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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
optimize the purication protocol to attain efcient capture and maximum recovery of the target.
The entire procedure depends upon exact interaction between the analyte and the opposite group which is covalently bound to the column packing. A column comprising an insoluble polymer or gel to which a specic competitive inhibitor or other ligand has been covalently bound is used for this purpose. The solution containing macromolecules to be puried is allowed to pass through this column. Those proteins that have high afnity with the ligand will be retained, whereas those with less afnity will pass unretarded through the column. Using certain procedures, such as washing the ligand–protein complex with a solution of displacing agent such as an inhibitor, or by changing the pH or ionic strength of the elution solvent to favor dissociation, exactly retained proteins can then be eluted. Various insoluble supports are used for enzyme purication by afnity chromatography, such as hydrophilic cellulose derivatives, polystyrene gels, cross-linked dextrans, beaded agarose, glass beads and polyacrylamide gels. Some chemical derivatives of agarose and polyacrylamide have also been used for enzyme purication. The afnity matrices used for enzyme purication can be divided into two categories:
Those which are specic for the desired enzyme by the specicity of the ligand such as the substrate, substrate analogs, inhibitors or antibody of the enzyme.
Those which will interact with a related group of enzymes because of an immobilized general ligand such as a co-factor (5-AMP, 2,5-ADP, NAD and others), which is specic for a class of enzymes and hydrocarbon ligands or dyes. This will interact with a large number of different enzymes.
The total expense of enzyme isolation can be minimized by the incorporation of an afnity step. One of the most common examples is purication of tissue plasminogen activator (t-PA) from cultures of human kidney cells, which is puried using α­benzylsulfonyl-p-amino-Sepharose [84].
2.3.6.4.5 Electrophoresis and ultracentrifugation
During electrophoresis, a molecule with an overall charge will move in an electric eld. Electrophoresis offers a potential means of separating proteins and other macromolecules, such as DNA and RNA. The velocity of migration (
) of a protein (or any molecule) in an electric eld depends on the electric eld strength (E), the net charge on the protein (z) and the frictional coefcient ( f ). Considering all these, one can see that electrophoretic purication is based on the movement of proteins when placed in an electrical eld. This process is capable of the highest resolution of enzymes employing physico-chemical separation. This procedure is now mainly used as a technique for separation of isoenzymes and other enzymes linked for diagnostic purposes. The procedures often used at the laboratory scale for enzyme purication and characterization are: free-boundary electrophoresis; zone electrophoresis with the help of supports such as paper, starch, and cellulose powder; electrophoresis in agarose and acrylamide gels; and isoelectric focusing. Recently, a continuous electrophoretic separator has been developed for large-scale separation of proteins. The separator can draw as many as 29 separate fractions. This application can be
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utilized for the isolation and purication of high value pharmaceutical enzymes and other proteins.
Electrophoresis-based separations are always performed in gels as the gel assists as a molecular sieve that enhances separation. Molecules that are small in comparison with the pores in the gel easily move from the gel, while molecules with a larger size than the pores are almost immobile. Intermediate-sized molecules move from the gel with different degrees of ease. Electrophoresis is performed in a thin, vertical slab of polyacrylamide and the direction of ow is from top to bottom [85]. One of the most suitable supporting media for electrophoresis is polyacryla­mide gels, formed by the polymerization of acrylamide and cross-linked by methylenebisacrylamide, as they are chemically inert and are readily formed. Electrophoresis is the reverse of gel ltration, in that all of the molecules, irrespective of size, are forced to move through the same matrix [85]. In a solution, proteins have the tendency to sediment at high centrifugal force, so it is tricky to separate a protein from a mixture by centrifugation. Enhanced separation of proteins has been accomplished by density gradient centrifugation. In this process, sucrose or glycerol is used as density gradient [85]. With more sophisticated continuous ow ultra­centrifugation, whole cells, viruses and subcellular components can be separated. With ultracentrifugation, we can determine parameters such as mass and density, study something about the shape of a molecule and examine the interactions between molecules [85].
2.3.7 Finishing operations
Its well-known that all enzymes are antigenic. After issues occurred in the late 1960s when workers manufacturing enzymes suffered from severe allergic responses after breathing enzyme dusts, methods have now been applied to reduce dust formation [3]. These include delivering enzymes as liquids wherever possible, or enhancing the particle size of dry powders from 10 μm to 200–500 μm by either prilling (mixing the enzyme with polyethylene glycol and preparing small spheres by atomization) or marumerizing (mixing the enzyme with a binder and water, extruding long laments, converting them into spheres in a marumerizer, drying them and covering them with a waxy coating) [3]. Finishing operations such as enzyme desalting, concentration, purity control and storage are of greater importance [3].
