Industrial enzyme

Historical background

Most of the reactions in living organisms are catalyzed by protein molecules called enzymes. Enzymes can rightly be called the catalytic machinery of living systems. The real break through of enzymes occurred with the introduction of microbial proteases into washing powders. The first commercial bacterial Bacillus protease was marketed in 1959 and major detergent manufactures started to use it around 1965.

The industrial enzyme producers sell enzymes for a wide variety of applications. The estimated value of world market is presently about US$ 2 billion. Detergents (37%), textiles (12%), starch (11%), baking (8%) and animal feed (6%) are the main industries, which use about 75% of industrially produced enzymes.

Enzyme classification

Presently more than 3000 different enzymes have been isolated and classified. The enzymes are classified into six major categories based on the nature of the chemical reaction they catalyze:

1. Oxidoreductases catalyze oxidation or reduction of their substrates.

2. Transferases catalyze group transfer.

3. Hydrolases catalyze bond breakage with the addition of water.

4. Lyases remove groups from their substrates.

5. Isomerases catalyze intramolecular rearrangements.

6. Ligases catalyze the joining of two molecules at the expense of chemical energy.

Only a limited number of all the known enzymes are commercially available . More than 75 % of industrial enzymes are hydrolases. Protein-degrading enzymes constitute about 40 % of all enzyme sales. More than fifty commercial industrial enzymes are available and their number is increasing steadily.

Enzyme production

Some enzymes still extracted from animal and plant tissues. Enzymes such as papain, bromelain and ficin and other speciallity enzymes like lipoxygenase are derived from plants and enzymes pepsin and rennin are derived from animal. Most of the enzymes are produced by microorganisms in submerged cultures in large reactors called fermentors. The enzyme production process can be divided into following phases:

1. Selection of an enzyme.

2. Selection of production strain.

3. Construction of an overproducing stain by genetic engineering.

4. Optimization of culture medium and production condition.

5. Optimization of recovery process.

6. Formulation of a stable enzyme product.

Criteria used in the selection of an industrial enzyme include specificity, reaction rate, pH and temperature optima and stability, effect of inhibitors and affinity to substrates. Enzymes used in the industrial applications must usually tolerant against various heavy metals and have no need for cofactors.

Microbial production strains

In choosing the production strain several aspects have to be considered. Ideally the enzyme is secreted from the cell. Secondly, the production host should have a GRAS-status. Thirdly, the organism should be able to produce high amount of the desired enzyme in a reasonable life time frame. Most of the industrially used microorganism have been genetically modified to overproduce the desired activity and not to produce undesired side activities.

Enzyme production by microbial fermentation

Once the biological production organism has been genetically engineered to overproduce the desired products, a production process has to be developed. The optimization of a fermentation process includes media composition, cultivation type and process conditions. The large volume industrial enzymes are produced in 50 -500 m3 fermentors. The extracellular enzymes are often recovered after cell removal (by vacuum drum filtration, separators or microfiltration) by ultrafiltration.

Protein engineering

Often enzymes do not have the desired properties for an industrial application. One option is find a better enzyme from nature. Another option is to engineer a commercially available enzyme to be a better industrial catalyst. Another option is to engineer a commercially available enzyme to be a better industrial catalyst. Two different methods are presently available: a random method called directed evaluation and a protein engineering method called rational design.

Enzyme technology

This field deals with how are the enzymes used and applied in practical processes. The simplest way is to use enzymes is to add them into a process stream where they catalyze the desired reaction and are gradually inactivated during the process. This happens in many bulk enzyme applications and the price of the enzymes must be low to take their use economical.

An alternative way to use enzymes is to immobilize them so that they can be reused. Enzyme can be immobilized by using ultra filtration membranes in the reactor system. The large enzyme molecule cannot pass through the membrane but the small molecular reaction products can. Many different laboratory methods for enzyme immobilization based on chemical reaction, entrapment, specific binding or absorption have been developed.

