1. Adsorption
2. Microencapsulation
3. Ionic Exchange
4. Cross Linking
5. Adsorption and Cross Linking
6. Problems When Using Immobilized Enzymes
7. Characteristics

  • pH profile
  • Kinetic
  • Thermal stability
  • Reactor for immobilized enzymes

    • 8. Practical Uses of Immobilized Enzymes

  • Enzymatic Electrodes
  • Glass electrodes
  • Electrodes made of liquid membrane
  • Electrodes made of crystal membrane



    • 1. Adsorption
    Some enzymes have been adsorbed by organic polymers, glass, mineral salts, metallic oxides andseveral silica compounds.


    2. Microencapsulation
    The encapsulation of enzymes within membranesmade of polymers avoid the lost of the enzymes and permits quantities ofsubstrates to reach the enzyme.


    3. Ionic Exchange
    It is a simple and inexpensive method. The first time this method was used comercially, the system used acilase to segregate a mixture of aminoacids.


    4. Cross Linking
    Generally, the product is in gel state andit is difficult to control.


    5. Adsorption and Cross Linking
    This immobilization method increases the stability of the enzymes a lot more than using one of them.


    6. Problems When Using Immobilized Enzymes
    The polymers may have undesirable physical characteristics. Their susceptibility to being attached by enzymes or microorganisms is a serious problem because a lot of them are hydrolized by microbial enzymes and can act like substrates which are used for the growth of other microorganisms.

    Most polymers are susceptible to solvents and the pH. The sites change configuration when the pH and the salt concentration or the solvents change. This affects the enzyme activity.

    The stability of a lot of enzymes may change when immobilized. When immobilized, some enzyme derivatives will increase in stability while others'stabilities will decrease. This is what determines the comercial value of an immobilized enzyme.

    The size of the site is very important because it is to be optimized for the system of reactors chosen.

    Small particles should not be used in big columns unless the diffusion of the substrate requires it.

    Big particles should not be used in batch reactors. This is because they are too large to fluidize constantly, and this will increase limitations on total diffusion.


    7. Characteristics

  • pH profile


    • Every enzyme has an optimum pH that changes when the enzyme is immobilized. This change is produced by the unequal ionic distribution.


  • Kinetics


    • See Michaelis-Menten It has been found that KM decreases when the substrate rate increases, that explains how the turbulence increases and the static layer of the liquid surrounding each particle decreases, which reduces the effect of mass transport.


  • Thermal Stability


    • Enzymes are susceptible to thermal degradation.
    If the temperature is increased, enzymes will begin to inactivate themselves when a certain temperature is reached. In a lot of cases, immobilized enzymes present an increase in their thermal stability so higher temperatures can be applied. Until now, it is not possible to predict what enzymes will show an increase or decrease in their thermal stability when immobilized.


  • Reactors for immobilized enzymes
    •  The kinetic behavior and mass transport of the reactor determine the enzymatic process and its operation.

    The use of paper and nylon: Enzymes have attached to layers of paper that are covered by a nylon net and rolled into a cylinder to conform a tubular reactor.

    The use of a tubular membrane for the hydrolysis of "almidon" with alpha-amilase. The enzyme and the "almidon" are retained by the membrane. The membrane is permeable to the product, maltose.
    Enzymes can be immobilized with magnetic particles that can be recovered with actual technology.

    All reactors used in comercial processes have fixed beds. An example is the japanese system DEAE-Sefadex-L-aminoacidacilase for the resolution of D-L aminoacids mixtures. The low quantity of enzyme per unity of reactor volume require big reactors of high envestment and expensive.

    The versatility and efficiency of a reactor with a fluidized bed are attributes that may expand their use. This type of reactor avoids any clogs, especially when the feed contains fine material in suspension. Small immobilized particles can cause inacceptable pressure drops in a reactor with a fixed bed, but in a reactor with a fluidized bed, it helps to decrease internal diffusional resistance.


    8. Practical Uses of Immobilized Enzymes

    Enzymatic Electrodes

    They have been used in laboratories to measure urea and glucose in the blood. The following enzymatic electrodes use a selective ionic membrane like a sensitive potentiometer:

    Substrate measured: Urea
    Sample: Plasma,blood,urine
    Enzyme: Urease
    Membrane: Glass

    Substrate measured: Glucose
    Sample: whey, plasma
    Enzyme: Glucose-oxidase
    Membrane: Glass

    Substrate measured: Phenylanine
    Sample: standard aqueous samples
    Enzyme: Aminoacid-oxidase

    Sample: drugs, fermentation broth
    Enzyme: Penicilinase
    Membrane: Glass


    • Glass electrodes

      • These are covered by a thin and rough membrane of silicate. For example, a mixture of Na2O-Al2O3-SiO2 is the active material of the sensor. The glass electrodes react to cations such as H+, Na+, K+, Li+, NH4+, Ag+, Cu+, Tl+, and very little to others. This selectivity is accomplished by changing the glass composition. There is no complete ion transport through the membrane during its normal use. Please see a Metrohm page for ion selective electrode.


    • Electrodes made of liquid membrane


      • The active liquid phase is retained by aninert membrane that acts as a glass support or porous plastic. Electrodes of this type have been prepared for Ca++, NO3-, CO3--, Cu++, Mg++, Mn++, Cl- and other organic radicals. The selectivity depends on the nature of theactive liquid phase. For this purpose, ion exchangers have been used for organic materials in compatible and natural solvents or synthetic neutral supports such as antibiotics.

    • Electrodes made of crystal membrane


      • The membrane is made of ionic materials of low solubility in water that is used as a sensor for the electrode.


      These three types of electrodes measure the ionic activity. and obey the Nernst equation:

      Fe = constant + R*T* ln(aM) / (Nui*F)



      Where:

      Fe = is the measured electromotive force
      R = is the universal gas constant
      T = is the absolute temperature
      F = is the Farday's constant
      NUI = is the ionic charge
      aM = is the ionic activity to be measured

      For an ionic mixture:

      Fe = constant + R*T* ln[(aM1++)*(K*aM2+)] / (Nui*F)


      K is the selectivity factor.

      (It is a quantitive measure of the electrode's capacity to choose ions, electrodes made of membranes have k values in the order of a million.)

    I hope this information has been useful to you and found it interesting. Thanks for your time.