. Bioimmobilization study showed that enzyme molecules could not only be adsorbed on the external surface of NTPS but also entrapped in their inner hollow cores, leading to higher enzyme loading capacities of NTPS (more than 350 mg/g silica) in a shorter time, as compared to common porous silica (less than 50 mg/g) and most conventional mesoporous silica materials (less than 100 mg/g).

Tyrosinase-modified carbon felt-based flow-biosensors:The role of ultra-sonication in shortening the enzyme immobilization time and improving the sensitivity for p-chlorophenol

Tyrosinase（TYR） was covalently immobilized onto amino-functionalized carbon felt surface via glutaraldehyde-coupling under ultrasonic treatment for 10 min.The resulting TYR-immobilized carbon felt was used as a working electrode unit of bioelectrocatalytic flow-through detector for TYR substrates（catechol,p-chlorophenol（p-CP）,p-cresol,phenol etc.）.Cathodic peak currents based on the electroreduction of enzymatically produced o-quinones were detected at-50 mV vs.Ag/AgCl.Compared with previous work in which TYR was immobilized onto amino-functionalized carbon felt for 16 hr without the ultrasonic treatment,we succeeded in（1） shortening the enzyme immobilization time from 16 hr to 10 min,（2） enhancing the sensitivity of p-CP,and（3） improving the operational stability of p-CP.The ultrasonic treatment during the TYR immobilization step would lead to certain changes in the structure of the immobilized TYR and the morphology of the immobilized TYR-layer on the carbon felt surface.

Enzyme immobilization on a pH-responsive porous polymer membrane for enzymatic kinetics study

Immobilization of enzymes onto carriers is a rapidly growing research area aimed at increasing the stability,reusability and enzymolysis efficiency of free enzymes.In this work,the role of phaseseparation and a pH-responsive"hairy"brush,which greatly affected the topography of porous polymer membrane enzyme reactors(PMER),was explored.The porous polymer membrane was fabricated by phase-separation of poly(styrene-co-maleic anhydride-acrylic acid)and poly(styrene-ethylene glycol).Notably,the topography and pores size of the PMER could be controlled by phase-separation and a pHresponsive"hairy"brush.For evaluating the enzymolysis efficiency of D-amino acid oxidase(DAAO)immobilized carrier(DAAO@PMER),a chiral ligand exchange capillary electrophoresis method was developed with D-methionine as the substrate.The DAAO@PMER showed good reusability and stability after five continuous runs.Notably,comparing with free DAAO in solution,the DAAO@PMER exhibited a17.7-folds increase in catalytic velocity,which was attributed to its tailorable topography and pHresponsive property.The poly(acrylic acid)moiety of poly(styrene-co-maleic anhydride-acrylic acid)as the pH-responsive"hairy"brush generated topography changing domains upon adjusting the buffer pH,which enable the enzymolysis efficiency of DAAO@PMER to be tuned based upon the well-defined architectures of the PMER.This approach demonstrated that the topographical changes formed by phaseseparation and the pH-responsive"hairy"brush indeed made the proposed porous polymer membrane as suitable supports for enzyme immobilization and fitting for enzymolysis applications,achieving high catalytic performance.

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.

Fabricating biopolymer-inorganic hybrid microspheres for enzyme immobilization:Connect membrane emulsification with biomimetic mineralization

The organic-inorganic hybrid composites displayed great potential for biotechnological and biomedical application.In this research,a gelatin/alginate/silica hybrid microsphere was developed by a synergy of membrane emulsification process and biomimetic mineralization method.The gelatin was mixed and complexed with alginate solutions(water phase).The water phase was extruded through a Shirasu Porous Glass(SPG)membrane,and then was crosslinked,which formed gelatin/alginate microspheres.The biomimetic mineralization was occurred in situ by immersing gelatin/alginate complex in a Na_(2)SiO_(3) solution,while silica was formed around the organic microspheres,resulting in the final gelatin/alginate/silica hybrid microspheres.These microspheres were characterized by SEM,TEM,EDS,TGA/DTA,and DSC.The hybrid microcapsules present a more than 40%mass fraction of the inorganic component,and displayed superior swelling resistance to biopolymer complex microspheres.Glycerol dehydrogenase(GlyDH)was immobilized in the obtained novel gelatin/alginate-silica hybrid microspheres as the model enzyme.Due to the protective effect of carriers,the pH tolerance stability,storage and recycling stability of the immobilized GlyDH were all improved in comparison with free GlyDH.