Large scale Enzyme applications

1] Detergents

Bacterial proteinases are still the most important detergent enzymes. Lipases decompose fats into more water-soluble compounds. Amylases are used in detergents to remove starch based stains.

2] Starch hydrolysis and fructose production

The use of starch degrading enzymes was the first large scale application of microbial enzymes in food industry. Mainly two enzymes carry out conversion of starch to glucose: alpha-amylase and fungal enzymes. Fructose produced from sucrose as a starting material. Sucrose is split by invertase into glucose and fructose, fructose separated and crystallized.

3] Drinks

Enzymes have many applications in drink industry. Lactase splits milk-sugar lactose into glucose and galactose. This process is used for milk products that are consumed by lactose intolerant consumers. Addition of pectinase, xylanase and cellulase improve the liberation of the juice from pulp. Similarly enzymes are widely used in wine production.

4] Textiles

The use of enzymes in textile industry is one of the most rapidly growing fields in industrial enzymology. The enzymes used in the textile field are amylases, catalase, and lactases which are used to remove the starch, degrade excess hydrogen peroxide, bleach textiles and degrade lignin.

5] Animal feed

Addition of xylanase to wheat-based broiler feed has increased the available metabolizable energy 7-10% in various studies. Enzyme addition reduces viscosity, which increases absorption of nutrients, liberates nutrients either by hydrolysis of non-degradable fibers or by liberating nutrients blocked by these fibers, and reduces the amount of faeces.

6] Baking

Alpha-amylases have been most widely studied in connection with improved bread quality and increased shelf life. Use of xylanases decreases the water absorption and thus reduces the amount of added water needed in baking. This leads to more stable dough. Proteinases can be added to improve dough-handling properties; glucose oxidase has been used to replace chemical oxidants and lipases to strengthen gluten, which leads to more stable dough and better bread quality.

7] Pulp and Paper

The major application is the use of xylanases in pulp bleaching. This reduces considerably the need for chlorine based bleaching chemicals. In paper making amylase enzymes are used especially in modification of starch. Pitch is a sticky substance present mainly in softwoods. Pitch causes problems in paper machines and can be removed by lipases.

8] Leather

Leather industry uses proteolytic and lipolytic enzymes in leather processing. Enzymes are used to remove unwanted parts. In dehairing and dewooling phases bacterial proteases enzymes are used to assist the alkaline chemical process. This results in a more environmentally friendly process and improves the quality of the leather . Bacterial and fungal enzymes are used to make the leather soft and easier to dye.

9] Speciality enzymes

There are a large number of specialty applications for enzymes. These include use of enzymes in analytical applications, flavour production, protein modification, and personal care products, DNA-technology and in fine chemical production.

10] Enzymes in analytics

Enzymes are widely used in the clinical analytical methodology. Contrary to bulk industrial enzymes these enzymes need to be free from side activities. This means that elaborate purification processes are needed.

An important development in analytical chemistry is biosensors. The most widely used application is a glucose biosensor involving glucose oxidase catalysed reaction.

Several commercial instruments are available which apply this principle for measurement of molecules like glucose, lactate, lactose, sucrose, ethanol, methanol, cholesterol and some amino acids.

11] Enzymes in personal care products

Personal care products are a relatively new area for enzymes. Proteinase and lipase containing enzyme solutions are used for contact lens cleaning. Hydrogen peroxide is used in disinfections of contact lenses. The residual hydrogen peroxide after disinfections can be removed by catalase enzyme. Some toothpaste contains glucoamylase and glucose oxidase. Enzymes are also studied for applications in skin and hair care products.

12] Enzymes in DNA-technology

DNA-technology is an important tool in enzyme industry. Most traditional enzymes are produced by organisms, which have been genetically modified to overproduce the desired enzyme. The specific order of the organic bases in the chain of DNA constitutes the genetic language. Genetic engineering means reading and modifying this language. Enzymes are crucial tools in this process.

