Covalent application immobilization

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Instruments that are presently being used in the industries and other areas for instant and rapid analysis of different processes are scarce and often limited to ph and conductivity variations. The application of these instruments to biological processes is further narrowed by the interference of variable concentrations of compounds in the measurement. In most cases, precise and accurate analysis of biological processes is costly and requires more sophisticated instruments. They require previous purification that is too much time consuming and expensive making the whole proceeding really complicated.

Fortunately in living organisms biological component like enzymes function as natural sensing and controlling devises. Enzymes extensive commercial availability in pure form and its quick isolation and purifying techniques has allowed their incorporation with physiochemical transduction devices to produce biosensors. [1] [2]


Biosensor is "a self-contained analytical device that incorporates biologically active material in intimate contact with an appropriate transduction element for the purpose of detecting the concentration or activity of chemical species in any type of sample". [3]


A biosensor has two Basic types of components:

  1. Biological
  2. Physical

Biological component first recognizes the specific analyte and interact with it in such a manner that it produces some kind of physical change detectable by the transducer. In this way a biosensor converts the chemical flow into electrical flow that can be measured. [4]

Main components of a biosensor

Schematic diagram showing the main components of a biosensor. The biocatalyst (a) converts the substrate to product. This reaction is determined by the transducer (b) which converts it to an electrical signal. The output from the transducer is amplified (c), processed (d) and displayed (e). [5]


Calorimetric biosensors Measures the heat released and absorbed in a reaction

Potentiometric biosensors Measures the production of an electrical potential due to changed distribution of electrons

Amperometric biosensors Measures movement of electrons due to redox reaction

Optical biosensors Measures light produced or absorbed during the reaction

Acoustic wave biosensors Measures change in mass of the biological component as a result of the reaction [6]



Biosensors can effectively be used to monitor the glucose levels in diabetic patients

  • Environmental applications e.g. the detection of pesticides and river water contaminants
  • Remote sensing of airborne bacteria e.g. in counter-bioterrorist activities
  • Determining levels of toxic substances before and after bioremediation
  • Detection and determining of organophosphate
  • Drug discovery and evaluation of biological activity of new compounds.[7]
  • Detection of pathogens.[8]
  • Detection of toxic metabolites such as mycotoxins [9]


In the food and beverage industries selective and reliable monitoring of various compounds is of increasing importance to assure good quality and possible traces of contaminants. Most of the classical analytical methods that are presently being used are expensive, time consuming, require previous separation, expensive materials, and well trained operators.

Amperometric enzyme based biosensors has emerged in the last decade as a useful measuring tool having promising application possibilities in food and beverage industry.

Amperometric biosensors are in general highly specific, relatively cheap, and easy to integrate in continuous analysis systems. [10]


Enzyme serves to catalyze different type of reactions. Enzymes display great specificity and is 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. If the enzymes are present with reactant or product in a same solution it is very difficult to separate it.

Therefore it is preferable to attach them to any support from where they can be used again and again for many rounds. Immobilized enzyme is an enzyme that is physically attached to a solid support over which a substrate is passed and converted to product.

Advantageous Features of Immobilization:

  1. A single batch of enzymes can be used multiple times, which ensures that the same catalytic activity is present for a series of analyses.
  2. Reaction can be stopped rapidly by removing the immobilized enzyme from the reaction solution.
  3. Immobilization stabilizes the enzyme
  4. Product is not contaminated with the enzyme(especially useful in the food and pharmaceutical industries) [11]
  5. The immobilized enzymes undergo inactivation with a period of time at steady and constant rate that is easily predictable.
  6. Immobilized enzyme systems are re-usable up to 10,000 times over a period of several months. It clearly shows that immobilized systems do considerable saving in terms of the enzymes' cost relative to the analytical usage of free soluble enzymes. [12]

The Economical aspect of immobilization

One of the important factors that must be considered before using any techniques is the expected cost of materials or the whole process. Many enzymes are commercially available at prices of about 1 mg-1, although some are much cheaper and many are much more expensive. These enzymes function as catalytic entities in a given reaction therefore they are not directly used up in a process. However with the passage of time they lose their activity due to denaturation. If these enzymes are stabilized against denaturation they could be used in an effective way.