2.3.7.1 Desalting
Ammonium sulfate (AS) fractionation is frequently used in protein and enzyme purication; however, the nal protein pellets have a high salt content and desalting by dialysis is required prior to subsequent analysis [86, 87]. Unnecessary amounts of inorganic salts must be eliminated from enzyme preparations. The salts existing in one enzyme are perhaps inhibitory to other enzymes present in clinical diagnostic reagents. Also, desalting may be crucial at intermediate stages of enzyme isolation, e.g. before ion-exchange adsorption. The simplest and oldest method of salt removal is dialysis [86, 87]. The main function of dialysis is to eliminate small molecules such as salts from larger ones, in particular the enzymes to be puried. Mixed bed ion
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exchange, the conventional method of removing small ions, has been applied to some of the more robust enzymes. Mixed bed cation and anion exchange resins are used to eliminate both positive and negative ions. Wang et al developed a phenol-based method for rapid desalting and concentration of proteins after ammonium sulfate fractionation of complex olive leaf protein extract [86, 87]. After re-dissolving, ammonium sulfate precipitates were desalted with phenol extraction and a lot of β-glucosidase was observed in each fraction using a specicantibody[86, 87].
2.3.7.2 Concentration of enzymes
Once extraction is performed, both intracellular and extracellular enzymes are received as a dilute solution and must be further concentrated prior to further use. On the pilot scale, precipitation and adsorption are employed to concentrate the enzymes. On the large scale ultraltration has been used.
2.3.7.2.1 Ultraltration
A semi-permeable membrane allows the separation of solvent molecules from larger enzyme molecules, as only the smaller molecules can enter the membrane when the osmotic pressure is exceeded. Ultraltration is performed to separate macromole­cules and colloidal particles by means of a membrane. Hydraulic pressure is employed as the active force for the solvent molecules to pass through the membrane, whereas the microporous membrane does not allow the passage of large solute molecules. Membranes with exclusion limits of 2000–300 000 Da are available for ultraltration. Ultraltration has a tendency to remove small molecules. The enzyme solution is fed into a cell tted with a membrane which retains the selected protein while being permeable to the solvent and small molecules. Negative pressure is applied to the collecting chamber and positive pressure to the solution, which acts as the driving force that causes the ow of solvent and small solute molecules across the membrane. Large volumes can be reduced to a few milliliters in only one or two hours. Dialtration is a very useful procedure for eliminating salts and other low molecular weight substances in the form of contaminants from dilute enzyme solution [88]. During this procedure the water is fed to the ultralter cell so that molecules other than water are successfully eliminated from the system. Cellulose acetate and organic polymers, e.g. polysulfone, have proven to be useful as membrane materials. With the exception of cellulose acetate, these membranes can easily be cleaned with alkali or acid and stream sterilized [88].
2.3.7.2.2 Drying
Drying procedures have long been established for food and pharmaceuticals and have now been applied successfully to enzymes. The extracellular enzymes in the food industry are often used as dried free-owing powders. The drying of enzymes mainly depends upon the thermal stability of enzymes. The drying of enzymes has commonly been carried out by spray-drying and freeze-drying [32]. Spray-drying is a common method for producing powder from a liquid, whereas lyophilization, or freeze-drying, is a procedure for preserving food and biological materials (enzymes, proteins, vitamins, etc), oral products, drugs and whole animals. Robust enzymes
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may be vacuum dried or spray-dried, while the delicate ones are freeze-dried or spray-dried. Spray-drying is extensively used for extracellular bacterial enzymes, while most intracellular enzymes are dried using freeze-drying processes [32].
2.3.7.2.3 Vacuum evaporation
Enzymes are labile molecules, so evaporation must be conducted under rather high vacuum conditions to prevent denaturation. During the production of extracellular enzymes, the diluted enzyme solution is reduced under vacuum pressure at temper­atures below 40 °C. At the industrial scale, enzymes are concentrated in a similar manner, with the same equipment as is used for the concentration of fruit juices at a reduced temperature [89]. In a characteristic application, an enzyme solution was reduced in two stages from 8% or 12% to 35% solids, then to 65% solids. Vacuum­based evaporation of enzymatic solution is always achieved by the addition of sugar alcohol such as sorbitol or sugars such as lactose in higher concentrations [89].
