Enzymes Prerna

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    GENERAL SCIENCE-ASSIGNMENT NO-8

    USE OF ENZYMES IN CHEMICAL PROCESSING

    -PRERNA AGRAWALF.P.TECH- 1

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    WHAT ARE ENZYMES?

    Enzymes are proteins and certain class of RNA (ribozymes) which enhance the rate

    of a thermodynamically feasible reaction and are not permanently altered in theprocess.

    In enzymatic reactions, the molecules at the beginning of the process are calledsubstrates, and the enzyme converts them into different molecules, called theproducts. Almost all processes in a biological cell need enzymes to occur atsignificant rates. Since enzymes are selective for their substrates and speed up onlya few reactions from among many possibilities, the set of enzymes made in a celldetermines which metabolic pathways occur in that cell.

    Like all catalysts, enzymes work by lowering the activation energy (Ea) for a

    reaction, thus dramatically increasing the rate of the reaction. Most enzyme reactionrates are millions of times faster than those of comparable un-catalyzed reactions.As with all catalysts, enzymes are not consumed by the reactions they catalyze, nordo they alter the equilibrium of these reactions. However, enzymes do differ frommost other catalysts by being much more specific. Enzymes are known to catalyzeabout 4,000 biochemical reactions. A few RNA molecules called ribozymes alsocatalyze reactions, with an important example being some parts of the ribosome.Synthetic molecules called artificial enzymes also display enzyme-like catalysis.

    ENZYMES CLASSIFICATIONACCORDING TO THE CHEMICAL

    REACTIONS THEY CATALYZE

    1. Oxidoreductases - oxidation/reduction

    (eg. dehydrogenases)2. Transferases- group transfer

    (eg. kinases)3. Hydrolases- hydrolysis

    (eg. proteases)4. Lyases- lysis, generating double bond

    (eg. synthases)5. Isomerases- rearrangement

    (eg. racemases)6. Ligases- ligation requiring ATP

    (eg. synthetases)

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    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 geneticengineering.

    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 andtemperature 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.

    ENZYMES ACTION

    Each reaction has a transition state where the substrate is in an unstable, short-livedchemical/ structural state. Enzymes act by lowering the free energy of the transitionstate.

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    The use of enzymes can lower the activation energy of a reaction (Ea).(Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates)

    LARGE SCALE ENZYME APPLICATIONS

    1] Detergents

    2] Starch hydrolysis and fructose production

    3] Drinks

    4] Textiles

    5] Animal feed

    6] Baking

    7] Pulp and Paper

    8] Leather

    9] Speciality enzymes

    10] Enzymes in analytics

    11] Enzymes in personal care products

    12] Enzymes in DNA-technology

    13] Enzymes in fine chemical production

    13 A] Chirally pure amino acids and aspartame

    13 B] Rare sugars

    13 C] Semisynthetic penicillins

    13 D] Lipase based reactions

    13 E] Enzymatic oligosaccharide synthesis

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    ENZYMES IN FINE CHEMICAL PRODUCTION

    Biocatalysis has been used in fine chemical production for a long time. Usually the

    catalyst has been a living organism. Ethanol, acetic acid, antibiotics, vitamins,pigments, solvents are but a few examples of biotechnical products. One of the

    reasons to use whole cell catalysts lies in the need to combine chemical energy

    source (in the form of ATP) or reducing/oxidising power (in the form of NAD(P)H) to

    the production process. This is elegantly done in a living cell. Candida yeasts can

    reduce the 5-carbon sugar xylose to a tooth-friendly polyol called xylitol by a xylose

    reductase enzyme:

    xylose + NADH xylitol + NAD

    The enzyme can be isolated and the reaction proceeds easily in a test tube.

    However, the reducing power of NADH has to be regenerated for the reaction to

    proceed. This is done in a living cell by other reactions, which reduce NAD back to

    NADH. One can isolate another enzyme, which does the same and couples two

    reactions together. One suitable enzyme is formate dehydrogenase:

    xylose + NADH xylitol + NAD

    formate + NAD CO2 + NADH

    Coupled enzymatic reactions have been extensively studied but only few commercial

    examples are known. Leucine dehydrogenase is used commercially to produce L-

    tert- leucine with a concomitant cofactor recycling using the formate reduction for

    cofactor regeneration. 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. We discuss here some of the most important

    examples.

    ENZYMES IN TEXTILE INDUSTRY

    The use of enzymes in textile industry is one

    of the most rapidly growing fields in industrial

    enzymology. Starch has for a long time been

    used as a protective glue of fibres in weaving

    of fabrics. This is called sizing. Enzymes are

    used to remove the starch in a process called

    desizing. Amylases are used in this process

    since they do not harm the textile fibres.

