Abstract The recent research progress inthe enzyme immobilized microgelsshowed that for the immobilization ofenzyme the microgels are used as carriermatrix. The immobilization of enzyme inmicrogels is of great importance becauseenzyme can used in the biotechnologicalapplications.
In the recent research thedifferent enzymes were immobilized inmicrogels. Because the microgels areprepared in the aqueous media and due tolow reaction temperature, the enzymes areimmobilized without the danger ofdenaturing. And denaturing of enzyme canbe controlled by changing the reactionparameters. The enzyme containingmicrogels were investigated in detailregarding their activity, particle size,swelling properties and zeta potential.
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Therecent studies showed that the microgelsstructure allows the increasing enzymeloading without reducing the enzymeactivity.1 IntroductionThe term microgels are consists of twowords and both these words have totallydifferent meanings. The micro word isassociated with the gel particle size. Theword gel of microgel describes the swollenpower of particles in suitable organicsolvent 1. So microgels contain verylarge molecular weight polymericorganization. Every particle of gel wasexisting as a particular polymer fragment.It is also define as colloidal scattering ofgel molecules.
The particle size ofmicrogels should be lie from 10-1000nm2. The microgels play an important rolein the area of drug delivery 3. Themicrogels also gains popularity in the fieldFig 1 pictorial representation of microgelRECENT RESEARCH PROGRESS IN ENZYME IMMOBILIZED MICROGELS 2of nanotechnology and it enhanced day byday 4. The greater change in their size isobserved by varying the ionic strength orby applying the electric and magneticfield, by temperature and pH 5.
Theparticles of microgels are rounded andTable 1Supportscontain a water swollen polymericorganization 6. The amount of water formicrogels are in the range from 10 to 90%which rely on the microgel diffusion che-Fig 2 A microgel colloidal dispersionmistry. The microgels have momentousbenefit over the macrogels due to theircolloidal quality. Microgels are alsoknown as the environmentally responsiveOrganicNatural polymers• Polysaccharides: cellulose, dextrans, agar, agarose, chitin, alginate• Proteins: collagen, albumin• CarbonSynthetic polymers• Polystyrene• Other polymers: polyacrylate polymethacrylates, polyacrylamide, polyamides,vinyl, and allyl-polymersRECENT RESEARCH PROGRESS IN ENZYME IMMOBILIZED MICROGELS 3systems 7. The microgels are grouped into two ways.
Firstly the grouping consists on thephysical and chemical nature of cross-linkswhich are the reason of their networkstructure and definite size. Secondly theygrouped due to their individual responsiveproperties which are depending upon thetypes of functional groups that are presentin the molecule and polymer configuration8-9. The properties of microgels alsoincludes that they are cripple and flexible10. Moreover the physically cross linkedmicrogels may lose their colloidal stabilitydue to the different factors such aspolymeric composition, temperature anddensities of cross linking. The idealutilization of physically cross linkedpolymers are in ecological systems 11.
The enzymes as biological catalysts havethe capability to enhance the rate ofchemical reactions 12. As a catalyst,enzymes are very active and workingunder mild conditions and due toselectivity and specificity, execute veryexplicit reactions 13. The production ofenzymes on board range is very expensiveand enzymes are not very stable. So if theenzyme is soluble then it adds theimpurities in the product of the reactionand it is very difficult to remove thesetypes of impurities from the reactionproduct. But this type of error or fault canbe removed by generating the insolubleenzymes in the reaction medium 14. Theimmobilized enzymes are used in thelarger ranges of pH values, organicsolvents and temperature 15.
Overallevery factor helps in attaining the greaterproduction during different processes. Forimmobilization purposes the efficientmatrices are those which is prepared fromthe hydrophilic materials. The supportswhich are used should be inert to restrictthe enzyme-support interactions orreactions. The supports which are used inimmobilized process should be organic orinorganic or may be both 16. Thepurpose of enzyme immobilization atupgraded their solubility, strength andRECENT RESEARCH PROGRESS IN ENZYME IMMOBILIZED MICROGELS 4recyclability by enhance their catalyticproperties 17. These points are verymuch of interest for the utilization ofenzymes in different technicalphenomenon. There are two types ofimmobilization as follows: the first isimmobilization by pairing and second isimmobilization by wrapping (enzyme issealed in carrier matrix).
