Enzymes are formed in all livingorganisms where they catalyze and regulate essential chemical reactions neededfor the life of organism (Nisha and Divakaran, 2014).

Enzymes are proteins in nature. Theyare fragile and large molecules. Hence enzymes are completely different from thewell-known organic and inorganic catalysts. Soluble enzymes are regarded as beinginstable and sensitive to process conditions (Biro et al., 2008; Buchholzet al., 2012).

Enzymes as biocatalystsEnzymes are biocatalysts which have differentapplications in industrial chemistry (Wohlgemuth, 2010). Thisapplication includes purified enzymes, immobilized enzymes or immobilized cellsas catalysts for the process mentioned above (Schmid et al., 2001; Gong et al.,2012). The development of biocatalysts is completely targeted to theprogress of protein expression, metabolic engineering, large-scale genomesequencing and detected evolution (Bornscheuer et al., 2012). Biocatalysts have a critical importance forprocesses of industrial, pharmaceutical and biotechnological application (Sanchezand Demain, 2010).

The success of enzyme application for any enzymaticprocesses depends on the cost competitiveness as well as the well-establishedchemical methods (Tufvesson et al.,2010).When being compared to chemicalcatalysts, it is noted that enzymes are more incline to be consequently and areused in performing molecular transformations which cannot be achievable by ordinarychemical catalysis (Liese et al.,2006).

Enzymes which are thermostable at hightemperatures are more desirable in industrial applications. The rate ofreaction typically increases every 10°C increase in temperature thus mostenzymes do not withstand high temperatures over higher than 40°C and they canbe denatured at extreme values of pH (Cornish-Bowden, 2004).When applied to the industrialbiocatalysts area, enzymes are proven to provide a great success.

Variousfactors may affect the application of biocatalysts, such factors are enzymepromiscuity, screening technologies as well as robust computational methods forimproving the properties of enzyme available for the applications (Adrio anddemain, 2014).In fact, the biotechnologicalprocesses have many advantages over well-established chemical processes such ashaving less catalyst waste, increased catalyst efficiency as well as a lowerenergy demand. They might be around 150 biocatalytic processes that are beingapplied in industry (Panke and Wubbolts, 2005). However, the newdevelopment in protein engineering made it easier to successfully useparticular enzyme characteristics in industrial purpose (Lutz, 2010).According to the fact that enzymesare involved in all aspects of biochemical conversion varying from the simpleenzyme or fermentation conversion leading to the complex techniques in geneticengineering, it is fair to say that enzymes are considered as a focal point ofbiotechnological processes (Ebbs, 2004).

Environmental and genetic manipulationscan be used to increase the enzyme levels. Thousand-fold increases have been observedfor catabolic enzymes, and biosynthetic enzymes have been increased severalhundred-fold (Burns and Dick, 2002).Many disadvantages have been noted inthe processes of different industries such as the production of pharmaceuticalsand chemicals. These disadvantages may include the need for high temperature, lowcatalytic efficiency, low pH and high pressure.

Not to mention that usingorganic solvents produces pollutants and organic waste. Enzymes such asbiocatalysts are more useful for the applications mentioned above because they havea long half-life, work under slight reaction conditions and they work onunnatural substrates (Johnson, 2013).Furthermore, enzymes can bechemically-modified or selected genetically for improving some characteristics suchas substrate specificity, stability as well as specific activity. However, somedisadvantages are found in enzymes including the requirement of certainco-factor by enzymes.

There are different ways that can be used in order tosolve such a problem among which using the whole cells as well as recycling ofcofactor (Baici, 2015).Reports show that enzymes isolatedfrom microbes are applied in pharmaceuticals as diagnostic reagents, asreagents for the production of chemicals, food additives, the manufacture ofdetergents, the treatment of industrial wastes and bioremediation (Baxterand Cummings, 2006).Stability of enzymeStability of enzymes is an importantconcern especially during thermal processing. Losing enzyme activity at hightemperature ranges is directly related to variations of enzyme conformation (Cuiet al., 2008, Fu et al., 2010). One can estimate this through thermodynamicparameters and Arrhenius equation (Marangoni, 2003).

