Enzyme immobilization techniques

              Enzyme immobilization techniques 

What is enzyme immobilization?

• imprisonment of cell or enzyme in a distinct support(matrix).
• The support/matrix allows change of medium.
• The medium contain substrate or inhibitor molecule.
• First immobilization technology : amino acylases by aspergillus oryzae for the production of L-amino acid japan.
• The main advantages of enzyme immobilization:    

       1.increase functional efficiency 

       2. enhanced reproducibility 

  Advantages-

• Reuse
• Continuous use 
• minimum reaction time 
• less chance of contamination 
• more stability 
• improve in process control 
• high enzyme substrate ratio

Disadvantages-

• loss of catalytic properties in sometime 
• some enzyme become unstable 
• some enzyme are inactivated by heat generation in system 
• high cost for isolation, purification and recovery of active enzyme 

Application-

• Industrial production: Eg. Antibiotics, beverage ,amino acid etc 
• Biomedical application: treatment, diagnosis and drug delivery .
• Food industry: Production of jams, jellies, and syrup.
• Research: blotting experiment, proteases for cell lysis 
• Waste water treatment: treatment of sewage & industrial effluents

    Support/matrix-

• The enzyme hold in support/matrix 
• The should be cheap and easy available 
• Their reaction with medium and enzyme should be minimums as possible 
• A wide range of matrix are used in immobilization of enzyme/ cell 
• Matrix categories into 3 grouped 

1.  Natural polymers
2. Synthetic polymer 
3. Inorganic polymer 

Types of immobilization-

1. Adsorption 
2. Covalent bonding 
3. Entrapment 
4. Copolymerization 
5. Encapsulation 

       (1).  Adsorption-

• Oldest method of enzyme immobilization 
• Simplest method of enzyme immobilization 
• Nelson and Griffin used charcoal to adsorb invertase 
• Enzyme are adsorbed to external surface of support 
• Support/ carrier may be ;

1. Mineral support (aluminum oxide, clay)
2. Organic support (starch)
3. Modified sephareose and ion exchange  resins               

     

Methods of adsorption ;

1.static process-

immobilization to be perform by the using solution which containing enzyme to contact with the carrier (without stirring)

2.dynamic batch process-

carrier is placed in a enzyme solution and mixed by using agitation 

3.Reactor loading process-

when enzyme solution is transfer to reactor the carrier will already present in the reactor 

4.Electrode position process-

 carrier is placed proximal to an electrode in enzyme bath and the current is put on , the enzyme migrate to the carrier and deposition on the surface 

Advantages-

• Easy to carry out 
• no reagent are required 
• minimum activation step is involve 
• comparatively cheap method 
• less disruptive to protein than chemical method 

Disadvantages-

• desoption of enzyme from the carrier 
• efficiency is less 

2.Covalent bonding;

• In this process involve the formation of covalent bond between the enzyme and support/ matrix
• widely used method for enzyme immobilization 
• chemical group in enzyme that form covalent bonds with support are;

1. Amino acid 
2. Hydroxyl group 
3. carboxyl group 
4. Thiol group and methyl group 
5. Guanidyl group and imidazole group 
6. phenol rings 

Matrix used for covalent bonding-

• Carbohydrates -cellulose, DEDE cellulose , agarose 
• synthetic agent - polyacrylamide 
• protein carrier 
• amino group bearing carrier -amino benzyl cellulose 
• inorganic carrier- porous glass , silica 
• cyanogen bromide (CNBr) - agarose and CNBr sepharose 
• hydroxyl and amino group form covalent bond more easily 

Methods of covalent bonding 

1. Diazoation ;bonding between amino groups of support and thyrosil or histidyl groups of enzyme 

2. Peptide bond;bonding between carboxyl /amino groups of support and enzyme 

3. Poly functional reagent ;use of bi-functional or multifunctional reagent (glutaradehyde) which form bonding between the amino group of the support and amino group of the enzyme

3. Entrapment-

• Enzyme are physically entrapped inside the support 
• Bonds are involve in entrapment such as covalent and non-covalent bond
• matrix used will be water soluble polymer like-

1. polyacrylamide gels 
2. cellulose triacetate 
3. agar 
4. gelatin
5. alginate 

• Form and nature of matrix varies 
• pore size of matrix is adjusted to prevent loss of enzyme 
• agar have larger pore sizes 
• easy to practice at small scale 

Method of entrapment-

1.Inclusion in the gels- enzyme are trapped in gels 

2.Inclusion of fibers- enzyme support in fiber format

3.Inclusion in microcapsules - enzyme are trapped in microcapsules formed by the using monomer mixture such as polyamine, calcium alginate .