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    Enzymes have replaced the use of volcanic lava stones in the preparation of Denim

    (special soft cotton based fibre where the dye has been partially faded away) from

    an indigo-dyed cotton fibre to achieve a high degree of dye fading. The stones

    caused considerable damage to fibres and machines. The same effect can beobtained with cellulase enzymes. The effect is a result of alternating cycles of

    desizing and bleaching enzymes and chemicals in washing machines.

    Recently, hydrogen peroxides have been tested as bleaching agents to replace

    chlorine-based chemicals. Catalase enzyme, which destroys hydrogen peroxide,

    may then be used to degrade excess peroxide. Another recent approach is to use

    oxidative enzymes directly to bleach textiles. Laccase a polyphenol oxidase from

    fungi - is a new candidate in this field.

    Laccases are produced by white-rot fungi, which use them to degrade lignin - the

    aromatic polymer found in all plant materials. Laccase is a copper-containing

    enzyme, which is oxidised by oxygen, and which in an oxidised state can oxidatively

    degrade many different types of molecules like dye pigments.

    'Amylases' isolated from bacteria, fungi, pancreas and malt are used in textile

    industry as softening agents for starched clothes. Starch is often added to cottonfibres as a stiffening agent, before weaving the fibre into cloth.

    Since, a starched cloth does not take good colour, the cloth is to be destarched

    before dyeing it. This is done with an amylase preparation, which hydrolyses starch).

    ENZYMES IN DETERGENTS

    Detergents are surfactants, which is a class of compounds that bind to hydrophobiccompounds and make them soluble in water. This is why detergents 'cut' grease ona pan: They bond to the lipids, forming a hydrophilic boundary between the pan andthe grease, allowing the normally hydrophobic lipids to be washed away by water.

    Enzymes are used in cleaning products as cleaning and fabric care agents. Most of

    the used enzyme types breakdown large, water-insoluble soils and stains which are

    attached to e.g. fabrics, into smaller, more water-soluble pieces. Subsequently, the

    smaller molecules are removed, e.g. from the fabric / chinaware, by the mechanical

    action of the (dish)washing machine or by the interaction of other detergent

    ingredients. The enzyme does not loose its functionality after having worked on one

    stain and continues to work on the next one. Some enzymes also deliver fabric care

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    benefits by e.g. better maintaining whiteness or keeping colours bright.

    The most important reasons to use enzymes in detergents are i) that a very small

    quantity of these inexhaustible bio-catalysts can replace very large quantity of man

    made chemicals and ii) enzymes can work at very low temperature at whichtraditional chemistry quite often is no longer effective iii) they are fully biodegradable.

    All these characteristics make enzymes - on top of their high efficiency -

    environmentally friendly ingredients.

    Several enzymes can be used in products; each one having its own very well defined

    target. Some enzymes are specialised to attack fat stains, others to attack food

    stains.

    ENZYMES IN FRUIT JUICE AND WINE PRODUCTION

    One of the major problems in the preparation of fruit juices and wine is cloudinessdue primarily to the presence of pectins. These consist primarily of a-1, 4-anhydrogalacturonic acid polymers, with varying degrees of methyl esterification.They are associated with other plant polymers and, after homogenization, with thecell debris. The cloudiness that they cause is difficult to remove except by enzymichydrolysis. Such treatment also has the additional benefits of reducing the solutionviscosity, increasing the volume of juice produce (e.g. the yield of juice from whitegrapes can be raised by 15%), subtle but generally beneficial changes in the flavourand, in the case of wine-making, shorter fermentation times.

    The enzymes used in brewing are needed for saccharification of starch (bacterialand fungal a-amylases), breakdown of barley a-1.4- and a-1,3-linked glucan (b-glucanase) and hydrolysis of protein (neutral protease) t increase the (later)fermentation rate, particularly in the production of high-gravity beer, where extraprotein is added. Cellulases are also occasionally used, particularly where wheat isused as adjunct to help break down the barley is b-glucans

    .Due to the extreme heat stability of the B. amyloliquefaciens a-amylase where this isused, the wort must be boiled for a much longer period (e.g. 30 minutes) to inactivateit prior to fermentation. Papain is used in the later post-fermentation stages of beer-making to prevent the occurrence of protein-and tannin- containing chill-hazeotherwise formed on cooling the beer.

    Recently, light beers, of lower calorific content, have become more popular. Theserequire a higher degree of saccharification at lower starch concentrations to reducethe alcohol and total solid content of the beer. This may be achieved by the use ofglucoamylase and/or fungal a-amylase during the fermentation.

    A new enzyme preparation of fungal pectin lyase was shown to be useful for theproduction of cranberry juice and clarification of apple juice in the food industry. A

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    comparative study showed that the preparation of pectin lyase is competitive withcommercial pectinase products. The molecular weight of homogeneous pectin lyasewas 38 kDa. Properties of the homogenous enzyme were studies. This enzyme wasmost efficient in removing highly esterified pectin.

