Enzyme Immobilization

Immobilization of Enzymes

• Immobilization limits the movement of biocatalysts via chemical or physical treatment
• Insoluble enzymes offer advantages as heterogeneous catalysts
• Convenient separation for reuse is achieved through filtration or centrifugation
• Continuous processes benefit from fixed-bed, fluidized-bed, and stirred-tank reactors
• Retention is ensured with a filter system

Reasons for Immobilization

• Enzyme reuse lowers costs
• Continuous processing enables facilitated process control
• Continuous processing enables low residence time and high volumetric activity
• Continuous processing allows optimization of product yield
• Product separation becomes easier
• Enzyme is more stable

Limitations of Immobilization

• Involves cost of carriers and immobilization
• Presents mass transfer limitations
• Raises problems with cofactor and regeneration
• Can cause problems with multienzyme systems
• Changes in selectivity may arise
• Enzyme activity can be lost

Principles

• Enzymes are immobilized by transformation into an insoluble form
• Method ensures retention in the reactor
• Immobilization can be achieved by inclusion in a definite space
• Method ensures retention in the reactor
• One-step reactions are suitable for immobilized enzymes

Binding to Porous Carriers

• This method is commonly used in laboratory and industry settings
• The properties of the external protein surface affects binding
• The functional groups accessible participate in binding
• Adsorption depends on hydrophilic and hydrophobic characteristics of the surface region
• Ionic Interaction relies on ionic groups and their pH-dependent charge and density
• Covalent Binding uses functional groups, mostly -NH2 of arginine/lysine or -COOH of aspartic acid
• Factors like carrier type, method, concentrations, pH, temperature, and reaction time affect the rate and yield
• Protein surface engineering influences interaction with surfaces and shifts isoelectric point

Properties and Classification of Carriers

• Hydrophilicity/Hydrophobicity impacts carrier performance
• Swelling properties must be low
• Chemical stability should be high
• Microbial stability needs to be good
• Particle size should range from 0.2-1 mm with narrow distribution
• Pore size should be between 30-60 nm
• Inner surface area for adsorption/binding needs to be large
• Resistance to pressure/compressibility should be good or low
• Elasticity must be sufficient
• Consider food grade for food industry applications
• Low cost is desired
• Inorganic carriers offer high pressure stability, but may undergo abrasion in stirred vessels
• Organic carriers from natural sources offer favorable compatibility with proteins
• Organic synthetic materials offer high chemical stability

Inorganic Carriers

• SiO2-based carriers are functionalized with amino groups via aminopropyl triethoxysilane (APTES)
• Covalent binding uses glutaraldehyde for activation
• Porous glass (Corning, Waters, Schuller) and silica (Grace, Solvay, Degussa) are used
• Celite (diatomaceous earth) stabilizes enzymes in organic media via adsorption
• Bentonite has excellent adsorption capacity to 1.5 g protein/g
• Bentonite is used for enzyme isolation via adsorption/desorption
• Cross-linking enzymes with glutaraldehyde prevents desorption
• Cross-linking enzymes carrier can be entrapped in alginate

Organic Carriers from Natural Sources

• Polysaccharides and derivatives are commonly used for immobilization
• They have wide networks
• Have hydrophilic properties and weak interactions with proteins
• Cellulose derivatives include DEAE-cellulose (diethylaminoethyl-)
• Dextran is widely used for enzyme immobilization
• Dextran is used for protein chromatography
• Dextran is activated by cyanogen bromide
• Dextran has mechanical stability limited
• Other polysaccharides include agarose, starch, pectin, and chitosan
• Proteins such as gelatine can be used

Organic Synthetic Carriers

• Offer a wide range with good capacity
• Cost-effective and simple to immobilize
• Ion-exchange resins are used
• Copolymerization can introduce functional groups like nitration, sulfonation, carboxylation, epoxidation
• Amberlite™ XAD™ is adsorbent with hydrophobic properties
• Polystyrene, Polyvinyl Acetate, and acrylic polymers can be used