In a nutshell, enzyme stability is absolutelyessential in basic and applied enzymology. Enzyme stabilization principlescould only be understood through illustrating how enzymes lose their activityfollowed by deriving the structure stability relationships existing inenzymatic molecules (Plou et al.,2009).The most important outcome of usingenzymes is to produce useful compounds. Since the fact that enzymes areunstable and can be quickly inactivated through different mechanisms, theycannot be the proper catalysts for industrial applications. Having a stableenzyme in soluble form is inevitable to achieve the storage of purified enzymesand the purification processes as well (Aehle, 2007).Different strategies have been used inorder to enhance enzyme stability.

The well-known methods for obtaining solublestable enzymes are: 1) chemical modifications of enzymes and 2) use ofadditives (Taravati et al., 2007, Shelley, 2011).Additives are soluble compounds thathave a particular effect on the thermostability of the enzyme protein. Remarkableeffect on the enzyme stability is noticed when particular compounds to enzymesolutions are added.

Such additives are polymers, polyhydrilic, sugar,alcohols, and other organic solvents (Polaina and Maccabe, 2007).Adding certain types of chemicalscould be used in avoiding such conformational changes of the enzyme. Thesechemicals include polyols which is mainly used to promote numerous hydrogenbonds or salt-bridge formation between amino acid residues. These bonds orbridges make the enzyme molecule more rigid, hence it becomes more resistant tothe thermal unfolding (George et al.,2001; Costa et al., 2002).However, the choosing of the appropriate additive depends on the enzymestructure.There are numerous methods used in inenzyme modification that can be mainly classified into three different types.

These types are: 1) attaching of the enzyme molecules to some water solublepolymers 2) polyfunctional substitutions with certain agents used to produceinterior intermolecular linkages and 3) substitutions of the amino-acid groupson the enzyme surface (Shanmugan and Sathishkumar, 2009).The methods mentioned above are usedfor identifying specific residues at the active site involved in substratebinding or chemical catalysis; however it has been used for tailoring thespecificities of enzymes (Qi et al.,2001; Davis, 2003; Svendsen, 2016).There are many ways that can be usedto achieve enzyme stabilization against thermal inactivation. One of these waysis cross-linking to a water insoluble carrier with a bi-functional reagent orcovalent coupling to natural and entrapment in gels and synthetic polymers (Najafiet al., 2005; Shelley, 2011).

Various purification procedures havebeen used to isolate proteins and some enzymes have been purified by using morethan one approach. Even though the process of purifying enzymes could becomplex at first sight, however it gets easier through the sequentialapplication of a few simple methods (Gupta et al., 2016).Purification of EnzymesProtein purification is a series ofprocesses intended to isolate one or a few protein from a complex mixture,usually cells, tissues or whole organisms. Protein purification is vital forthe characterization of the structure, function and interactions of the proteinof interest (Iqbal et al.

, 2016).Protein and non-protein parts of themixture are separated in the purification process, and finally separate thedesired protein from all others is typically the most laborious aspect ofprotein purification. Differences in protein size, binding affinity, physio-chemicalproperties, and biological activity are exploited in the separation steps (Kennedy,1990; Iqbal et al., 2016).

Analytical and preparative methodsare mainly the methods used in protein purification. However, the distinctionis not exact, but amount of protein that can practically be purified with thatmethod is the final deciding factor (Iqbal et al., 2016). The main goal of analytical methods isto detect and identify a protein in a mixture, whereas preparative methods aimto produce large quantities of the protein for other purposes, such as industrialuse or structural biology. Bottom line is, the preparative methods can be usedin analytical applications, but not the other way around (Regnier, 1983).

Techniques of Purification1.    Size exclusion chromatographyChromatography separates protein insolution or denaturing conditions through the use of porous gels. Such atechnique is known as “size exclusion chromatography”. The techniqueis based on the fact that smaller molecules have to traverse a large volume ina porous matrix. Therefore, proteins in a certain range in size will require avariable volume of eluent (solvent) before being collected at the other end ofthe column of gel (Kennedy, 1990).