2.3.7.3 Storage
Good storage conditions are of paramount importance in the industrial production of enzymes. One of the major concerns is that the loss of activity during storage will result in the loss of product in its purest and most expensive form. Most enzymes have a restricted shelf-life owing to inherent active-conformation liability in the minority of molecules present in high energy states, at any temperature at any particular time. This loss can be suspended by cross-linking, immobilization or by using enzyme stabilizers, e.g. sugars, certain ions and co-factors. Most enzyme products are marketed as dry powders; a few enzymes are available in a liquid or suspension form. These products usually contain compounds which decrease the loss of enzyme activity during storage and check microbial growth, i.e., enzyme stabilizers and co-factors. Proteins (in particular enzymes) must be stored at in a suitable temperature and pH range and frequently in the presence of concentrated (approximately 1 M) glycerol, sucrose, or a similar substance, for the proteins to retain activity and prevent aggregation [90]. The compounds generally used for these purposes are glycols, propylene glycol, ethylene glycol, sorbitol, mannitol, thiol, reducing agents, sodium chloride, salts of organic hydroxy acids and other salts of buffers, sodium benzoate, and esters of parahydroxy benzoic acid. Recently, compounds such as gelatin, dextrans, partially hydrolyzed collagen, gum arabic, albumin, polyamines, poly-l-lysine and glycerol monoethers have been used as stabilizers. Enzyme containers should be kept sealed to prevent the escape of the stabilizing atmosphere and also to maintain the low moisture content (below 50%) of the solid product. The storage temperature should be low but not freezing, as freezing may cause denaturation of certain enzymes [90].

2.4 Recombinant proteins from algae

As photosynthetic organisms, microalgae can efciently convert solar energy into biomass. Microalgae are currently used as an important source of valuable natural biologically active molecules, such as carotenoids, chlorophyll, long-chain poly­unsaturated fatty acids, phycobiliproteins, carotenoids and enzymes. Signicant
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Table 2.6. Recombinant proteins from algae.
Expression
Algae name Products
Amphidinium sp. β-Glucoronidase Nucleus C. reinhardtii Anti-HSV glycoprotein D Isc, Anti-CD22-gelonin sc, Anti-
CD22-ETA sc, VP1-CTB, 14FN3, Metallothionein-2, TRAIL, Allophycocyanin, Anti-PA 83 anthrax IgG1, E7GGG, VEGF, HMGB1
C. reinhardtii GBSS-PfMSP
GBSS-PbAMA
1–19
1-C
location
Chloroplast
Nucleus
advances have been achieved in microalgae biotechnology over the last decade, and the use of microalgae as bioreactors for expressing recombinant proteins is receiving increased interest. Compared with the bioreactor systems that are currently in use, microalgae may be an attractive alternative for the production of pharmaceuticals, recombinant proteins and other valuable products (table 2.6) Products synthesized via the genetic engineering of microalgae include vaccines, antibodies, enzymes, blood-clotting factors, immune regulators, growth factors, hormones, and other valuable products, such as the anticancer agent Taxol. Here, we briey compare the currently used bioreactor systems, summarize the progress in genetic engineering of microalgae, and discuss the potential for microalgae as bioreactors to produce pharmaceuticals.

2.5 Enzyme immobilization techniques

The process of immobilizing enzymes for usage in biotechnology requires the essential step of afxing soluble enzymes onto a solid substrate or matrix. This step is necessary since immobilization cannot occur without it. There are several benets to using this technique, including improved enzymatic activity and stability, versatility, and a straightforward separation procedure (table 2.7).
2.5.1 Advantages and applications of enzyme immobilization
Compared to the usage of set-free soluble enzymes, the process of immobilizing enzymes, which is a common approach in biotechnology, possesses many benets that, depending on the specics of the situation, make it an additionally advanta­geous choice [91]. The immobilization of enzymes onto solid substrates or matrices has enhanced their stability, reuse ability, and activity as catalysts. This objective may be accomplished because they are restricted to a rigid surface. The immobiliza­tion of enzymes is currently used in many production methods, including conven­tional and innovative biotechnological techniques [92]. This section presents a complete overview of the benets linked with immobilizing enzymes [93].
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Table 2.7. A comparative analysis of widely utilized techniques for enzyme immobilization.