Advantages-

• This is fast method and cheap compare to another method of immobilization 
• Mild condition are required 
• No conformation change in this method 

Disadvantages-

• chance of microbial contamination 
• leakage of enzyme 

4. Cross linking or co-polymerization ;

• In this method involve covalent bonding between various groups of enzyme via poly-functional reagent 
• there are not involve matrix or support
• Commonly used poly-functional reagents are -Glutaradehyde , Diazonium salt 

 5. Encapsulation 

• enzyme are enclosed in semi-permeable membrane capsule 
• capsule is made up of nitro cellulose or nylon 
• effectiveness depends on the stability of enzymes 

Advantages-

• Simple and cheap method , large quantity of enzyme can immobilized by the using this method 

Disadvantages-

• Pore size is limitation 
• only small size of substrate is able to pass through the membrane 

Enzymes

                                              Introduction-

• Enzymes are work as a biological catalysts that increase the rate of reaction(biochemical reaction). 
• Mainly enzymes are in 3D structure globular proteins(tertiary and quaternary structure)
• Enzyme are macromolecular biological catalysts, enzyme accelerate or catalyze chemical reaction.
• During the reaction substrate bind with the enzyme at active site of enzyme , converts into different molecule called products.
• microbial enzyme are the biological catalysts for the biolchemical reaction leading to microbial growth and respiration ,as well as formation of fermentation products. 

Structure of enzyme-

  The active site of an enzyme is a region that binds substrates ,co-factors and prosthetic groups. Active site is less than 5% of the total surface area of enzyme.active site have tertiary structure of proteins.

active site further divided into two types 

Binding site- it choose the substrate and binds to active sites 

Catalytic site- it performs the catalytic action of enzyme 

Characteristics;

• Enzyme increase the rate of reaction 
• The presence of enzyme does not effects the nature and properties of end products
• Small amount of enzyme accelerate the chemical reaction 
• Enzyme are sensitive to change in pH, temp, and substrate concentration\.

Culture medium or Growth medium

                                              Definition  

A liquid or solid preparation made for the growth, storage, or transport of microorganisms or other types of cells such as animal or plant cells. 

A growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms or cells, or small plants like the moss Physcomitrella  patens.

History-

• Robert Koch (1843-1910) could be considered the father of culture media.

• His first success in bacteriology was in the isolation of Bacillus anthracis which at the time was causing the disease anthrax in cattle.
• This was the first time that any pathogenic organism had been isolated and studied outside of the host's body.
• It was after this discovery that Koch wrote his famous postulates regarding the causative agents of infectious disease, which were published in 1882:
1. The organism must be consistently associated with the disease.
2. It must be isolated and grown in pure culture.
3. The pure culture must be shown to induce the disease when injected into experimental animals.
• Although Louis Pastuer (1822-1895) and Koch are both considered to be responsible for the establishment of the science of microbiology, Koch and his coworkers systematically identified all the agents of the major bacterial infectious diseases of the 19th century.

Requirements-

 A microbiological culture medium must contain available sources of:
• Carbon
• Nitrogen
• Inorganic phosphate and sulfur
• Trace metals
• Water
• Vitamins

Cultivation of microorganisms depends on a number of important factors:

 Proper nutrients must be available.

 Oxygen or other gases must be available, as required.
 Moisture is necessary.
 The medium must have an appropriate pH.
 Proper temperature relations must prevail.
 The medium must be free of interfering bioburden.
 Contamination must be prevented.

CONSTITUENTS	SOURCE
Amino-Nitrogen	Peptone, protein hydrolysate, infusions and extracts
Growth Factors	Blood, serum, yeast extract or vitamins, NAD (Nicotinamide adenine dinucleotide)
Energy Sources	Sugar, alcohols and carbohydrates
Buffer Salts	Mineral Salts and Metals
Mineral Salts and Metals	Phosphate, sulfate, magnesium, calcium, iron
Chemicals, antimicrobials and dyes	Chemicals, antimicrobials and dyes
Indicator Dyes	Phenol red, neutral red
Gelling agents	Agar, gelatin, alginate, silica gel

Media ingredients -

PEPTONE, PROTEIN HYDROLYSATES, INFUSIONS

AND EXTRACTS –

                                    Are the major sources of nitrogen and vitamins in culture media. Peptones are water-soluble ingredients derived from proteins by hydrolysis or digestion of the source material; e.g., meat, milk.

CARBOHYDRATES-are employed in culture media as energy sources and may be used for differentiating genera and identifying species.

BUFFERS- maintain the pH of culture media.

SELECTIVE AGENTS-include bile salts, dyes and antimicrobial agents.

• Bile salts and desoxycholate are selective for the isolation of gram-negative microorganisms,               inhibiting gram positive cocci.

DYES AND INDICATORS -are essential in the preparation of differential and selective culture           media.

• In these formulations, dyes act as bacteriostatic agents or indicators of changes in acidity or                alkalinity of the substrate.

ANTIMICROBIAL AGENTS are used in media to inhibit the growth of bacteria, yeasts and fungi.

SOLIDIFYING AGENTS, including agar, gelatin and albumin, can be added to a liquid medium in    order to change the consistency to a solid or semisolid state.