Binding Methods - Adsorption

• Binding occurs onto silica, clay, or ion-exchange materials
• Binding involves weak interactions like ionic, electrostatic, and hydrophobic forces
• Selection of an appropriate carrier and optimal conditions is necessary
• Adsorption affected process conditions (ratio of enzyme to carrier, pH, temperature, ionic strength and hydrophobicity)
• Process is cost-effective, reversible and may cause unfolding
• The adsorbed enzyme may be stabilized by cross-linking with glutaraldehyde

Binding Methods - Covalent Binding

• Carrier functional groups and enzyme surface groups have major role
• Optimal conditions determined empirically for each enzyme and system
• Functional groups (amino, epoxy, thiol, cyanide) are introduced to both carrier and enzyme
• Derivatization, activation, and enzyme binding are the steps

Enzyme Cross-linking and Polymeric Network Inclusion

• Soluble enzymes are cross-linked using linkers
• Glutaraldehyde is one such linker, working with lysine residues
• Other techniques exist like cross-linking enzyme crystals (CLEC®) and enzyme aggregates
• Whole, dead cells containing enzymes can be cross-linked
• Inclusion into a polymeric network is a convenient method for whole-cell immobilization
• With diameter problems and leakage, combination with cross-linking or binding to nanoparticles of beads can assist

Properties of Immobilized Enzymes

• Temperature or chemical stability may vary upon immobilization
• Depends on how the new micro-environment affects the enzyme.
• Optimum pH can change by up to 2 pH units
• Km is affected by the electrostatic field of the carrier
• Km value decreases significantly when the carrier is opposite the charge of the substrate
• Diffusion factors may also affect the Km value

Membrane Systems and Processes

• Membrane systems retain or recycle enzymes to the reactor
• Semi-permeable membranes allow product molecules to freely pass, but keeps the enzyme
• Membrane systems offer advantages for cofactor-dependent reactions when coenzyme must be regenerated
• Low molecular weight cofactors need to be coupled to polymers of sufficient molecular weight
• Membrane reactor systems are used for small-scale production
• This is especially true for multi-enzyme pathways or coenzyme regeneration
• The high cost and need to replace the membranes routinely is a major disadvantage

Industrial Applications of Immobilized Enzymes

• Examples for industrial enzymes in major commercial processes (2002 estimate):
• Glucose Isomerase for glucose-fructose syrup (10 million t per year)
• Thermolysin for Aspartame (10,000 t per year)
• Nitrilase for Acrylamide and Nicotinamide (>95,000 t/a and > 5,000 t/a respectively)
• Aminoacylase for L-Amino acids (several 1000 t/a)
• Hydantoinases for D-amino acids (ca. 100 t a-1)
• Lipases for Pharmaceutical and agrochemical intermediates (multi-kg; >1000 ta-1)
• Sucrose Mutase for Isomaltulose (ca. 100,000)
• β-Galactosidase for Glucose-galactose syrup (>6000 ta-1)
• Penicillin acylase for 6-APA (>10000 ta-1)
• D-amino acid oxidase and Glutaryl amidase for 7-ACA

Immobilization of Microorganisms and Cells

• First example took place in 1823, Acetobacter adsorbed to wood chips for acetic acid production
• Immobilizing cells is useful for:
  • Production of secondary metabolites.
  • Cofactor regeneration and multienzyme systems (e.g., alcohol production) If difficult to isolate or unstable, low-activity enzymes are used
• Continuous processing with (re)synthesis is possible
• Industrial application of immobilized viable cells
• beer maturation with yeast cells.
  • anchorage-dependent mammalian cell is used in the production of vaccines.
  • environmental technologies using mixed cultures can be applied

Advantages and Limitations of Immobilization

• Advantages
• No enzyme isolation and purification needed
• Useful in multienzyme complex reactions
• Cofactor regeneration in native system is possible Application of achorage-dependent cells can be used.
• Syntrophic mixed cultures can be used
• Limitations
• Insufficient stability, low resistance
• Mass transfer limitation
• Side reactions, degradation of product
• risk of Byproducts from lysis of cell or toxic metabolites
• Low productivity