    ENZYMES IN BAKING

    Alpha-amylases have been most widely studied in connection with improved breadquality and increased shelf life. Both fungal and bacterial amylases are used.Overdosage may lead to sticky dough so the added amount needs to be carefullycontrolled.One of the motivations to study the effect of enzymes on dough and bread qualities

    comes from the pressure to reduce other additives. In addition to starch, flour

    typically contains minor amounts of cellulose, glucans and hemicelluloses like

    arabinoxylan and arabinogalactan. There is evidence that the use of xylanasesdecreases the water absorption and thus reduces the amount of added water

    needed in baking. This leads to more stable dough. Especially xylanases are used in

    whole meal rye baking and dry crisps common in Scandinavia.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.

    ENZYMES IN PERSONAL CARE PRODUCTS

    Personal care products are a relatively new area for enzymes and the amounts used

    are small but worth to mention as a future growth area. One application is contact

    lens cleaning. Proteinase and lipase containing enzyme solutions are used for this

    purpose. Hydrogen peroxide is used in disinfections of contact lenses. The residual

    hydrogen peroxide after disinfections can be removed by a heme containing catalase

    enzyme, which degrades hydrogen peroxide.

    Some toothpaste contains glucoamylase and glucose oxidase. The reasoning behind

    this practise is that glucoamylase liberates glucose from starch-based oligomersproduced by alpha-amylase and glucose oxidase converts glucose to gluconic acid

    and hydrogen peroxide which both function as disinfectants.

    Dentures can be cleaned with protein degrading enzyme solutions. Enzymes are

    studied also for applications in skin and hair care products.

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    ENZYMES IN RARE SUGARS

    Non-natural monosaccharides are needed as starting materials for new chemicalsand pharmaceuticals. Examples are L-ribose, D-psicose, L-xylose, D-tagatose and

    others. Some of the sugars are presently produced by chemical isomerization orepimerisation. 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. Recently it has been shown that it can catalyse previously unknown

    conversions. For example L-arabinose is isomerised to L-ribulose and slowly also to

    L-ribose. D-xylose is isomerised to D-xylulose and slowly to D-lyxose. Also 4-carbon

    sugars are good substrates. Enzymatic methods are an important tool in production

    of rare sugars.

    ENZYMES IN THE PULP AND PAPER INDUSTRY

    The pulp and paper industry processes huge quantities of lignocellulosic biomassevery year. The technology for pulp manufacture is highly diverse, and numerousopportunities exist for the application of microbial enzymes. Historically, enzymeshave found some uses in the paper industry, but these have been mainly confined toareas such as modifications of raw starch. However, a wide range of applications inthe pulp and paper industry have now been identified. The use of enzymes in the

    pulp and paper industry has grown rapidly since the mid 1980s. While manyapplications of enzymes in the pulp and paper industry are still in the research anddevelopment stage, several applications have found their way into the mills in anunprecedented short period of time. Currently the most important application ofenzymes is in the prebleaching of kraft pulp. Xylanase enzymes have been found tobe most effective for that purpose. Xylanase prebleaching technology is now in useat several mills worldwide. This technology has been successfully transferred to fullindustrial scale in just a few years. The enzymatic pitch control method using lipasewas put into practice in a large-scale paper-making process as a routine operation inthe early 1990s and was the first case in the world in which an enzyme wassuccessfully applied in the actual paper-making process. Improvement of pulp

    drainage with enzymes is practiced routinely at mill scale. Enzymatic deinking hasalso been successfully applied during mill trials and can be expected to expand inapplication as increasing amounts of newsprint must be deinked and recycled. TheUniversity of Georgia has recently opened a pilot plant for deinking of recycledpaper. Pulp bleaching with a laccase mediator system has reached pilot plant stageand is expected to be commercialized soon. Enzymatic debarking, enzymaticbeating, and reduction of vessel picking with enzymes are still in the R&D stage buthold great promise for reducing energy. Other enzymatic applications, i.e., removalof shives and slime, retting of flax fibers, and selective removal of xylan, are alsoexpected to have a profound impact on the future technology of the pulp and paper-making process.

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    ENZYMES IN MEDICINE

    Enzyme applications in medicine are as extensive as in industry. Pancreaticenzymes have been used in digestive disorders since nineteenth century. Mostenzymes are used extracellularly for

    (i) topical applications, e.g., col1agenase,

    (ii) removal of toxic substances, e.g., rhodonase, or in

    (iii) disorders within blood circulation system, e.g., streptokinase, urokinase, etc. Theenzyme preparations must be of high purity and free from unwanted contamination;therefore, they are generally from animal, sources and very costly.

