SESSION 2011-2012

ENZYME IMMOBILIZATION

HEAD OF THE DEPT. OF PHARMACY: SUBMITTED TO:

DR. A K PHATHAK MISS NISHTHA SAHU

SUBMITTED BY:

PUNIT DUBEY

CERTIFICATE

This is to certify that PUNIT DUBEY student of B.Pharma 7th sem has successfully completed this project work titled as “ENZYME IMMOBILIZATION” in partial fulfillment for award of degree in Bachelor of Pharmacy in the year 2012 by Barkatullah University, Bhopal.

Under the Guide of: Head of Dept.

Miss Nishtha Sahu Dr. A.K. Pathak

DECLARATION

I hereby declare that the project report which is being presented on the ENZYME IMMOBILIZATION is an authentic record of my own work carried under the guidence of Miss Nishtha Sahu.

I hereby declare that above statement is correct to the best of my knowledge.

PUNIT DUBEY

ACKNOWLEDGEMENT

We wish to acknowledge our profound sense of gratitude to our project guide Miss Nishtha

Sahu, Department of Pharmacy, Barkatullah University, Bhopal for his guidance and continued

encouragement during the preparation of this project. Indeed it was a matter of great felicity and

privilege for us to work under his aegis. We express our thankfulness to her for dedicated

inspiration, lively interest and patience through our errors, without which it would have been

impossible to bring the project to near completion.

We would like to thank Dr. A.K. Pathak H O D of department of pharmacy, Barkatullah

University, Bhopal for all the encouragement and facilities provided to us. Last but not the least

we would like to thank our parents for their support and cooperation.

Regardless of the source we wish to express our gratitude to those who may have

contributed to this work, even though anonymously.

INTRODUCTION

Enzyme engineering is a fast-growing application in the pharmaceutical market. Enzymes are keys to new processes because they are environmentally friendly and reduce hazardous waste. Enzymatic reactions can occur under milder conditions, at a faster rate, while being highly specific. Therefore, enzymatic process allows to minimize process steps.

Enzymes can be operated in the liquid form or immobilized on various supports. Immobilized enzymes enhance process robustness; allow longer duration of activity of enzymes, and re-use of the same enzymes in multiple cycles.

The use of immobilized enzyme eliminate the enzyme separation step from the main process thus simplifying and increasing the overall process yield.

 

Easy separation from reaction mixture, providing the ability to control reaction times and minimize the enzymes lost in the product.

Re-use of enzymes for many reaction cycles, lowering the total production cost of enzyme mediated reactions.

Ability of enzymes to replace multiple standard chemical steps and provide enatomerically pure products.

What is an Immobilized Enzyme?

       Enzymes are protein molecules which serve to accelerate the chemical reactions of living cells.  Without enzymes, most biochemical reactions would be too slow to even carry out life processes.  Enzymes display great specificity and are not permanently modified by their participation in reactions.  Since they are not changed during the reactions, it is cost-effective to use them more than once.  However, if the enzymes are in solution with the reactants and/or products it is difficult to separate them.  Therefore, if they can be attached to the reactor in some way, they can be used again after the products have been removed.  The term "immobilized" means unable to move or stationary.  And that is exactly what an immobilized enzyme is:  an enzyme that is physically attached to a solid support over which a substrate is passed and converted to product.

Methods of Immobilization

        When immobilizing an enzyme to a surface, it is most important to choose a method of attachment that will prevent loss of enzyme activity by not changing the chemical nature or reactive groups in the binding site of the enzyme.  In other words, attach the enzyme but do as little damage as possible. It is desired to avoid reaction with the essential binding site group of the enzyme. Alternatively, an active site can be protected during attachment as long as the protective groups can be removed later on without loss of enzyme activity. In some cases, this protective function can be fulfilled by a substrate or a competitive inhibitor of the enzyme.

        The surface on which the enzyme is immobilized is responsible for retaining the structure in the enzyme through hydrogen bonding or the formation of electron transition complexes. These links will prevent vibration of the enzyme and thus increase thermal stability. The micro environment of surface and enzyme has a charged nature that can cause a shift in the optimum pH of the enzyme of up to 2 pH units.

