For the immobilization of enzymes, thecovalent bonding is very common methodsto employ. The covalent bonded enzymesactivity depends upon the carrier materialsize and shape, composition, nature ofcoupling method and on the specificconditions during coupling. The veryimportant factor in the covalent bindingbetween the enzyme and support is thedirection of the enzyme binding and it alsofound its stability.
The active center aminoacids is not include in the binding withsupport in order to get the or attains thehighest enzyme activity level. Thecoupling with support is take place in twomanners, which is mainly depend upon theactive groups found in molecules which isRECENT RESEARCH PROGRESS IN ENZYME IMMOBILIZED MICROGELS 10to be immobilized. The matrices which aregenerally used in these methods are suchas: agrose, cellulose, porous glass and ionexchange resins. The powerful relation isdeveloped between the enzyme andsupport due to the covalent bonds and itdoes not allowed the enzyme release intothe reaction surrounding. The advantagesof this method include the increase in theenzyme thermal stability and half-life bycoupling with matrix like chitosan,mesoporous silica 52.Figure 4. Schematics of the three most common enzyme immobilization techniques: (A)physical adsorption, (B) entrapment and (C) covalent attachment/cross-linking4 PropertiesAs a result of enzyme immobilized onmicrogels, few characteristics of theenzyme molecule like stability change ascompared to its soluble counterpart. Thisalteration of properties might be inducingdue to variation in the immobilizedenzyme activity or due to the immobilizedenzyme interaction with microgels in themicroenvironment which is totallydistinctive.
Due to the immobilization ofenzymes on microgels the variation inmicroenvironment which is totallydistinctive. Due to the immobilization ofenzymes on microgels the variation incatalytic properties are also seen becauseof binding of enzyme to the matrix.distinctive. Due to the immobilization ofenzymes on microgels the variation inRECENT RESEARCH PROGRESS IN ENZYME IMMOBILIZED MICROGELS 11catalytic properties are also seen because of binding of enzyme to the matrix.Table 2Technological Properties of Immobilized Enzyme SystemsAdvantages DisadvantagesCatalyst reuse Loss or reduction in activityEasier reactor operation Diffusional limitationEasier product separation Additional costWider choice of reactorThe working stability of the enzymeenhanced due to the immobilization on themicrogels. The idea of stabilization is avery important for the immobilizing of theenzymes.
The process becomes diffusioncontrolled due to the loading of excessenzymes on microgels 53. The limitationof the utilization of immobilized enzyme isthat its catalytic activity becomes lost andparticularly when it’s immobilized onmacromolecular substrates. And due to thelost in activity the characteristic array ofproducts are also varied which is formeddue enzyme immobilized microgels.Table 3Major Products Obtained Using Immobilized EnzymesEnzyme ProductGlucose isomerase High-fructose corn syrupAmino acid acylase Amino acid productionPenicillin acylase Semi-synthetic penicillinsNitrile hydratase Acrylamide?-Galactosidase Hydrolyzed lactoseRECENT RESEARCH PROGRESS IN ENZYME IMMOBILIZED MICROGELS 12This problem is overcome by carefulchoice of the enzymes residue involved inimmobilization and utilization of thehydrophilic and unreactive spacer arms.The temperature and time are the factorson which immobilized enzymes stabilityrely or depend. Microgels also effected bycross linking. When the cross linkingreduced, the maximum current alsodecreases which shows that the effusion ofenzymes from microgels take place andthis is due to the microgels large poroussize. So the microgels which have 4%cross linking are best and also chosen asenzyme immobilized system.
In order tocheck the behavior of enzyme immobilizedon microgels the effect of enzyme loadingwas seen by two different perspective, thefirst is varying the immobilized enzymeamount into the microgel and secondlychanging the microparticles amount.5 CharacterizationsFor the characterization of enzymeimmobilized on microgels there aredifferent types of techniques availiable54. The techniques are such as lightscattering, differential scanningcalorimetry, uv-visible spectrophotometry,FTIR, electron microscopy, XRD, atomicforce microscopy.
In characterization therelevant characteristics involves themolecular weight, particle size and amountof charge as function of temperature, pHand ionic strength. The technique which ismostly used is dynamic light scattering55. The advantages of this techniqueinclude accuracy, quickness andreproducibility. Due to the immobilizedenzyme on microgel there should becharge created which is measured anddetermine as a function of pH 56.5.
1 FTIR spectroscopyThe functional groups that are present inthe microgels are determined by FTIRspectroscopy. This technique also verifiesthe fruitful interaction between theenzymes and microgels as after theimmobilized into the matrix of microgelsand spectra recorded. FTIR spectra arevery efficient tool for the determination ofRECENT RESEARCH PROGRESS IN ENZYME IMMOBILIZED MICROGELS 13microgels molecular structure and thepower of interaction between the enzymesand microgels because the bond frequencyis greatly influenced by the interaction57.
5.2 UV-Visible spectroscopyThe microgels absorb the light in thevisible region due to the development ofresonance oscillation at the junction ofpolymer matrix and enzymes. This takesplace due to the process of surfaceplasmon resonance (SPR). And thisphenomenon is greatly rely on the pH andtemperature.