2.    Ion exchange chromatographyIon exchange chromatography is usedto separate compounds according to the nature and degree of their ionic charge.The column to be used is chosen based on type and strength of charge. Anionexchange resins have a negative charge and are used to retain and separatepositively charged compounds, while cation exchange resins have a positivelycharged compounds, while cation exchange resins have a positive and are used forthe separation of negatively charged molecules (Kennedy, 1990). Immobilization of enzymesWhile free enzymes are unstable andcannot be used to meet the economical requirements for an industrial purpose, immobilizedenzymes are used in industrial bioprocesses especially in food, nutritional,and technology of pharmaceuticals (Sheldon, 2007).Immobilized enzyme is used in manyways because of several factors.

First, enzyme could be handled easily, second theability to reuse costly enzymes, with longer half-lives and less degradation (Shiet al., 2011), third it helpspreventing the contamination of the substrate with enzyme?protein or othercompounds which decreases purification costs, forth its facile separation fromthe product (Spahn and Minteer, 2008).Among the supports used for enzymesimmobilization are hydrogels and inorganic beads, synthetic organic polymers, smartpolymers and biopolymers (Sheldon, 2007; Salemi, 2010).

Enzyme immobilization uses waterinsoluble polysaccharides including agarose, cellulose, chitosan and starch.Also some proteins including albumin and gelatin have been reported as beadsfor the immobilization of enzyme (Krajewska, 2004; Spahn and Minteer, 2008).Also, some biomaterial such as eggshell membrane, has been found to be an effective and stable enzymeimmobilization substrate (Choi and Yiu, 2004; Wu et al., 2004).  Enzymeimmobilization has been implemented on a larger scale, in the food industry andin the manufacture of fine chemicals and pharmaceuticals (Krajewska, 2004).

During the process of immobilization,retention of the activity and the stability must be taken in consideration. Ithas been reported that some enzyme activity is lost during immobilization. Theimmobilization procedure should be chosen carefully because of the interactionbetween enzyme, matrix as well as protein modification (Chen et al.

, 2014).Thermostability and pH stabilityindicate the capability of the conjugate of enzyme support to resist highertemperatures or pH at alkaline or acidic sides before occurring denaturation (Shelley,2011).Storage stability is the ability ofthe enzyme to keep its activity under some certain condition of storage.However, the operational stability does only represent the enzyme function butit represents the durability of the carrier and concentrations of the inhibitorin the solution under assay (Raafat etal., 2012).The general methods used for theimmobilization of enzymesThere are various methods used forthe immobilization of enzymes. These methods could be classified mainly intothe five groups as shown in Fig.

2: (1) Covalent binding of the enzyme to areactive insoluble carrier. (2) Adsorption of enzyme onto support. (3)Cross-linking of the enzyme protein with glutaraldehyde as a bifunctionalreagents. (4) Entrapment. (5) Encapsulation.   1.

    Adsorption:The easiest way for enzymeimmobilization is the physical adsorption of the enzyme protein onto a solidcarrier. Such a method depends on the physical interaction between the surfaceof the carrier and the enzyme protein. This can be done through mixing enzymewith the carrier (Johnson et al.,1996; Jegannathan et al., 2008).

Adsorption is characterized by thefact that it does not demand reagents but only little activation steps (Nishaet al., 2012). Hence, adsorptionis less distributive and cheap for enzyme protein when compared to chemicalmethods of attachment. The binding occurs by hydrogen bonds, salt linkage aswell as Vander Waals forces (Brady and Jordan, 2009; Zucca and Sanjust,2014).Because of the week bonds involved inthe process, adsorption of the protein resulting from changes in pH, temperature,ionic strength or even the more presence of substrate is often considered as adisadvantage (Zhang et al., 2013).One more disadvantage is non-specificadsorption of other proteins since the immobilized enzyme is being used (Zuccaand Sanjust, 2014). This may result in changing the properties of the substrateor the immobilized enzyme and the velocity of enzyme catalysis may be decreaseddepending on the mobility of enzyme and substrate (Rao et al.