13] Enzymes in fine chemical production

In spite of some successes, commercial production of chemicals by living cells using pathway engineering is still in many cases the best alternative to apply biocatalysis. Isolated enzymes have, however, been successfully used in fine chemical synthesis. Some of the most important examples are represented here.

13 A] Chirally pure amino acids and aspartame

Natural amino acids are usually produced by microbial fermentation. Novel enzymatic resolution methods have been developed for the production of L- as well as for D-amino acids. Aspartame, the intensive non-calorie sweetener, is synthesized in non-aqueous conditions by thermolysin, a proteolytic enzyme.

13 B] Rare sugars

Recently enzymatic methods have been developed to manufacture practically all D- and L-forms of simple sugars. Glucose isomerase is one of the important industrial enzymes used in fructose manufacturing.

13 C] Semisynthetic penicillins

Penicillin is produced by genetically modified strains of Penicillium strains. Most of the penicillin is converted by immobilised acylase enzyme to 6-aminopenicillanic acid, which serves as a backbone for many semisynthetic penicillins.

13 D] Lipase based reactions

In addition to detergent applications lipases can be used in versatile chemical reactions since they are active in organic solvents. Lipases used in transesterification and also used for enantiomeric separation of alcohols and separate racemic amine mixtures. Lipases have also been used to form aromatic and aliphatic polymers.

13 E] Enzymatic oligosaccharide synthesis

The chemical synthesis of oligosaccharides is a complicated multi-step effort. Biocatalytic syntheses with isolated enzymes like glycosyltransferases and glycosidases or engineered whole cells are powerful alternatives to chemical methods. Oligosaccharides have found applications in cosmetics, medicines and as functional foods.

Future trends in industrial enzymology

Industrial enzyme market is growing steadily. The reason for this lies in improved production efficiency resulting in cheaper enzymes, in new application fields. Tailoring enzymes for specific applications will be a future trend with continuously improving tools and understanding of structure-function relationships and increased search for enzymes from exotic environments.

New technical tools to use enzymes as crystalline catalysts, ability to recycle cofactors, and engineering enzymes to function in various solvents with multiple activities are important technological developments, which will steadily create new applications.

Dr. Prashantkumar Kudli Shrinivas has a vast industrial experience of Aroma chemicals, Essential oils, Perfumery and Flavour formulations. He is a permanent member of Indian Institute of Chemical Engineers & NMR Association of India.

Article Source: http://EzineArticles.com/890345

effect of temperature

Effect of Temperature on Enzymes

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Due to proteinaceous nature, enzymes are very sensitive to heat. The rate of an enzyme action increases with rise in temperature, the rate of action increases 2 to 3 times for a rise in temperature of 10°C, i.e., the value of temperature quotient or Q₁₀ is 2 to 3. But at higher temperatures, the value of coefficient does not remain constant and decreases rapidly. Above 60°C, the enzymes coagulate and thus become inactivated due to irreversible change in their chemical structure.
With certain exceptions, the rates of enzymes catalyzed reaction are increased as the temperature is raised. By raising temperature, the number of activated molecules is increased which ultimately results in velocity of the reaction. The enzyme catalyzed reactions show the increase in the velocity between 25°C to 35°C. At 0°C or below 0°C the enzymes become inactivated but they are not destroyed. At 60°C to 70°C, in a liquid medium the enzymes are inactivated and destroyed. This destruction of enzymes at high temperature results in coagulation and denaturation. Thus as the temperature is raised, the reaction rate increases upto a certain limit and above that the enzymes get denatured. The temperature, at which the rate of reaction in maximum, is known asoptimum temperature.

Effect of temperature on enzymes-Graph (temperature vs enzyme activity)

Factors affecting enzymatic reaction 2

a)      pH

·         Enzymes are sensitive to pH.pH is a measure of the hydrogen concentration. It is measured on a scale of 1-14 with pH 7 being the neutral point. A pH less than 7 is acidic wheareas greather than 7 is alkaline.