If the hybrid microgels areused then this property is used todetermine the loading of enzyme into thematrix and also very crucial in the study ofmicrogels volume phase transition. Thisproperty greatly relies on the distancebetween the enzyme and polymer withinthe matrix and which basically depend onpH and temperature 58.5.3 X-ray diffraction analysisThe purity, characterization and presenceof enzymes are confirmed and structure iscertifying by XRD analysis as the everyenzyme constituent gives particular signalintensity.
So the size of particle isdetermined by debye-scherrer formula59. The degree of cross linking and %age are find with the technique which isvery advanced and called as energydispersive XRD.5.4 Electron microscopyThe study of internal surface of microgelsmade possible by the use of highresolution techniques which are calledscanning electron microscopy andtransmission electron microscopy.
Thecharacteristics of high importance arestudied such as shape, size, poly dispersityand appearance. These properties alsodepend upon the conditions of preparationof microgels. And the transmissionelectron microscopy used in the study ofbehavior of swelling and deswelling ofmicrogels 60.5.5 Atom force microscopyThe peculiarity of this wonderfultechnique is to provide the threeRECENT RESEARCH PROGRESS IN ENZYME IMMOBILIZED MICROGELS 14dimensional structure with greaterresolution, cross sectional analysis andalso provides pictures of microgels. Thevolume phase transitions of microgels arealso studied by these techniques.5.
6 Dynamic light scatteringIn the case of hybrid microgels the veryimportant characteristics is thehydrodynamic study which is rely on thepH and temperature. So by the swellingand de-swelling the variation in volume isalso determined by scattering method. Thistechnique is also helpful in determiningthe size hydrodynamic diameter and sizedistribution. This technique is basicallyused for the photon depend upon the anglecorrelate spectroscopy. The hydrodynamicworking of microgels is also determinedby this technique 61.
At angle which isfixed scattering angle of 60 all the thermaldependent measurements occur.6 ApplicationsThe enzyme immobilized in microgels hasmany applications in different fields suchas drug delivery, chemical separation,biosensing, optics and catalysis. Thethermoresponsive microgels are also usedin the biotechnology. Over the last tenyears the microgels rapidly attainedstrength or power as intelligent materialsbecause of their responsive behavior.Many new applications of microgels haveappeared. In the preparation of inorganicnanoparticles they are used asmicroreactors.
Also used as photoniccrystals and mostly used in drug deliverysystems.Due to their simplest synthesis microgelsare used as microreactors for thepreparation of nanoparticles. Theimportant characteristics of microgel arethe relatively greater size comparable tothe visible light wavelength.
The enzymeimmobilized microgels have potentialapplication in the photonic crystals. Theimmobilized enzymes in microgels areused in medicine. It is used for thediagnosis and treatment in the medicalfield. The inborn metabolic loss can becontrolled by the replacement ofRECENT RESEARCH PROGRESS IN ENZYME IMMOBILIZED MICROGELS 15encapsulated enzymes. For the externalenzyme drugs the RBC works as a carrierand there is no immune response due tothe biocompatible nature of enzyme.
Theeasier way of the enzyme immobilizationis the electroporation and this process isreversible from which enzymes canrecreated. The enzymes gives biologicaland functional systems when attached withthe biomaterials. In tissue engineeringapplications the biomaterials are used torepair the defect. The benefit of theenzyme immobilization is that the freeenzymes are not available for use andhence the stability of immobilizedenzymes remains same. The purifiedenzymes are used in food industries but thedenaturing of enzyme occur due to thepurification of enzyme. And enzymesbecome stable due to the immobilizedtechniques.In the production of syrups theimmobilized enzymes are used. Theenzyme attached with matrix by covalentbonding.
But this method is costly that’swhy enzyme was attached by crosslinking. And for the different purposessuch as confectionaries and ice-creams thismethod is used. Due to the mixing of freshwater with polluted water the treatment ofthis problem are of very importance atpresent. To overcome this problemimmobilized enzymes are used.
The dyesfrom the different industries are mixedwith water but immobilized enzyme hasthe power to degrade the dyes. Because ofeasy regeneration the adsorption methodare greatly used. The lipase enzyme hasthe capacity to hydrolyze the oil and fats.
The enzymes are used for different textileapplications such as desizing, denimfinishing, treating wool and bio polishing.Instead of harsh chemicals the industriesused enzymes now days. The fabricationwith enzyme may require higher pH andtemperature. For the removal of stains thedetergent industries also used enzymes.
The protease is used in detergent industryto remove the stains of egg, blood andhuman sweat. The oil and fats stains areRECENT RESEARCH PROGRESS IN ENZYME IMMOBILIZED MICROGELS 16removed by the used of lipase. Forimprove softening the cellulose are used.
The minute quantity of enzyme baseddetergents is used in comparison withsynthetic detergent 62.ConclusionsWe studied the recent research progress ofimmobilization of enzyme in microgels.The immobilization of enzyme is allowedby the mild conditions during theformation of microgel without the dangerof denaturing of enzyme. In microgels theenzyme amount is controlled by thechanging of the concentration of enzymein aqueous droplets. Functionalization ofmicrogels with enzyme cause greatermicrogel particles swelling and due tohydrophilic character of the enzyme thevolume phase transition temperature shiftsto the higher temperature.
The recentstudies explained that the enzyme loadingand activity can be enhanced by enhancingthe enzyme content in the microgels, andthis is describes by the electrostaticinteractions which are very strong betweenthe enzyme and the polymer network