Carrier-Binding : the binding of enzymes to water-insoluble carriers Cross-Linking : intermolecular cross-linking of enzymes by bi-functional or

multi-functional reagents. Entrapping : incorporating enzymes into the lattices of a semi-permeable gel

or enclosing the enzymes in a semi-permeable polymer membrane

CARRIER BINDING

        The carrier-binding method is the oldest immobilization technique for enzymes. In this method, the amount of enzyme bound to the carrier and the activity after immobilization depend on the nature of the carrier. The following picture shows how the enzyme is bound to the carrier:

        The selection of the carrier depends on the nature of the enzyme itself, as well as the:

Particle size Surface area Molar ratio of hydrophilic to hydrophobic groups Chemical composition

        In general, an increase in the ratio of hydrophilic groups and in the concentration of bound enzymes, results in a higher activity of the immobilized enzymes. According to the binding mode of the enzyme, the carrier-binding method can be further sub-classified into:

PHYSICAL ADSORPTION

IONIC BINDING

COVALENT BINDING

PHYSICAL ADSORPTION :- This method for the immobilization of an enzyme is based on the physical adsorption of enzyme protein on the surface of water-insoluble carriers. Hence, the method causes little or no conformational change of the enzyme or destruction of its active center. However, it has the disadvantage that the adsorbed enzyme may leak from the carrier during use due to a weak binding force between the enzyme and the carrier. The processes available for physical adsorption of enzymes are:

Static Procedure Electro-deposition Reactor Loading Process Mixing or Shaking Bath Loading

IONIC BINDING :- The ionic binding method relies on the ionic binding of the enzyme protein to water-insoluble carriers containing ion-exchange residues. Polysaccharides and synthetic polymers having ion-exchange centers are usually used as carriers. The binding of an enzyme to the carrier is easily carried out, and the conditions are much milder than those needed for the covalent binding method. Hence, the ionic binding method causes little changes in the conformation and the active site of the enzyme.  Therefore, this method yields immobilized enzymes with high activity in most cases.

COVALENT BINDING :- The covalent binding method is based on the binding of enzymes and water-insoluble carriers by covalent bonds. The functional groups that may take part in this binding are listed below:

                Amino group                Carboxyl group                    Sulfhydryl group,                 Hydroxyl group            Imidazole group                    Phenolic group                 Thiol group                  Threonine group                    Indole group

CROSS-LINKING

        Immobilization of enzymes has been achieved by intermolecular cross-linking of the protein, either to other protein molecules or to functional groups on an insoluble support matrix. Cross-linking an enzyme to itself is both expensive and insufficient, as some of the protein material will inevitably be acting mainly as a support. This will result in relatively low enzymatic activity. Generally, cross-linking is best used in conjunction with one of the other methods. It is used mostly as a means of stabilizing adsorbed enzymes and also for preventing leakage from polyacrylamide gels.          for example carbamy phosphokinase cross-linked to alkyl amine glass with glutaraldehyde lost only 16% of its activity after continuous use in a column at room temperature for fourteen days.           The most common reagent used for cross-linking is glutaraldehyde. Cross-linking reactions are carried out under relatively severe conditions. These harsh conditions can change the conformation of active center of the enzyme; and so may lead to significant loss of activity.

ENTRAPPING ENZYMES

        The entrapment method of immobilization is based on the localization of an enzyme within the lattice of a polymer matrix or membrane. It is done in such a way as to retain protein while allowing penetration of substrate. It can be classified into lattice and micro capsule types.

        This method differs from the covalent binding and cross linking in that the enzyme itself does not bind to the gel matrix or membrane.  This results in a wide applicability. The conditions used in the chemical polymerization reaction are relatively severe and result in the loss of enzyme activity. Therefore, careful selection of the most suitable conditions for the immobilization of various enzymes is required.

        Lattice-Type: entrapment involves entrapping enzymes within the interstitial spaces of a cross-linked water-insoluble polymer. Some synthetic polymers such as polyarylamide, polyvinylalcohol, etc. and natural polymer (starch) have been used to immobilize enzymes using this technique.