·         The precise three-dimensional molecular shape which is vital to the functioning of enzymes is partly the result of hydrogen bonding between positive and negative charges in the enzyme molecule. By breaking the hydrogen bonds which give enzyme molecules their shape any change in pH can effectively denature enzyme.

·         These bonds may be broken by the concentration of hydrogen ion present. In other words a change of pH environment of the protein alters the ionic charge of the acidic and basic groups and causes the protein to change. This changes the shape of the active site of the enzyme and the enzyme may no longer have the correct shape to bind to its substrate

·         Each enzyme has its own range of pH in which it functions most efficiently. Most intracellular enzymes function best at around neutral. If the pH is too low or too high the activity will fall. pH sensitivity is due to acidic and basic side groups of amino acids.

·         Enzymes are adapted to the pH of their environment. They have optimal activity at a certain pH.

 D) Temperature

·         As the temperature increases the kinetic energy of the substrate and enzyme molecule increase and so they move faster. The faster these molecules move, the more often they collide with one another and the greater the rate of reaction.

·         Enzymes function best at a particular temperature known as their optimum temperature.

·         Enzymes are inactive by excessive heat. Up to about 40^oC, the rate of an enzyme-controlled reaction increases smoothly, a 10^oC rise in temperature approximately doubles the rate of the action. Above 40^oC the rate begins decrease and at about 60 ^oC the reaction stops.

·         This is caused by the denatured of protein which constitutes the enzyme. As the temperature increases the more the atoms which make up the enzyme molecule vibrate. Above a critical temperature, enzyme activity falls rapidly because the enzyme unfolds. This break the hydrogen bonds which hold the molecules in their precise shape.

The three-dimensional shape of the enzyme molecule is altered to such an extent that their active sites no longer fit the substrate. The enzyme is said to be denatured and it loses its catalytic properties. Heat sterilization

Factors affecting enzymatic reaction

Factors affecting Enzymatic reaction

a)      Concentration of enzyme

b)      Substrate concentration

c)      pH

d)     Temperature

a)      Concentration of enzymes

·         The rate of an enzyme- catalysed reaction is proportional to the concentration of the enzyme.

·         The higher the enzyme concentration, the faster will be the reaction rate for a certain period.

b)      Substrate concentration

·         For a given amount of enzyme, the rate of an enzyme controlled reaction increases with an increases in substrate concentration up to a certain point.

·         With a fixed enzyme concentration an increase of substrate will result at first in a very rapid rise in velocity or reaction rate.

·         At lo substrate concentration the active sites on the enzyme are noy saturated by the substrate and thus enzyme rate varies with substrate concentration.

·         As the substrate concentration is increased more and more active site come into play. The more substrate molecule there are, the greater will be the chances of substrate and enzyme molecules colliding in the correct manner. The ate of reaction continues to increases.

·         When substrate concentration is high enough, the enzymes becomes saturated with substrate. At this point, all the active sites in all enzyme molecules are engaged.

·         When all active sites are being used, increasing the substrate concentration further cannot increase the rate of reaction. At this point the amount of enzyme is the limiting factor. This plateau in the graph which indicates that when the substrate concentration reaches a certain level the system becomes saturated and all the enzymes molecule are working at full capacity.

·         The only way to increase the rate of the reaction is to increase the concentration of the enzyme.

Activation energy ( EA )

Activation energy ( E_A )

·         To understand how an enzyme speeds up a chemical reaction, it is necessary to define the activation energy of a reaction. There is usually an energy barrier to every chemical reaction which prevents the reaction from spontaneously occurring, even if the reaction is exergonic. This energy barrier is called the activation energy (E_A).

·         Activation energy ( E_A ) is the minimum energy which is needed to break the bonds in a molecule and start a reaction so that an effective reaction can occur.

·         Enzymes speed up metabolic reactions by lowering energy barriers and increasing the number of molecule with a sufficient energy content to react and form a product.