Immobilization by Adsorption, Adhesion, and Entrapment

• Adsorption and growth of microorganisms surface is a natural phenomenon
• Vinegar production uses adsorbed of Acetobacter sp. in wood chips (Conversion 90%)
• In beer manufacture, Saccharomyces cerevisiae is immobilized by adsorption in porous silica particles
• Antibodies, therapeutic proteins and vaccines can also be applied
• Adhesion follows adsorption and microorganisms produce secondary matrix of polymers
• Environmental tech applications include waste/nitrogen elimination
• For entrapment, polymeric networks are applied for cell retention
• Conditions combine with variety of low-cost materials
• For ionotropic gels: Water soluble polyelectrolytes (eg: Ionic polysacharides) for solid polymeric networks (gels by crosslinking
• lonic polysacharides are carboxy- or sulfonyl groups (alginate - pectin - carrageenan)
• Ionotropic gels are low cost and non-toxic
• Gels made more complex with alginate, adding alginate polymerized, adding water soluble sodium

Industrial Whole Cell Immobilizations

• Ethanol is produced by Saccharomyces cerevisiae with alginate carrier
• Ethanol is produced by Zymomonas mobilis with alginate carrier
• 2,3-butanediol is produced by Enterobacter aerogenes with alginate carrier
• L-isoleucine is produced by Serratia marcescens with alginate carrier
• 11β-hydroxy-progesterone is produced by Acetobacter phoenicis with alginate carrier
• Prednisolone is produced by Acetobacter globiformis with Polyacrylamide carrier
• L-DOPA is produced by Erwinia herbicola with Carrageenan carrier
• Thienamycin is produced by Streptomyces cattleya with Celite carrier

Production of High Fructose Corn Syrup (HFCS)

• Produced at 10 million tons per year, using 1500 tons of immobilized glucose isomerase
• Contains 53% glucose, 42% fructose, and 5% other products
• Alternatively, HFCS contains 40% glucose, 55% fructose, and 5% other products
• Advantages include that it is 10-20% cheaper than sucrose at matching sweetener levels
• Further, exhibits lesser tendency to crystallize in a variety of products
• HFCS is an alternative that is mostly used in Western colas, sauces, and juices
• The sweetener and the taste are almost equal to that of sucrose
• At 45°C, 48% fructose and at 85°C, 55% fructose

Development of Glucose Isomerase

• Glycolytic enzymes (phosphohexose isomerase/ATP) ruled out
• An isomerase must be commercially useful
• Late 1950s conversion of D-xylose to D-xylulose with cobalt ions showed to isomerise to D-fructose
• Since then, enzymes will produce isomerase with glucose, genetics will modify stability of Mg2+
• Production is 20 t/kg with life of 80–150 days

Isolation of Glucose Isomerase and Methods

• Two methods for the application of Glucose Isomerase
• First Method
• Cross linked via Glutaraldehyde
• Second method
• Isolated , then purified
• Finally adsorbed by an ion exchange matrix

Methods and Comparison

• Batch is costly, long residence in fructose
• Lead to significant production rates
• Added with Mg2+ and Co2+ before it is isolated

Use of Immobilized Lactase

• Lactase is one of few enzymes that can be used free in processes
• Yeast is immobilized by incorporation with cellulose to be used with 5°C STR Process
• Fungal have been put in porous silica using glutar and g-aminopropyl
• Yeast and fungal have ph optima

Production of Antibiotics

• G and V are used for antibiotics
• The hydrolyzation is done with amidase
• A method must be done to have ph
• Supports must be able to have the proper carriers

Use of Enzymes in Industrial Applications.

• In food, in pharms
• All have separate methods for reactions

Enzymes are Required in New Industries

• For fine chems
• Abx
• Paper production
• Drugs
• Glycodylation
• Environments