A Critical Study On Enzymes

A Critical Study On Enzymes

Chapter One

Introduction And Literature Review

1.1 Enzyme

Enzymes are large biological molecules responsible for thousands of chemical inter-conversions that sustain life (Smith, 1997). All known enzymes are proteins. They are high molecular weight compounds made up principally of chains of amino acids linked together by peptide bonds, they are denatured at high temperature and precipitated with salts, solvents and other reagents. They have molecular weights ranging from 10,000 to 2,000,000 units. Enzymes do not cause reactions to take place, but rather they enhance the rate of reactions that would have been slower without their presence and still remains unused and unchanged.

Many enzymes require the presence of other compounds – cofactors – before their catalytic activity can be exerted. This entire active complex is referred to as the holoenzyme; i.e. apoenzyme (protein portion) plus the cofactor (coenzyme, prosthetic group or metal-ionactivator) is called the holoenzyme (Alexopoulos et al., 1996)

The living cell is the site of tremendous biochemical activity called metabolism. It is the process of chemical and physical change which goes on continually in the living organism involving the build-up of new tissues, replacement of old tissue, conversion of food to energy, disposal of waste materials, reproduction – all the activities that we characterize as “life.”Thephenomenon of enzyme catalysis makes possible biochemical reactions necessary for all life processes. Catalysis is defined as the acceleration of a chemical reaction by some substance which itself undergoes no permanent chemical change. Synthetic molecules called artificial enzymes also display enzyme like catalysis (Grovesm, 1997).

The catalysts of biochemical reactions are enzymes and are responsible for bringing about almost all of the chemical reactions in living organisms. Without enzymes, these reactions take place at a rate far too slow for the pace of metabolism(Bairoch, 2000).

Enzymes actually work by lowering the activation energy of a reaction. This is achieved when it creates an alternative pathway which is faster for the reaction hence speeding it up such that products are formed faster. Enzyme catalysed reactions are million times faster than uncatalysed reactions, they alter the rates but not the equilibrium constant of the reaction being catalysed (Ashokkumar et al., 2001). A few RNA molecules called ribozymes also catalyse reactions, with an important example being some parts of ribosome (Lilley, 2005).

1.1.1 Types of enzymes

Metabolic enzymes: These have been called the spark of life, the energy of life and the vitality of life. These descriptions are not without merit. Metabolic enzymes catalyse and regulate every biochemical reaction that occurs within the human body, making them essential to cellular function and health (Sangeethaet al.,2005). Digestive enzymes turn the food we eat into energy and unlock this energy for use in the body. Our bodies naturally produce both digestive and metabolic enzymes as they are needed. They either speed up or slow down the chemical reactions within the cells for detoxification and energy production. The enable us to see, hear, and move and think. Every organ, every tissue and all 100 trillion cells in our body depend upon the reaction of metabolicenzymes and enjoy their energy factor. Without these metabolic enzymes, cellular life would beimpossible.

Food enzymes:These are introduced to the body through the raw foods we eat and throughconsumption of supplemental enzyme products. Raw foods naturally contain enzymes providing asource of digestive enzymes when ingested(Hossainet al., 1984). However, raw food manifests only enough enzymesto digest that particular food, not enough to be stored in the body for later use (the exceptionsbeing pineapple and papaya, the sources of the enzymes bromelain and papain). The cooking andprocessing of food destroys all of its enzymes. Since most of the foods we eat are cooked orprocessed in some way and since the raw foods we do eat contain only enough enzymes toprocess that particular food (Persike et al., 2002) our bodies must produce the majority of the digestive enzymes werequire, unless we use supplemental enzymes to aid in the digestive process. A variety ofsupplemental enzymes are available through different sources. It is important to understand thedifferences between the enzyme types and ensure that one is using an enzyme product which willmeet one’s particular needs.

Plant based enzymes:These are the most popular choice of enzymes. They are grown in a laboratorysetting and extracted from Aspergillus species. The enzymes harvested from Aspergillusspecies are called plantbased, microbial and fungal. Of all the choices, plant based enzymes are the most active. Thismeans they can break down more fat, protein and carbohydrates in the broadest pH range than any other sources (Ashokkumar et al., 2001).

1.1.2 Characteristics of enzymes

Protein nature:

Enzyme is a protein. The main components of an enzyme is protein.