The adsorbedenzymes are simply detached from thecarrier matrix due to the phenomenon ofdesorption which is activate due to thevariations of pH, temperature and ionicstrength. The desorption complication canbe control by the covalent connections ofenzymes but due to this the enzymeactivity is greatly decreased. The phraseimmobilized enzymes assign to “enzymesare physically restricted in a specific areawith holding their catalytic movementswhich can be utilized frequently andconstantly 18. The enzyme, matrix andapproach of attachment are the mostimportant part of immobilized enzymesystems. The enzymes are organiccatalysts and encourage the shifting ofchemical species in the body.2 Classification of microgels2.
1 Physically cross-linkedIn physically cross linked microgels, thenetwork formation is developing due to thevan der waal forces of attraction 19.These are also very helpful for the drugencapsulation which is discharge ondisintegration of polymer layer network.The factors on which the physically crosslinked microgels are depends are polymercomposition, ionic strength of the mediumand on temperature. The physically crosslinked microgels are prepared from thebiopolymers such as dextron, agarose andalginate 20.
This is also very importanttype of microgels used in many fields.2.2 Chemically cross linkedThey are covalent in nature and thisproperty enhance their stability than thephysically cross linked counterparts. Themonomers are copolymerizing for thepreparation of chemically cross linkedRECENT RESEARCH PROGRESS IN ENZYME IMMOBILIZED MICROGELS 5microgels in the presence ofmultifunctional cross linking agent 21.
2.3 Thermoresponsive microgelsWhen the temperature is varying slightlyand microgel respond to this slightvariation in temperature is calledthermoresponsive microgels 22. Thegreat change in the volume is alsodisplayed by thermoresponsive microgelsat volume phase transition temperature. Ifthe temperature is below the lower criticalsolution temperature (LCST) the microgelsparticles converted into hydrophilic andbecomes swell, while at temperaturegreater than the (LCST) the microgelparticles are occur in reduced form.
Because of these characteristics microgelsbecomes more useful for their use in drugdiagnosis and in the field of controlleddrug release 23.2.4 pH responsive microgelsWhen the very small pH is changed andthose microgels which respond to thisminute change in pH are called pHresponsive microgels. Some pH responsivemicrogels are poly (acrylic acid)/poly(vinyl amine) (PAAc/PVAm) 24.2.5 Salt responsive microgelsThe microgels which have the capability togive response against the concentration ofthe salt, is called salt responsive microgels.
The hybrid microgel radii greatly relyupon the type and concentration of theelectrolyte. The microgels zeta potentialradius and colloidal stabilization are alsodisturbed by the electrolyte. If thedispersion medium is present than it wasseen that with higher the ionic strength ofelectrolyte the hydrodynamic radiusbecomes lessen 25.
2.6 Multiresponsive microgelsAt the same time period if the microgelgives response two or more than twostimuli, is called multiresponsivemicrogels. These microgels give respondto temperature and pH at the same time.Some examples of multiresponsivemicrogels, poly (N-isopropylacrylamideco-acrylic acid). The microgel whichrespond to external temperature, pH,RECENT RESEARCH PROGRESS IN ENZYME IMMOBILIZED MICROGELS 6organic solvent and salt concentration arepoly–N-morpholino) ethyl methacrylate26.2.7 Electric signal sensitivemicrogelsWhen the electric field is present somemicrogels showed change in their volumeis called electric signal sensitive microgels27. The examples of these gels arechitosan based gels.
2.8 Light sensitive microgelsIn some gels volume change is occurringdue to exposure of light is called lightsensitive microgels. These are greatlysensitive towards light. In cartilage tissuesengineering these types of gels are used.The classification of microgels is basedupon the type of organic and inorganicmaterial used and type of polymerization.