    For example, urokinase is isolated from human urine and costs nearly $ 200/mg; the

    annual market for this enzyme is nearly $150 million. Enzymes have a majorpotential application in treatment of cancer, e.g., asparagenase in the treatment oflymphocytic leukaemia.

    Tumour cells are unable to synthesize L-asparagine due to an enzyme deficiency,and obtain this amino acid from body fluids. Asparaginase drastically reduces thelevels of free L-asparagine in the blood stream, creating starvation in tumour cells forthis amino acid; normal cells are not affected since they can synthesize L-asparagine.

    Asparaginase is injected intravenously, shows half-life of about 1 day (in dog), and

    may lead to complete recovery in 60% of the cases. Enzyme applications inmedicine are limited by and suffer from certain limitations.

    1. Their large molecular size interferes with their distribution among body cells.

    2. Enzymes are antigenic, and can elicit immune response in the patient, especiallyon prolonged use.

    3. Most enzymes have short effective life in the circulatory system; e.g., of fewminutes.

    ENZYMES USED IN INDUSTRY

    Lactose is hydrolysed to its constituent monosaccharides, glucose and galactose, bylactase. Glucose itself can be removed from some foodstuff, for example, prior to

    drying where glucose would cause discoloration, by fungal glucose oxidase.

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    Enzyme Uses

    Bacterial glucose isomerase Glucose ----> Invert sugar (i.e., fructoseformation)

    Bacterial a-Amylase Fungalamyloglucosidase

    Starch -----> Glucose

    Fungal a-Amylase Partial degradation of starch in supplementationof amylase-deficient flour for bread making

    Microbial rennets k-casein -----> para-casein (in milk curdling forcheese manufacture)

    Bacterial protease Removal of protein-based stains and laundering (inbiological washing powders)

    Papain (from papaya melon) Several protease applications including meattenderization and dehazing of beer

    Cellulose Cellulose -----> Glucose

    Fungal pectinase Pectin degradation (in fruit and vegetable processing)

    Aminoacylase (immobilized) Resolution of DL-amino acids to produce L-aminoacids for food supplementation

    Glucose isomerase(immobilized)

    Production of invert sugar and of high fructose syrupsfrom glucose

    Penicillin acylase (immobilized) Hydrolysis of penicillin-G to make 6-aminopenicillanicacid for production of new penicillins

    ENZYMES IN FOOD INDUSTRY

    Proteases may be used at various pH values, and they may be highly specific in theirchoice of cleavable peptide links or quite non-specific. Proteolysis generally increasesthe solubility of proteins at their isoelectric points.

    The action of rennet in cheese making is an example of the hydrolysis ofa specific peptide linkage, between phenylalanine and methionineresidues (-Phe105Met106-) in the k-casein protein present in milk. Calfrennet, consisting of mainly chymosin with a small but variable proportionof pepsin, is a relatively expensive enzyme and various attempts havebeen made to find cheaper alternatives from microbial sources

    These have ultimately proved to be successful and microbial rennets areused for about 70% of US cheese and 33% of cheese production

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    worldwide. The development of unwanted bitterness in ripening cheese isan example of the role of proteases in flavour production in foodstuff. Theaction of endogenous proteases in meat after slaughter is complex but"hanging" meat allows flavour to develop, in addition to tenderizing it. Ithas been found that peptides with terminal acidic amino acid residues

    give meaty, appetizing flavours akin to that of monosodium glutamate.

    The presence of proteases during the ripening of cheese is not totally undesirable anda protease from Bacillus amyloliquefaciens may be used to promote flavour productionin Cheddar cheese. Lipases from Mucor miehei or Aspergillus niger are sometimesused to give stronger flavours in Italian cheese by a modest lipolysis, increasing theamount of free butyric acid

    Meat tenderization by the endogenous proteases in the muscle after slaughter is acomplex process which varies with the nutritional, physiological and even psychological(i.e., frightened or not) state of the animal at the time of slaughter.Meat of older animals

    remains tough but can be tenderized by injecting inactive papain into the jugular vein ofthe live animals shortly before slaughter. Proteases are also used in the bakingindustry.

    A quick review of the different enzyme applications in chemical processing.

    Industry Effect

    Detergent

    proteinase protein degradation

    lipase

    cellulase

    fat removal

    color brightening

    Textile

    cellulase microfibril removal

    laccase color brightening

    Animal feed

    xylanase fiber solubility

    phytase release of

    phosphate

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    Starch

    amylases glucose formation

    glucose isomerase fructose formation

    Pulp and paper

    xylanase biobleaching

    Fruit juice

    pectinase

    cellulase,

    xylanase

    juice clarification,

    juice extraction

    Baking

    xylanase dough conditioning

    alpha-amylase

    glucose oxidase

    loaf volume; shelf-

    life

    dough quality