Method Principle Advantages Examples
Entrapment Isolation inside a gel or
porous matrix
Covalent
bonding
Adsorption Weakly interacting
Encapsulation Getting stuck inside a
Cross-linking Enzyme-enzyme or
Establishment of
permanent enzyme­support chemical bonds
connection to a supporting surface
capsule or membrane that lets some light through
enzyme-support­enzyme covalent link formation
Simple to put
together; keeps the action going
Strong ties,
permanent solidity
Easy to set up,
flexible
Enzyme prevention
and sustained activity
Improvements in
stability and resistance to enzyme leaching
Glucose isomerase is used to
manufacture high-fructose corn syrup; Lactase is used in the dairy sector to hydrolyse lactose.
Pharmaceutical companies rely
on penicillin G acylase in the production of antibiotics.
Choline oxidase for choline
sensing biosensor study and creation
Enzymes that have been
immobilized for use in enzyme microcapsules for timed-release
Binding enzyme with
glutaraldehyde for better stability
2.5.1.1 Advantages of enzyme immobilization
The immobilization of an enzyme enhances resilience against several factors, such as uctuations in temperature or pH. Additionally, this technique facilitates enzymes immobilization, enhancing their ease of separation and reusability. Immobilized enzymes have a more extended period during which they may catalyze reactions, contributing to their increased catalytic efciency [94]. Immobilizing an enzyme allows it to be retrieved and used several times, which is one of the most signicant benets of this technique [95]. In contrast to loosened enzymes, which may get denatured or destroyed during reactions, immobilized enzymes can be removed from the reaction mixture, regenerated, and reintroduced into subsequent processes with little loss of activity. This reusability factor affects the efciency and longevity of biocatalytic processes [96]. Immobilized enzymes may be readily extracted from the reaction mixture using simple procedures like ltration or centrifugation. The ease of product recovery and further processing upstream means that elaborate purication techniques may be avoided [97]. Multiple enzymes may be attached to a single support via various immobilization methods, allowing for the co-immobilization of enzyme cascades or multi-enzyme systems. Synergistic enzymatic reactions result from this approach, expanding the scope of possible uses and boosting the efciency of the process overall [98]. Immobilization simplies the construction of continuous ow reactors, in which the immobilized enzyme remains stationary inside the reactor
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Figure 2.6. Applications of enzymatic reactions in food and beverage industries, highlighting the use of amylase, lactase, pectinase, lipase, and invertase.
Table 2.8. The utilization of enzyme immobilization in several industrial sectors.
Industry Enzyme Application
Food stuff and
drinks
Pharmacological Chymotrypsin Peptide creation Bioremediation Urease The breakdown of urea in water treatment plants The manufacturing
of biodiesel
Biosensors Glucose
Invertase The manufacture of speciality sugars, such as inverted
sugar, is a topic of interest in food science and technology Lactase Making dairy products that do not include lactose Penicillin G
Acylase
Lipases Making biodiesel from vegetable oils using
oxidase
Antibiotic making
transesterification.
Medical gadget glucose monitoring
while the reaction substrates pass over it. The advantages of this design include better reaction control, less enzyme loss, and enhanced enzymatic process stability [99]. Several enzymatic reactions in food and beverages used in industries are mentioned in gure 2.6 (table 2.8).
2.5.2 Methods of enzyme immobilization
Enzymes are turned into immobilized forms by using the central biotechnology technique of enzyme immobilization, in which they are attached to a stable substrate or matrix. Improved stability, reusability, and ease of separation from reaction mixtures make this technique essential for biocatalysts, gure 2.7.
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Figure 2.7. Comparison of reversible and irreversible enzyme immobilization methods such as adsorption, ionic binding, and encapsulation.
Table 2.9. Examples of enzymes immobilized by entrapment and their applications.
Enzyme Application
Glucose isomerase Making corn syrup sweetened with high fructose Lactase Hydrolysis of lactose for the dairy sector and urea for wastewater treatment Urease The creation of a choline-detecting biosensor Choline oxidase The making of biodiesel Lipases Making corn syrup sweetened with high fructose
2.5.2.1 Entrapment
One non-covalent method of immobilizing enzymes is physically conning them within a porous matrix or gel. The gel formation process isolates the enzyme in a protective matrix, allowing substrate and product transfer. Matrixes for trapping are commonplace, including agar gel, polyacrylamide, and calcium alginate. Immobilized glucose isomerase produces high-fructose corn syrup [100], while immobilized lactase is used for lactose hydrolysis in the dairy industry. Table 2.9 provides some other instances of how enzymes that have been captured and immobilized might be put to use.