       Microcapsule-Type: entrapping involves enclosing the enzymes within semi permeable polymer membranes. The preparation of enzyme micro capsules requires extremely well-controlled conditions and the procedures for micro capsulation of enzymes can be classified as:

Interfacial Polymerization Method:   In this procedure, enzymes are enclosed in semi permeable membranes of polymers. An aqueous mixture of the enzyme and hydrophilic monomer are emulsified in a water-immiscible organic solvent. Then the same hydrophilic monomer is added to the organic solvent by stirring. Polymerization of the monomers then occurs at the interface between the aqueous and organic solvent phases in the emulsion. The result is that the enzyme in the aqueous phase is enclosed in a membrane of polymer.

Liquid Drying:   In this process, a polymer is dissolved in a water-immiscible organic solvent which has a boiling point lower than that of water. An aqueous solution of enzyme is dispersed in the organic phase to form a first emulsion of water-in-oil type. The first emulsion containing aqueous micro droplets is then dispersed in an aqueous phase containing protective colloidal substances such as gelatin, and surfactants, and a secondary emulsion is prepared. The organic solvent in then removed by warming in vacuum. A polymer membrane is thus produced to give enzyme micro capsules.

Phase Separation:   One purification method for polymers involves dissolving the polymer in an organic solvent and re-precipitating it.  This is accomplished by adding another organic solvent which is miscible with the first, but which does not dissolve the polymer.

The solid supports used for enzyme immobilization can be inorganic or organic . Some organic supports include: Polysaccharides, Proteins, Carbon, Polystyrenes, Polyacrylates, Maleic Anhydride based Copolymers, Polypeptides, Vinyl and Allyl Polymers, and Polyamides.

TYPE OF REACTORS

In an enzyme reactor, the highest specific enzyme activity is desirable.  It is considered an added bonus if the support that is used also aides in separation. One approach is to use a molecular sieve as the support and pulse the reactor bed with the alternating passage of substrate solution and water.  The result is that bands of unused substrate and product progress down the column. It so happens that the enzymes for which this technique would be useful are also those which in some cases benefit in having the enzyme immobilized on a porous support.         For an industrial reactor, it is preferable to use supports that are non-biodegradable such as glass, silica, Celite, Bentonite, alumina, or titanium oxide, if possible. Even the linkages between enzyme and support can be non-biodegradable, as they are in the case of titanium. In some of these supports the physical nature of the surface becomes a major problem. Thus, some supports that form excellent packed beds fail to do so when coated with enzyme. Particles which ideally self-suspend in a fluid bed may form aggregates during use which will require more power to pump through substrate. Many problems were encountered using porous glass supports until someone realized that the glass itself could dissolve. This problem has been eliminated by treatment of the glass surface with zirconium.  

Many types of reactors have been proposed including the following:

Batch reactors may include:

Stirred Tank for Soluble Enzymes Stirred Tank for Immobilized Enzymes Stirred Tank with Immobilized Enzyme Basket Paddles Stirred Tank with Immobilized Enzyme Basket Baffles Total Recycle Packed Bed Reactor Total Recycle Fluidized Bed Reactor

 

Continuous reactors may include:

Stirred Tank Reactor with Filtration Recovery Stirred Tank Reactor with Settling Tank Recovery Stirred Tank Reactor with Immobilized Enzyme Basket Paddles Stirred Tank Reactor with Ultra filtration Recovery PACKED BED REACTOR (same link as above) Packed bed with recycle Flat Bed Reactor Filter Bed Reactor FLUIDIZED BED REACTOR, SAME BUT BETTER DESIGN (EXPANDED TOP

SECTION) Membrane Reactor using hollow fibers

APPLICATION OF IMMOBILIZED ENZYME

Isomerization of glucose to fructose

D-glucose looks like:

D-fructose looks like:

The isomerization of glucose to fructose is part of the glycolysis cycle that converts glucose to pyruvate. The way this is done is to isomerize the aldehyde (hemiacetal) glucose to the ketone (as a hemiacetal) fructose,and make another phosphate ester. It is noted that this bond involves the carbon next to the carbonyl carbon of fructose. This cleavage would not have been possible without the isomerization of glucose to fructose, because the carbonyl group of glucose is too far from carbons three and four to make that bond breakable. 