Some characteristics such as magnetic andplasmonic properties are also used toclassify the hybrid microgels 28.3 Fabrication methodsThe very vital part of immobilizationprocess is the choice of properimmobilization method because it plays anessential part in finding the activity andproperties of enzyme in a specific reaction.There are two types of immobilizationmethods which include physical andchemical methods. The properties ofphysical methods include weakmonovalent interaction, affinity binding,ionic binding or enzyme mechanicallycontainment with in the support 29. Inthe chemical methods the development ofcovalent bonds can be attained by amide,ether, thioether or carbamate bondsbetween the support material and enzymes.For the enzyme immobilization there arefour basic methods such as entrapment,adsorption, cross linking and covalent30.3.
1 Physical adsorptionOne of the simplest methods ofimmobilization is the physical adsorption.It includes the physical attachment of theenzymes on the support material. So theadsorption can be take place by weakforces which is van der waals,RECENT RESEARCH PROGRESS IN ENZYME IMMOBILIZED MICROGELS 7hydrophobic interaction and by thehydrogen bonds 31. The method is usedalso for the elimination of the reversiblyimmobilized enzymes from the supportand the support can be recreated andreloaded with fresh enzymes 32. Theessential requirement of physicaladsorption includes the support soakedinto the solution of the enzyme and breedto give time for physical adsorption to takeplace.
The other method contains thedrying of enzyme solution on the electrodesurfaces and then washed away theunadsorbed enzymes 33. One of the mainlimitations of this method is the enzymeleakage from the matrix. The enzymeimmobilization can also be take place dueto the presence of ionic forces between theenzyme and support and this is also knownas reversible immobilization of enzymeswhich are easily used in ion exchangers34. The on the surface of the enzymedepend upon the pH of the solution andisoelectric point 35. In physical methodsthe affinity binding is also used forenzyme immobilization 36. The mainbenefit of this method is the high retentionof immobilized molecule activity 37.There are also a two ways in which theaffinity binding attains between supportand enzyme 38.
The development ofaffinity binding method is the bioaffinitylayering due to which the enzyme bindingcapability is enhanced 39. Thehydrophobic interactions are very useful inthe attachment of enzymes to the supportsurfaces. In the immobilization due to thedisturbance of greater number of watermolecules by one enzyme molecule fromsupport and his surface cause entropy gainto develop hydrophobic interactions 40.
The hydrophobic interactions also dependupon the pH modifications, temperatureand salt concentration during enzymeimmobilization process. The advantages ofenzyme immobilization through physicaladsorption include very low cost, verysimple and greater activity ofenzymes 41. But physical bonding isRECENT RESEARCH PROGRESS IN ENZYME IMMOBILIZED MICROGELS 8quiet weak for the enzymes to bind tocarrier and percolation of enzyme maytake place 42.3.
2 EntrapmentEntrapment is defined as the nonreversibleenzyme immobilization methodin which the enzymes are caught in asupport which passes through the productsbut holds the enzymes 43. Entrapment, inother words is the enzyme is physicallyblocked with in confined area 44. Themain advantage of this method, incude theimprovement of mechanical stability andreduce leaching of enzymes and enzymesremain inert with polymer 45. Theassociation of support material pore sizeand adsorption is describes that in the caseof very small pore size the adsorption takeplace 46.
One of the easy and frequentenzyme entrapment methods includes thegelation of polymers.Some polymers which may be used asmatrix are alganite, carrageenan, gelatin,silicon rubber and polyvinyl alcohol 47.Because of the mild gelling characteristicsthe alganites are most commonly usedpolymers. But this method also suffer fromthe drawback of leakage of enzyme whichtakes place due to the large pore size ofsupport matrix, abrasion, low loadingcapability and deactivation 48.
3.3 Cross linkingThe non-reversible method for theimmobilization of enzymes is cross linkingand it does not need a support to avoid lossof enzyme. This method is also calledsupport-free immobilization 49. Thecross linking is achieved due to thedevelopment of intermolecular crosslinkages between the enzyme moleculesdue to the multifunctional reagent. Theglutaraldehyde is very frequently used as across linking reagent because it is of lowcost and available in huge amounts 50. Inthese methods the immobilizationachieved through three different ways, firstis the prepolymer mixing with thephotosensitizer, mixing with solution ofenzyme and gelling by introduce to thenear UV radiations.