Temperature:

Enzymes are sensitive to temperature. Many work best at temperatures close to body temperatures and most lose their ability to catalyse if they are heated above 60 or 70o C. (Ashokkumar et al., 2001).

Acidity and alkalinity:

Many enzymes work best at a particular pH and stop working if the pH becomes too acidic or alkaline.

Catalytic effect:

It acts as catalyst, enzyme functions in accelerating chemical reaction, but the enzyme itself does not change after the reaction ends.

Specificity:

It functions specifically. The enzyme only catalyzes one kind of substrate and cannot function for many substrates. The term is called one enzyme one substrate.

Reversibility:

It means the enzyme does not determine the direction of reaction, but it only functions in accelerating reaction rate until it reaches equilibrium. The enzyme also functions in substance synthesis and substance breaking down reaction.

Small quantity:

It is required, in small amount. A small amount of enzyme is able to catalyze a chemical reaction (Nason, 1968).

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Origin, purification and uses of enzymes

Enzymes are ubiquitous

Enzymes are essential components of animals, plants and microorganisms, due to the fact that they catalyse and co-ordinate the complex reactions of cellular metabolism.

Up until the 1970s, most of the commercial application of enzymes involved animal and plant sources. At that time, bulk enzymes were generally only used within the food-processing industry, and enzymes from animals and plants were preferred, as they were considered to be free from the problems of toxicity and contamination that were associated with enzymes of microbial origin. However, as demand grew and as fermentation technology developed, the competitive cost of microbial enzymes was recognized and they became more widely used.
Compared with enzymes from plant and animal sources, microbial enzymes have economic, technical and ethical advantages, which will now be outlined.

Economic advantages

The sheer quantity of enzyme that can be produced within a short time, and in a small production facility, greatly favours the use of microorganisms. For example, during the production of rennin (a milk-coagulating enzyme used in cheese manufacture) the traditional approach is to use the enzyme extracted from the stomach of a calf (a young cow still feeding on its mother’s milk). The average quantity of rennet extracted from a calf’s stomach is 10 kg, and it takes several months of intensive farming to produce a calf. In comparison, a 1 000-litre fermenter of recombinant Bacillus subtilis can produce 20 kg of enzyme within 12 h. Thus the microbial product is clearly preferable economically, and is free from the ethical issues that surround the use of animals. Indeed, most of the cheese now sold in supermarkets is made from milk coagulated with microbial enzymes (so is suitable for vegetarians).

A further advantage of using microbial enzymes is their ease of extraction. Many of the microbial enzymes used in biotechnological processes are secreted extracellularly, which greatly simplifies their extraction and purification. Microbial intracellular enzymes are also often easier to obtain than the equivalent animal or plant enzymes, as they generally require fewer extraction and purification steps.

Animal and plant sources usually need to be transported to the extraction facility, whereas when microorganisms are used the same facility can generally be employed for production and extraction. In addition, commercially important animal and plant enzymes are often located within only one organ or tissue, so the remaining material is essentially a waste product, disposal of which is required.

Finally, enzymes from plant and animal sources show wide variation in yield, and may only be available at certain times of year, whereas none of these problems are associated with microbial enzymes.

Technical advantages

Microbial enzymes often have properties that make them more suitable for commercial exploitation. In comparison with enzymes from animal and plant sources, the stability of microbial enzymes is usually high. For example, the high temperature stability of enzymes from thermophilic microorganisms is often useful when the process must operate at high temperatures (e.g. during starch processing).

Microorganisms are also very amenable to genetic modification to produce novel or altered enzymes, using relatively simple methods such as plasmid insertion. The genetic manipulation of animals and plants is technically much more difficult, is more expensive and is still the subject of significant ethical concern, especially in the U.K.
Enzymes may be intracellular or extracellular

Although many enzymes are retained within the cell, and may be located in specific subcellular compartments, others are released into the surrounding environment. The majority of enzymes in industrial use are extracellular proteins from either fungal sources (e.g. Aspergillus species) or bacterial sources (e.g. Bacillus species). Examples of these include α-amylase, cellulase, dextranase, proteases and amyloglucosidase. Many other enzymes for non-industrial use are intracellular and are produced in much smaller amounts by the cell. Examples of these include asparaginase, catalase, cholesterol oxidase, glucose oxidase and glucose-6-phosphate dehydrogenase.
Enzyme purification