2.5.2.2 Covalent bonding
When an enzyme is covalently immobilized, strong chemical linkages are formed between the enzyme and the support matrix. The approachs high adherence ensures
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
the immobilized enzymeslong-term stability and reusability. Bifunctional reagents or linkers are often used to promote covalent bonding. Covalent immobilization has been used in the pharmaceutical industry to immobilize penicillin G acylase and chymotrypsin to produce antibiotics and peptides [99]. Table 2.10 displays some more examples of covalently immobilized enzymes and their respective applications.
2.5.2.3 Adsorption
Adsorption is a simple and widely used method in which enzymes are bound to the surface of a solid support material through weak contacts such as hydrophobic interactions, van der Waals forces, and hydrogen bonds. Common adsorption support materials include silica gel, activated carbon, and ion exchange resins. Adsorption has been employed in a wide variety of biotechnological contexts. Choline oxidase, for instance, may be detected in some diagnostic tools thanks to its adsorption onto support materials for biosensor development. In addition, lipases have been absorbed into activated carbon for use in biodiesel production [101] (table 2.11).
2.5.2.4 Encapsulation
During encapsulation, enzymes are enclosed in semi-permeable membranes or capsules. The encapsulating medium allows substrates and products to circulate, preventing enzyme leakage and protecting sensitive enzymes. This method is suitable when working with enzymes that must be shielded from harsh reaction conditions. The regulated release of enzymes has led to their encapsulation into
Table 2.10. Examples of enzymes immobilized by covalent bonding and their applications.
Enzyme Application
Penicillin G acylase Biosynthesis of antibiotics Chymotrypsin Manufacturing of peptides Catalase Breakdown of hydrogen peroxide Glucose oxidase Glucose sensing biosensor study and creation Urease Biological waste treatment
Table 2.11. Examples of enzymes immobilized by adsorption and their applications.
Enzyme Application
Choline oxidase The creation of a choline-detecting biosensor Lipases The making of biodiesel Glucose oxidase Biomedical gadget glucose monitoring Invertase Manufacturing of inverted sugar and other specialized sugars Catalase Catalysis in a natural setting
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Table 2.12. Examples of enzymes immobilized by encapsulation and their applications.
Enzyme Application
Various enzymes Methods of medication administration with controlled release Glucose isomerase Making syrup from corn sweetened with high fructose Urease Wastewater treatment by urea hydrolysis Lipases Biological catalytic membranescarefully regulated enzyme secretion Catalase Biological waste treatment
Table 2.13. Examples of enzymes immobilized by cross-linking and their applications.
Enzyme Application
Various enzymes Pharmaceutical and specialized chemical manufacturing Glucose isomerase Making corn syrup sweetened with high fructose Lipases Pharmaceutical intermediate synthesis Invertase Manufacturing of inverted sugar and other specialized sugars Urease Wastewater treatment by urea hydrolysis
microcapsules. Microencapsulated enzymes that have been immobilized offer a highly accurate and prolonged enzymatic response inside formulations that allow for regulated release. Encapsulation research has also focused on developing biocata­lytic membranes for utilization in diverse biotechnology and environmental reme­diation applications [102] (table 2.12).
2.5.2.5 Cross-linking
Chemical agents are utilized in cross-linking to facilitate covalent connections between enzymes and the support matrix. This method generates enzymes with immobilization that demonstrate endurance and lifespan due to increased stabiliza­tion of enzymes and the avoidance of enzyme leach. The use of glutaraldehyde as a cross-linker in the enzymatic immobilization process is widely employed. An instance of a method used in biocatalysts to augment the activity and stability of enzymes is the application of glutaraldehyde bridging. Several commercial uses, such as the manufacture of speciality chemicals and pharmaceutical intermediates, have been found to use cross-linked enzymes [103] (table 2.13).

2.6 Enzyme engineering for enhanced stability and activity

Enzyme manufacturing is signicant to biotechnology, given its capacity to improve the stability and function of enzymes, which may boost their economic and therapeutic usefulness [104]. This phenomenon may be attributed to the limited number of approaches capable of achieving this objective. Researchers may engage in structural and property alterations to improve the functioning of an enzyme under certain environmental circumstances to improve its catalytic stability and
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