Lactose hydrolysis An important application of immobilized enzymes

The main purpose of using immobilized enzymes here is to convert the disaccharide lactose via hydrolysis into its monosaccharide components, glucose and galactose. Lactose is a disaccharide that occurs naturally in both human and cow's milk. It is widely used in baking and in commercial infant-milk formulas.

One large problem with lactose is that many people are lactose intolerant - meaning that their body is incapable of digesting lactose. So it must be hydrolyzed into its monosaccharide components, allowing digestion which is the purpose of products today such as LACTAID.

Like cellobiose and maltose, lactose is a reducing sugar. It exhibits muta - rotation and is a 1,4'-beta-linked glycoside. Unlike cellobiose and maltose, however, lactose contains two different monosaccharide units. Acidic hydrolysis of lactose yields 1 equiv of D-glucose and 1 equiv of D-galactose; the two are joined by a beta-glycoside bond between C1 of galactose and C4 of glucose. In other words, 100 g of lactose will produce 50g each of galactose and glucose.

Lactose, a 1,4'-beta-glycoside[4-0 -(beta-D-Galactopyranosyl)-beta-D-glucopyranose]

Penicilline acaylase`

The natural penicillins are excellent antibiotics, their properties depend on the side chain. For

example, simply changing the side chain to the phenoxymethyl of Penicillin V confers much

better acid stability so that oral administration is not so badly beset by the low pH in the

stomach. In other words, Penicillin V has relatively good acid stability.

In the search for better penicillins, only those side chains accepted by the producing organisms

(usually Penicillium notatum) could be made. A great advance was using the enzyme penicillin

acylase to cleave off the side chain. New side chains could be attached, and many dozens of

new penicillin call semi-synthetic have been made and evaluated. Commercial manufacturing

relies on immobilizing the enzyme for economic reuse.

REFERENCES o Ichiro Chibata, “Immobilized enzyme” halsted press kodansha Ltd., 1978

o Bungay R. henry, “basic bio chemical engineering” Biline Associate, 1989

o Voet, Donald and Judith, “Biochemistry” John Wiley & Sons Inc. , 1995

o Mesing Ralph, “Immobilized enzyme for industrial reactors” , Academic Press, 1995

o Gaur R. , Pant H. , Jain R. , Khare S.K. , “Galacto oligosaccharide synthesis by immobilized

aspergillus oryzae β-galactosidase” food chemistry, 2006

o Rajendhran J. , Gunasekaran P. , “Application of cross-linked enzyme aggregates of Bacillus

badius penicillin G acylase for the production of 6-aminopenicillanic acid” Letters in applied

Microbiology,2007

o Honda T. , Miyazaki M. , Nakamura H. , Maeda H. ,”Immobilization of enzymes on micro

channel surface through cross-linking polymerization” AIChE spring national meeting,

conference proceedings, Orlando, FL, United States, april 2006

o Wilsion L. , Illanes A. , Pessela B.C.C. , Abian O. , Femandez-Lafuente R. , Guisan

J.M. ,”Encapsulation of cross linked penicillin G Acylase aggregates in Lentikats: Evaluation of

a novel Biocatalyst in organic media” Biotecnology and Bioengineering, 2004

o Turkiewicz M. , Makowski K. , “New methods for enzyme immobilization” Biotecnologia, 2004

o Cao L. , Van Langen L. , Sheldon R.A. , “Immobilized enzymes: carrier-bound or carrier-free”

Curropin Biotechnol, 2003

CONTENTS

INTRODUCTION

IMMOBILIZED ENZYME

METHODS OF IMMOBILIZATION

CARRIER BINDING

CROSS LINKING

ENTRAPPING ENZYME

TYPE OF REACTORS

APPLICATIONS OF IMMOBILIZED ENZYME

REFERENCES