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 and Properties of Lipase from Candida rugosa on Electrospun Nanofibrous Membranes with Biomimetic Phospholipid Moities

Reported here is a protocol to fabricate a biocatalyst with high enzyme loading and activity retention, from the conjugation of electrospun nanofibrous membrane having biomimetic phospholipid moiety and lipase. To improve the catalytic efficiency and activity of the immobilized enzyme, poly（acrylonitrile-co-2-methacryloyloxyethyl phosphorylcholine）s（PANCMPCs） were, respectively, electrospun into nanofibrous membranes with a mean diameter of 90 nm, as a support for enzyme immobilization. Lipase from Candida rugosa was immobilized on these nanofibrous membranes by adsorption. Properties of immobilized lipase on PANCMPC nanofibrous membranes were compared with those of the lipase immobilized on the polyacrylonitrile（PAN） nanofibrous and sheet membranes, respectively. Effective enzyme loading on the nanofibrous membranes was achieved up to 22.0 mg/g, which was over 10 times that on the sheet membrane. The activity retention of immobilized lipase increased from 56.4% to 76.8% with an increase in phospholipid moiety from 0 to 9.6%（molar fraction） in the nanofibrous membrane. Kinetic parameter Km was also determined for free and immobilized lipase. The Km value of the immobilized lipase on the nanofibrous membrane was obviously lower than that on the sheet membrane. The optimum pH was 7.7 for free lipase, but shifted to 8.3-8.5 for immobilized lipases. The optimum temperature was determined to be 35 ℃ for the free enzyme, but 42-44℃ for the immobilized ones, respectively. In addition, the thermal stability, reusability, and storage stability of the immobilized lipase were obviously improved compared to the free one.

Potentiometric Urea Biosensor Based on Ion-Selective Electrode by Different Immobilization Procedures

Enzyme was immobilized on an ammonium ion-selective electrode by different methods.An ion-selective electrode is not completely ion-specific,and interfering ions react with the ion-selective electrode membrane,altering the measured potential.Therefore,the characteristics of the effect of other ions on ammonium ion-selective electrode-based urea biosensors are considered.Based on the experimental results,the urea biosensor based on entrapment had a high response voltage of around 189 mV and fast response time of around 16 sec.Moreover,selectivity of the urea biosensor in different interfering ions was considered to elucidate the characteristics of ammonium ion-selective electrode-based biosensors.

Immobilization of laccase from Myceliophthora thermophila on functionalized silica nanoparticles: Optimization and application in lindane degradation

This work is focused on immobilization of laccase from Myceliophthora thermophila expressed in Aspergillus oryzae(Novozym 51003?laccase)on amino modified fumed nano-silica(AFNS)and the possible use in bioremediation.Hereby,for the first time,factors affecting the immobilization of Novozym 51003?laccase on AFNS were investigated for defining the immobilization mechanism and optimizing the utilization of AFNS as support for laccase immobilization.The highest specific activity(13.1 IU·mg-1 proteins)was achieved at offered 160 mg per g of AFNS and for the same offered protein concentration the highest activity immobilization yield,reaching68.3%after the equilibrium time,at optimum pH 5.0,was obtained.Laccase immobilization occurs by adsorption as monolayer enzyme binding in 40 min,following pseudo-first-order kinetics.The possible use of obtained immobilized preparation was investigated in degradation of pesticide lindane.Within 24 h,lindane concentration was reduced to 56.8%of initial concentration and after seven repeated reuses it retained 70%of the original activity.

Covalent Immobilization of Lipase on Poly(acrylonitrile-co-maleic acid) Ultrafiltration Hollow Fiber Membrane

Lipase from Candida rugosa was covalently immobilized on the surface of an uhrafihration hollow fiber membrane fabricated from poly （ acrylonitrile-co-maleic acid） （ PANCMA ） in which the carboxyl groups were activated with 1-ethyl-3-（ dimethylaminopropyl ） carbodiimide hydrochloride （ EDC ） and dicyclohexyl carbodiimide （ DCC ）/ N-hydroxyl succinimide（NHS）, respectively. The properties of the immobilized lipase were assayed and compared with those of the free enzyme. The maximum activities were observed in a relatively broader pH value range at high temperatures for the immobilized lipase compared to the free one. It was also found that the thermal and pH stabilities of lipase were improved upon immobilization and at 50 ℃ the thermal inactivation rate constant values are 2. 1 × 10^ -2 for the free lipase, 3.2 × 10^-3 for the immobilized lipase on the EDC-activated PANCMA membrane and 3.5 × 10^-3 for the immobilized lipase on the DCC/NHS-activated PANCMA membrane, respectively.

Partial purification,immobilization and preliminary biochemical characterization of lipases from Rhizomucor pusillus

Lipases have important applications in biotechnological processes, motivating us to produce, purify, immobilize and perform a biochemical characterization of the lipase from Rhizomucor pusillus. The fungus was cultivated by solid state fermentation producing lipolytic activity of about 0.5 U/mL(4U/g). A partial purification by gel filtration chromatography in Se-phacryl S-100 allowed obtaining a yield of about 85% and a purification factor of 5.7. Our results revealed that the purified enzyme is very stable with some significant differences in its properties when compared to crude extract. The crude enzyme extract has an optimum pH and temperature of 7.5 ° C and 40 ° C, respectively. After purification, a shift of the optimum pH from 7 to 8 was observed, as well as a rise in optimumtemperature to 60 ° C and an increase in stability. The enzyme was immobilized on CNBr-Agarose and Octyl-Agarose supports, having the highest immobilization yield of 94% in the second resin. The major advantage of immobilization in hydrophobic media such as Octyl is in its hyper activation, which in this case was over 200%, a very interesting finding. Another advantage of this type of immobilization is the possibility of using the derivatives in biotechnological applications, such as in oil enriched with omega-3 as the results obtained in this study display the hydrolysis of 40% EPA and 7% DHA from sardine oil, promising results compared to the literature.

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.

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.

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.

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.

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.