Within the cell, enzymes are generally found along with other proteins, nucleic acids, polysaccharides and lipids. The activity of the enzyme in relation to the total protein present (i.e. the specific activity) can be determined and used as a measure of enzyme purity. A variety of methods can be used to remove contaminating material in order to purify the enzyme and increase its specific activity. Enzymes that are used as diagnostic reagents and in clinical therapeutics are normally prepared to a high degree of purity, because great emphasis is placed on the specificity of the reaction that is being catalysed. Clearly the higher the level of purification, the greater the cost of enzyme production. In the case of many bulk industrial enzymes the degree of purification is less important, and such enzymes may often be sold as very crude preparations of culture broth containing the growth medium, organisms (whole or fragmented) and enzymes of interest. However, even when the cheapest bulk enzymes are utilized (e.g. proteases for use in washing powders), the enzyme cost can contribute around 5–10% of the final product value.
Pretreatment

At the end of a fermentation in which a microorganism rich in the required enzyme has been cultured, the broth may be cooled rapidly to 5°C to prevent further microbial growth and stabilize the enzyme product. The pH may also be adjusted to optimize enzyme stability. If the enzyme-producing organism is a fungus, this may be removed by centrifugation at low speed. If the enzyme source is bacterial, the bacteria are often flocculated with aluminum sulfate or calcium chloride, which negate the charge on the bacterial membranes, causing them to clump and thus come out of suspension.

Treatment

Extracellular enzymes are found in the liquid component of the pretreatment process. However, intracellular enzymes require more extensive treatment. The biomass may be concentrated by centrifugation and washed to remove medium components. The cellular component must then be ruptured to release the enzyme content. This can be done using one or more of the following processes:

• Ball milling (using glass beads)
• Enzymic removal of the cell wall
• Freeze–thaw cycles
• Liquid shearing through a small orifice at high pressure (e.g. within a French press) • osmotic shock
• Sonication.

Separation of enzymes from the resulting solution may then involve a variety of separation processes, which are often employed in a sequential fashion.

The first step in an enzyme purification procedure commonly involves separation of the proteins from the non-protein components by a process of salting out. Proteins remain in aqueous solution because of interactions between the hydrophilic (water-loving) amino acids and the surrounding water molecules (the solvent). If the ionic strength of the solvent is increased by adding an agent such as ammonium sulfate, some of the water molecules will interact with the salt ions, thereby decreasing the number of water molecules available to interact with the protein. Under such conditions, when protein molecules cannot interact with the solvent, they interact with each other, coagulating and coming out of solution in the form of a precipitate. This precipitate (containing the enzyme of interest and other proteins) can then be filtered or centrifuged, and separated from the supernatant.

Since different proteins vary in the extent to which they interact with water, it is possible to perform this process using a series of additions of ammonium sulfate, increasing the ionic strength in a stepwise fashion and removing the precipitate at each stage. Thus such fractional precipitation is not only capable of separating protein from non-protein components, but can also enable separation of the enzyme of interest from some of the other protein components.

Subsequently a wide variety of techniques may be used for further purification, and steps involving chromatography are standard practice.

Ion-exchange chromatography is often effective during the early stages of the purification process. The protein solution is added to a column containing an insoluble polymer (e.g. cellulose) that has been modified so that its ionic characteristics will determine the type of mobile ion (i.e. cation or anion) it attracts. Proteins whose net charge is opposite to that of the ion-exchange material will bind to it, whereas all other proteins will pass through the column. A subsequent change in pH or the introduction of a salt solution will alter the electrostatic forces, allowing the retained protein to be released into solution again.

Gel filtration can be utilized in the later stages of a purification protocol to separate molecules on the basis of molecular size. Columns containing a bed of cross-linked gel particles such as Sephadex are used. These gel particles exclude large protein molecules while allowing the entry of smaller molecules. Separation occurs because the larger protein molecules follow a path down the column between the Sephadex particles (occupying a smaller fraction of the column volume). Larger molecules therefore have a shorter elution time and are recovered first from the gel filtration column.