·         Enzymes lower (E_A) so that the transition state can be reached at cellular temperatures. Reactant must absorb enough energy (E_A) to reach the transition state ( uphill portion of the curve )

·         The enzymes act by forming an unstable enzyme-substrate complex with the substrates. This complex is subsequently transformed into the product with the release of the enzyme ( downhill portion of the curve )

Keyword:

Transition state : The unstable condition of reactant molecules that have absorbed sufficient free energy to react.

Enzyme With Industrial Applications Characterized

The industrial sector is increasingly concerned with reducing the energy consumption of key processes, producing less waste and contaminants, and improving the quality of end products through the application of sustainable technologies. One example is the bleaching of wood pulp in paper manufacturing, which has traditionally produced large amounts of toxic residues due to the lignin content of the raw materials. To clean up the procedure, enzymes called xylanases are now used to degrade the xylan that traps the residual lignin responsible for the unwanted dark coloration of the pulp. The use of these enzymes reduces the amount of chlorine required by 20-25%, lowering the volume of organochlorine contaminants generated. According to Dr. F. Javier Pastor of the Department of Microbiology, "the enzyme (Xyn10B) we have characterized and protein-engineered for the study is particularly notable for its thermostability, which makes it more resistant to the bleaching process, and for its ability to improve the catalytic efficiency of xylan degradation."

In 1993, Dr. Pastor's group isolated a bacteria in a rice paddy in the Ebro delta which was found to be an effective degrader of polysaccharides and lipids. In 2005, following an extensive taxonomic study, the group was able to establish that they had in fact discovered an entirely new species of bacteria, which they named Paenibacillus barcinonensis (Paenibacillus is a bacteria genus, and barcinonensis was the specific name the group agreed upon for the new species). Gene expression analysis revealed that the bacteria contained a number of different enzymes (cellulases, xylanases and lipases) with degrading properties. Potentially, one of the most useful of these enzymes is the xylanase Xyn10B, "since it is one of the few intracellular xylanases to have been discovered to date," explains Dr. Pastor.

The article describes the new xylan-degrading enzyme and the identification of its highly specific properties through purification and a crystallographic study of its structure. In the second part of the study, two mutations were combined to produce a highly thermostable and more active enzyme. Modern enzyme technology is largely geared towards use in biorefineries, with the aim of creating an integrated process enabling plant waste and other biomass components to be reused in new applications. This technology can also be used to obtain new added-value products from xylan, ranging from prebiotic ingredients for functional foods to bioethanol, which can be obtained by depolymerization of the xylan. Other researchers involved in the project include scientists from the Institute of Agrochemistry and Food Technology (Valencia) and the Rocasolano Institute of Chemical Physics in Madrid, both affiliated to the Spanish National Research Council (CSIC).

from : http://www.sciencedaily.com

Properties of enzyme

Enzymes are normally complex there-dimensional globular proteins and therefore subjected to the same controls as all protein. The following describe some important properties of enzymes:

ü  Enzymes are specific. Each particular enzyme binds only to specific substrate. An enzyme must recognize the correct substrate then take it or merge it with another substrate to form one or more specific products.

ü  Most enzymes have high turnover numbers. Enzymes are very efficient. They generally work very rapidly.

ü  Enzymes are reusable as they are not destroyed while catalyzing reactions.

ü  An enzyme can catalyses in a forward and backward direction. The direction of metabolic reactions which are reversible depends on the relative concentrations of the substrate and the products.

ü  As enzymes are proteins, they can be denatured. High temperatures of 40 ^oC or above can denature most enzymes. Upon denaturation, the structure of enzymes is permanently altered resulting in an irreversible loss of activity.

ü  Enzymes also affected by and can be denatured by changes in pH. The rate at which the inactive form become active or vice versa is determined by the chemical environment inside the cell.

Keyword :

Turnover number : The number of molecules of substrate which acted upon by a molecule of enzyme per second.