When these enzymes are used in soluble form, after a certain reaction they retain some activity which can not be recovered and is thus wasted.This activity residue remains to contaminate the product and its removal may involve extra purification costs. . Simple and economic methods must be used which enable the separation of the enzyme from the reaction product In order to overcome this wastage and enhance productivity. The most economical way of achieving this goal is the usage of two phase system i.e. 1 phase having enzyme and other phase having product. Reactants are able to move freely between the two phases. This can be achieved by immobilizing enzyme on some solid support.

The easy separation of the enzyme from the products minimizes downstream processing costs and possible effluent handling problems, particularly if the enzyme is noticeably toxic or antigenic.It also allows continuous processes to be practicable, with a considerable saving in enzyme, labor and overhead costs. In this way immobilization of enzyme is more economical than the freely soluble enzyme system.[13]


Two things that must be considered while using enzymes in biosensors are:

  1. Operational stability
  2. Long term use

Both of these factors are dependent on the use of immobilization strategy. Therefore the selection of immobilization technique is very important and critical step. Following are the methods or enzyme immobilization:-




  • Gentle;
  • No direct chemical modification;
  • Specificity and analyte interaction retained
  • Gentle treatment of enzyme
  • No modification of enzyme
  • Matrix can be regenerated
  • Used in conjugation with entrapment to reduce loss of enzyme
  • Low diffusion resistance
  • Strong binding forces between enzyme and matrix
  • Resistance to adverse condition of pH, toxic strength


  • High diffusion barrier
  • Only good for small analysis
  • Continuous loss of enzyme
  • Very weak bonds
  • Susceptible to changes in pH, temp, ionic strength
  • Matrix not regenerable
  • May involve toxic chemicals [14]


Covalent immobilization is a chemical method of immobilizing enzymes on solid support matrix. Among the 4 available techniques of immobilization it is the most efficient and advantageous one. It's also very suitable from economical as well as technical point of view. It allows easy and frequent replacement of enzyme membrane which extends the life and versatility of the biosensors. [15]

Covalent bonding

In covalent immobilization technique a covalent bond is formed between the chemical groups of enzyme and the surface of the carrier support. Covalent bonding can occur by four different mechanisms. They are;


Amino group of the support (like amino benzyl, cellulose, aminosilanized porous glass, amino derivatives) and a tyrosyl or histydyl group of the enzyme are engaged in bonding.

Peptide bond formation

A covalent bond formed between amino or carboxyl group of the support and amino or carboxyl group of the enzyme.

Group activation

Cyanogen bromide is attached to a support containing glycol group, e.g. cellulose, sephadex, sepharose, etc.

Polyfunctional reagents

Bifunctional or polyfunctional molecules like glutaraldehyde which can bond to the amino group of the support and also to the amino group of the enzyme. [16]


There are six major classes of enzymes: Oxidoreductases, Transferases, Hydrolases, Lyases, Isomerases, and Ligases. [17] "This enzyme belongs to the family of oxidoreductases, specifically those acting on the CH-OH group of donor with oxygen as acceptor. The systematic name of this enzyme class is alcohol: oxygen oxidoreductase. This enzyme is also called ethanol oxidase." [18]

"Alcohol oxidase is an oligomeric enzyme with eight identical subunits, each containing a non-covalently bound flavin adenine dinucleotide molecule (FAD) as a cofactor."[19] [20]. During the reaction the reaction FAD is first reduced to its hydrogenated form and re-oxidized to its native form by molecular oxygen resulting in the formation of hydrogen per oxide.[21] Alcohol oxidase is involved in the methanol oxidation pathway of methylotrophic yeasts and is also able to oxidize alcohols other than methanol. [22]


Alcohol oxidase catalyses the following reaction

A primary alcohol + O2 \rightleftharpoonsan aldehyde + H2O2

Thus, the two substrates of this enzyme are primary alcohol and O2, whereas its two products are aldehyde and H2O2. [23

Alcohol oxidase catalyses the oxidation of primary low molecular weight alcohols with oxygen as electron acceptor. This enzyme can be therefore used to do alcohol assays by using biosensors. [24]


"Alcohol oxidase is produces by methylotrophic yeasts (e.g. Hansenula, Pichia, Candida) located and assembled in peroxisomes." [25]


  • Generation of hydrogen per oxide in liquid detergent systems is very useful in Laundry industry.
  • Immobilized alcohol oxidase can be used for the oxidation of alcohols or in the absence of catalase activity for the production of hydrogen per oxide.
  • It can be used for the production of formaldehyde from methanol.[26]
  • Monitoring of ethanol in beverages and fermentation industries, clinical chemistry and forensic analysis.