Affinity chromatography procedures can often enable purification protocols to be substantially simplified. Typically, with respect to enzyme purification, a column would be packed with a particulate stationary phase to which a ligand molecule such as a substrate analogue, inhibitor or cofactor of the enzyme of interest would be firmly bound. As the sample mixture is passed through the column, the enzyme interacts with, and binds, to the immobilised ligand, being retained within the column as all of the other components of the mixture pass through the column unrewarded. Subsequently a solution of the ligand is introduced to the column to release (elute) and thereby recover the bound enzyme from the column in a highly purified form.

Nowadays numerous alternative affinity chromatography procedures exist that are able to separate enzymes by binding to areas of the molecule away form their active site. Advances in molecular biology enable us to purify recombinant proteins, including enzymes, through affinity tagging. In a typical approach the gene for the enzyme of interest would be modified to code for a further short amino acid sequence at either the N- or C- terminal. For example, a range of polyhistidine tagging procedures are available to yield protein products with six or more consecutive histidine residues at their N- or C- terminal end. When a mixture containing the tagged protein of interest is subsequently passed through a column containing a nickelnitrilotriacetic acid (Ni-NTA) agarose resin, the histidine residues on the recombinant protein bind to the nickel ions attached to the support resin, retaining the protein, whilst other protein and non-protein components pass through the column. Elution of the bound protein can then be accomplished by adding imidazole to the column, or by reducing the pH to 5-6 to displace the His-tagged protein from the nickel ions.

Such techniques are therefore capable of rapidly and highly effectively isolating an enzyme from a complex mixture in only one step, and typically provide protein purities of up to 95%. If more highly purified enzyme products are required, other supplemental options are also available, including various forms of preparative electrophoresis e.g. disc-gel electrophoresis and isoelectric focusing.

Finishing of enzymes

Enzymes are antigenic, and since problems occurred in the late 1960s when manufacturing workers exhibited severe allergic responses after breathing enzyme dusts, procedures have now been implemented to reduce dust formation. These involve supplying enzymes as liquids wherever possible, or increasing the particle size of dry powders from 10 μm to 200–500 μm by either prilling (mixing the enzyme with polyethylene glycol and preparing small spheres by atomization) or marumerizing (mixing the enzyme with a binder and water, extruding long filaments, converting them into spheres in a marumerizer, drying them and covering them with a waxy coat).

Industrial enzymology

Although many industrial processes, such as cheese manufacturing, have traditionally used impure enzyme sources, often from animals or plants, the development of much of modern industrial enzymology has gone hand in hand with the commercial exploitation of microbial enzymes. These were introduced to the West in around 1890 when the Japanese scientist Jokichi Takamine settled in the U.S.A. and set up an enzyme factory based on Japanese technology. The principal product was Takadiastase, a mixture of amylolytic and proteolytic enzymes prepared by cultivating the fungus Aspergillus oryzae on rice or wheat bran. Takadiastase was marketed successfully in the U.S.A. as a digestive aid for the treatment of dyspepsia, which was then believed to result from the incomplete digestion of starch.

Bacterial enzymes were developed in France by August Boidin and Jean Effront, who in 1913 found that Bacillus subtilis produced a heat-stable α-amylase when grown in a liquid medium made by extraction of malt or grain. The enzyme was primarily used within the textile industry for the removal of the starch that protects the warp in the manufacture of cotton.

In around 1930 it was found that fungal pectinases could be used in the preparation of fruit products. In subsequent years, several other hydrolases were developed and sold commercially (e.g. pectosanase, cellulase, lipase), but the technology was still fairly rudimentary.

After World War Two the fermentation industry underwent rapid development as methods for the production of antibiotics were developed. These methods were soon adapted for the production of enzymes. In the 1960s, glucoamylase was introduced as a means of hydrolysing starch, replacing acid hydrolysis. Subsequently, in the 1960s and 1970s, proteases were incorporated into detergents and then glucose isomerase was introduced to produce sweetening agents in the form of high-fructose syrups. Since the 1990s, lipases have been incorporated into washing powders, and a variety of immobilized enzyme processes have been developed (see section on enzyme immobilization), many of which utilize intracellular enzymes.

Currently, enzymes are used in four distinct fields of commerce and technology (Table 6):

• As industrial catalysts
• As therapeutic agents
• As analytic reagents
• As manipulative tools (e.g. in genetics).