Introduction of enzyme

1.      A catalyst is a substance that speeds up a chemical reaction without being affected chemically in the reaction. The addition of a catalyst enables the reaction to reach equilibrium at a faster rate compared to similar reactions occurring without catalysts.

2.      Enzymes are biological or organic catalysts, which speed up a reaction without undergoing any permanent changes themselves.

3.      Most enzymes are made up from globular proteins that are produced by living cell. In the few special cases in which RNA demonstrates enzymatic activity the term ribozyme is used.

4.      Before a reaction can occur, the reactants must absorb energy to break chemical bonds. The amount of energy required for a reaction to proceed is called the activation energy. In our body, enzymes lower the activation energy by forming a complex with particular molecules.

5.      The reactant in an enzymatic reaction are substrate. Enzyme are often name after their substrate. For instance, maltase is the enzyme that digest or hydrolyses maltose into glucose.

6.      Enzymes are normally larger than the substrate molecule. Only a small a part the enzyme molecule actually comes into contact with the substrate. This region is called active site, where the reaction occurs.

7.      An enzyme ( E ) combine with its substrate ( S ) to form an enzyme-substrate complex, which then dissociates into products ( P ) and enzyme ( E ), as stated in the following equation :

Enzyme ( E ) + Substrate ( S )       Enzyme-substrate complex           Product ( P ) + Enzyme ( E )

This digestive enzyme is a powerful agent in the fight against cancer

(NaturalNews) Bromelain is a natural digestive enzyme extracted from pineapples. The nutrient is rapidly taking its place aside many of the most powerful natural agents in the war on cancer and other chronic conditions that take the lives of millions each year. Research published in Cancer Letters explains the protease enzyme exhibits multiple actions including anti-inflammatory and immune cell activation that can deal a powerful blow to cancer development. Include bromelain in your supplemental regimen to benefit from the potent anti-cancer properties now attributed to this amazing pineapple enzyme.

Similar to super nutrients such as resveratrol, curcumin and green tea extract, bromelain is a potent compound that fights cancer by dissolving unnecessary tissue throughout the body. Bromelain breaks down scar tissue and other debris created from the natural processes of stress and physical wear and tear that develop with normal cellular function. As we age, our body has a reduced capacity to eliminate these garbage byproducts from within our cells and we undergo the process of advanced aging.

Enzymes such as bromelain are critical to break down proteins used to drive metabolic functions within the body. Processed and overcooked foods have been stripped of the natural enzymes that originally existed, and the pancreas is forced to work beyond its capacity to break down digested proteins. Cancer cells also use a protein shield to cloak themselves and avoid detection from the immune system. Protease enzymes help destroy the protein bond around cancer clusters so the body can destroy developing tumor wall structures and thwart cancer initiation.

Evidence developed in European clinical trials and published in the journal Integrative Cancer Therapies explains that natural enzymes such as bromelain and papain provide a significant improvement in the outcome of alternative therapies to treat breast and colon cancers. The authors concluded that the enzyme mixture demonstrated potent anti-inflammatory, anti-infectious and antitumor/anti-metastatic activity.

Further scientific documentation is published in the journal Molecular Carcinogenesis to support the cancer-killing ability of bromelain, independent of other therapies. Cancer is fueled by systemic inflammation and out of control gene activity from the protein complex, NF-KappaB (nuclear factor kappa beta). Bromelain inhibits the activity of NF-KappaB, significantly lowering cellular free radical damage causing cancer cells to undergo normal cell death (apoptosis).

Research continues to uncover the amazing health-promoting benefits of natural enzymes such as bromelain in the fight against inflammatory and immune-deficiency diseases like cancer. You can dramatically lower your risk by supplementing with 50 mg of bromelain extract daily, taken with meals to aid protein digestion and on an empty stomach to uncloak rogue cancer cell development.



from :http://www.naturalnews.com/032951_cancer_digestive_enzymes.html