To determine the alcohol content in beers, wines, saliva, blood and urine Alcohol biosensors are extensively being used in many industries. Functioning of these biosensors can be further enhanced by the addition of stabilizers.

Protein Stabilisation Technology

Applied Enzyme Technology Ltd. has now launched stabilizer molecules which give further stability to proteins and more versatility for the wider application of stabiliser formulations in industry. [27] The examples of these stabiliser molecules are polyalcohol, polyelectrolyte, etc.


The addition of stabilisers:

  • Prevents inactivation during dehydration
  • Maintains high levels of enzyme activity for extended periods of time in the dry state at room temperature and almost indefinitely ay 4°C
  • Results in increased accuracy and reliability of sensor readings [28]

Long term Dry Stability[29]

By analyzing these graphs it is quiet evident that the use of the stabilizer molecules makes the enzyme more stable. There is a remarkable difference in the enzymatic activity with and without using stabilizer molecules. Hence the addition of stabilizer molecules ensures better performance of enzymes.

Long Term Shelf Stability of Alcohol Biosensors

While manufacturing any commercial product its shelf life is a key factor in terms of its success. Prolonged shelf life increases the demand of product in market and hence makes it more valuable. By the addition of these stabilizer molecules enhanced shelf life of alcohol biosensors have been observed.

When the enzymatic activity of two trains of yeast i.e. Hansenula and Pichia was compared with or without using stabilizers it showed that the stability of these strains increases with the addition of stabilizers and Hansenula showed even more stability than Pichia.

Comparative Stability of Hansenula and Pichia alcohol oxidases at 37°C. (Dry Preparations)


For clinical and forensic purpose the quantitative measurement of alcohol is very important for the analyses of human breath and blood. On the other hand food and beverage industries are very much interested in rapid analytical method to control product quality. The determination of alcohol especially ethanol has importance in Environmental and Agricultural areas. [30]

Over the years many analytical methods have been developed for the determination of alcohol contents. They include redox titration, colorimetric methods, specific gravity, chromatography, refractive index measurement, spectroscopic methods. Although some of these methods are precise and reliable but they are complex, time consuming, require expensive instruments and conditions.[31]


The thermal stability of multimeric alcohol oxidase from three different sources i.e. Candida boidinii, Hansenula sp. and Pichia pastoria was previously evaluated. Enzyme from Hensenula sp. was the most stable amongst the three. When this enzyme obtained from Hensenula sp. was immobilized using various immobilization methodologies the best results in terms of stability were obtained when the enzyme was covalently immobilized. [32] Hence alcohol oxidase for the biosensor application was selected by Hensenula polymorpha source.

Characteristics of Hansenula polymorpha

  • Rapid growth at the expense of methanol as the sole carbon source and energy
    1. Remarkable heat tolerant permitting growth at temp up to 49 degree centigrade.
  • Easy interconversion between haploid and diploid state. [33]


For enzyme immobilization various supports have been used. The support that was previously used for the immobilization of alcohol oxidase was of organic nature. [34]

Inorganic materials like silica beads, alumina, controlled pore glass, and zeolite have been used as enzyme supports, in biosensors. [35]


The usage of inorganic support has some advantages over organic supports in enzyme immobilization.

  • They are mechanically stable
  • They are thermally resistant
  • They are chemically inert
  • They are non toxic
  • They do not swell during reaction
  • Resistant towards pH changes
  • Resistant to microbial attack [36-38]

The above mentioned features clearly shows that the use of inorganic support for enzyme immobilization will surely be a better choice as compare to organic support. Therefore the previously used organic support is replaced by more useful inorganic support.

Perlite is an amorphous porous aluminium silicate with a high content of silica of more than 70 %. Commercially, the term perlite is used to describe either natural or expended perlite which is formed by quickly heating. [39] When compared to other inorganic supports, perlite is inexpensive [40] & because of its hydrophilic character, provides a desirable microenvironment to decrease resistance to mass transfer. [41]

In the present study, alcohol oxidase from Hansenula polymorpha was induces, purified and immobilized by using the most efficient and relatively cheaper technique of covalent immobilization. The enzyme was immobilized on readily available and inexpensive inorganic perlite support. The stability of enzyme and shelf life of alcohol biosensor was further increased by using stabilizer molecules. The biochemical properties of immobilized enzyme were compared with those of free enzyme.