, 1998; Shelley, 2011).2.    Encapsulation:Another method for enzyme immobilizationis encapsulation; which means the confinement of a particular enzyme insidelattices for polymerized gels (Zhang etal., 2013). This accelerates the free diffusion of substrate with low molecularweight substrate as well as the products of reaction.  The known polymerization of the hydrophilicmatrix in aqueous enzyme solution is then followed by the breakage of thepolymeric mass into certain particle of the desired size (Lam and Malikin,1994).

The entrapment of biocatalyst usuallyuses calcium alginate hydrogel beads as carriers (Li and Li, 2010;Shen et al., 2011). This method hasmany advantages such as its simplicity of preparation, high porosity and lowcost, however this material still has some limitations since it has large poresize, high bimolecule leakage and biocompatibility (Li and Li, 2010; Zuccaand Sanjust, 2014).Since no bond formation in occlusionprocess between the polymer matrix and the enzyme it is applicable method thatin theory, involves no disruption of the protein molecules (Sassolas et al., 2012).

Other disadvantagefor this method is that only substrate of low molecular weight can diffusequickly to the enzyme protein. This method is absolutely suitable for otherenzymes such as ribonuclease, dextranase and trypsin (Marangoni, 2002).A leakage in some entrapped enzyme mightoccur due to the pore size for synthetic gels of the polyacrylamide, sometimesafter prolonged washing (Grosova etal., 2008; Zucca and Sanjust, 2014).

3.    Cross-linking:Enzyme immobilization has beencarried out by intermolecular cross-linking of the protein, either to bind toother protein molecules or to bifunctional reagents on an insoluble supportmatrix (Sheldon, 2007; Tran and Belkus, 2011).Cross-linking is used in conjugationwith one of other methods (Lam and Malikin, 1994; Zucca and Sanjust, 2014).

The covalent cross-linking with polymers, such as glutaraldehyde have been usedto increase the encapsulation efficiency and control release of enzyme from thegel matrix (Li and Li, 2010; Zucca and Sanjust, 2014).Using cross-linking method for enzymeimmobilization is relatively cheap. Many aldehydes and other cross-linkingagent are used for this purpose (Kurby, 1990; Zucca and Sanjust, 2014).4.    Covalent binding:The formation of covalent bondsbetween the enzyme and the support matrix is used as immobilization method. Thechoice is limited by the fact do not cause loss of enzymatic activity and theactive site of the enzyme must be unaffected by the used reagent (Copeland,2004; Zhang et al., 2013).

The suitable functional groups ofproteins suitable for covalent binding include : 1) the indole group oftryptophan 2) the imidazole group of histidine, 3)  ?-amino groups of the chain and amino groupsof lysine and arginine , 4) –SH group of cysteine, 5) the phenol ring oftyrosine, 6) –OH groups of serine and threonine, and 7) ?-carboxyle group ofthe chain end and ?- and ?- carboxyl groups of aspartic and glutamic acid (Marangoni,2002;  Singh,2009).Aminoethyl cellulose has beenattached to the carboxylic acid residues of enzyme protein in the presence ofcarbodiimide. It has been reported that SH residues of enzyme protein have beenlinked to the thiol groups present in the cross-linked copolymer of acrylamideand non-acrylol cysteine (Copland, 2000; Nisha et al., 2012).This method has one disadvantage whichis that it often causes the low activity recovery which is resulted from thedestruction of enzyme active conformation during immobilization reaction. Themultipoint attachment of the enzyme to the supports or steric hindrance ofenzyme or the strong strength of covalent binding causes low activity recovery (Zhanget al.

, 2013). 5.    Entrapment:The last method used for the immobilizationof enzymes is entrapment. In this method enzymes are physically entrappedinside a porous matrix. Bonds used in stabilizing the enzyme to the matrix maybe covalent or non-covalent. The matrix used will be a water-soluble polymer.The form and nature of matrix varies with different enzymes. Pore size ofmatrix could be adjusted to prevent the loss of enzyme.

Pore size of the matrix can beadjusted with the concentration of the polymer used. Agar-agar and carrageenanhave comparatively large pore sizes. However, the main disadvantage of thismethod is that there is a possibility of leakage of low molecular weightenzymes from the matrix. Such matrixes used for entrapment are: carrageenan, polyacylamidegels, agar, cellulose triacetate, gelatin, and alginate (Dwevedi, 2016).