Downstream processing in fermentation

                                   Downstream process 

  • The various stages of downstream  processing that occur after the completion of the fermentation,          including separation, purification, and packaging of the product.

Stages;

 Removal of Insoluble

 Product Isolation 

 Product Purification 

 Product Polishing 

 A few product recovery methods may be considered to combine two or more stages. For example,      expanded bed adsorption accomplishes removal of insoluble and product isolation in a single             step.  Affinity chromatography often isolates and purifies in a single step.

•   Removal of insoluble 

• Separation of cells, cell debris or other particulate matter 

• Typical operations to achieve this:

 1) Filtration

 2) Centrifugation 

3) Sedimentation 

4) Flocculation a process where a solute comes out of solution in the form of floc or flakes.

5) Gravity settling

1. Filtration -

• It is a type of mechanical operation used for the separation of solids from fluids (liquids or gases)         by a porous membrane through which the fluid can pass, but the solids in the fluid are hold in the       porous membrane.  

• The solid particles deposited on the filter form a layer, which is known as filter cake. 

• All the solid particles from the feed are stopped by the cake ,and the cake grows at the rate at which     particles are bought to its surface. 

• All of the fluid goes through the cake and filter medium.

2.Centrifugation-

• Centrifugation is a technique used to separate the solid  particles (100 – 0.1 micrometer) from         liquid by using  centrifugal forces. 

• It depends on particles size,density difference between the cells and the broth and broth viscosity.

• Use of the centrifugal force for the separation of particles from the  mixtures of component. 

• More-dense components migrate away from the axis of the centrifuge(ruter).

• Less-dense components  migrate towards the axis. 

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3.Chromatography-

• Separation of mixtures on the basis of their colour.

• Passing a mixture dissolved in a "mobile phase" through a stationary phase, which separates the            analyte to be measured from other molecules in the mixture and allows it to be isolated.

Types of chromatography 

1. Ion-exchange chromatography 
2. Affinity chromatography 
3. HPLC chromatography 
4. Paper chromatography 
5. TLC chromatography 
6. Size exclusion chromatography 

4.Ultrafiltration-

• UF is governed by a screening principle and dependent on particle size.

• UF membranes have a pore size between 1 nm and 100 nm (10 and 2000 Å), thus allowing                   retention of compounds with a molecular weight of 300 to 500 000 Dalton. 

• Typically, the process is suitable for retaining biomolecules, bacteria, viruses, polymers, colloidal particles and sugar molecules

5.Flocculation-

• Process where a solute comes out of solution in the form of flocs or flakes. 

• Particles finer than 0.1 µm in water remain continuously in motion due to electrostatic charge              which  causes them to repel each other.

• Once their electrostatic charge is neutralized (use of coagulant) the finer particles start to collide         and combine together . 

• These larger and heavier particles are called flocs.

Product isolation-

• Removal of those components whose properties vary markedly from that of the desired product. 

• Water is the chief impurity 

a) Isolation steps are designed to remove it (i.e.dialysis) 

b) Reducing the volume 

c) Concentrating the product. 

d) Liquid –liquid extraction, adsorption, ultrafiltration, and precipitation are some of the unit operations involved.

Upstream & Downstream processing in fermentation

      UPSTREAM &DOWNSTREAM  PROCESSING 

1. Upstream processing ;The upstream processing in bio-process or fermentation  involves identifying and extracting the raw materials. This forms the initial process of fermentation.

     upstream process- following steps 

     • Inoculate preparation which includes screening or microorganisms and selection          of suitable strain and genetic modification of the organism if needed

    • Preparation of culture media having suitable growth parameters at laboratory          

     • Scale up of the entire process. 

•  Inoculation

2.Downstream processing ; when products are subjected to forms in the bioreactor including separation, purification it is also known as downstream processing 

Downstream processing -following steps 

• Solid-liquid separation • Release of intracellular  products 

• Concentration • Purification • Formulation

Upstream & downstream process of 

• Antibiotics 
• Hormones 
• Vaccines 

Antibiotics;It is a chemical drugs used to treat bacterial infections. 

A medicine (such as penicillin or its derivatives) that inhibits the growth of or               destroys microorganisms. 

Originally, an antibiotic was a substance produced by one microorganism that                selectively inhibits the growth of another.  

Example : penicillin, streptomycin

Hormones;A chemical substance produced in the body that controls and regulates the activity of certain cells or organs.

 it act like messenger molecules in the body. 

It is carried out by blood.

Hormones are essential for every activity of life, including the processes of digestion, metabolism, growth, reproduction, and mood control. 

Example: thyroid hormone,insulin.

Vaccines-A vaccine is a biological preparation that improves immunity to a particular disease.

a preparation of killed microorganisms, living attenuated organisms, or living fully virulent organisms that is administered to produce or artificially increase immunity to a particular diseases. 

 Example: Diphtheria vaccine, Polio vaccine, 

• Upstream process

• Fermentation 
• Downstream process