Enzyme Technology

For thousands of years natural enzymes made by microorganisms have been used to make products such as cheese, bread, wine, and beer. Enzymes are now used in a wide range of industrial processes. The study of industrial enzymes and their uses is called enzyme technology. The advantages and disadvantages of using enzymes are directly related to their properties: Advantages They are specific in their action and are therefore less likely to produce unwanted byproducts Disadvantages They are highly sensitive to changes in physical and chemical conditions surrounding them. They are easily denatured by even a small They are biodegradable and therefore cause less environmental pollution increase in temperature and are highly susceptible to poisons and changes in p . Therefore the conditions in which they work must be tightly controlled. They work in mild conditions, i.e. low temperatures, neutral pH and normal atmospheric pressure, and therefore are energy saving The enzyme substrate mixture must be uncontaminated with other substances that might affect the reaction. icroorganisms produce enzymes

icrobes are still the most common source of industrial enzymes.

inside their cells !intracellular enzymes" and may also secrete enzymes for action outside the cell !e!tracellular enzymes". The microorganisms selected are usually cultured in large fermentation chambers !known as fermenters see later" under controlled conditions to maximise enzyme production. The microorganisms may have specific genes introduced into their #$% through genetic engineering, so that they produce enzymes naturally made by other organisms & this is explained in further detail under the genetic engineering section of this unit.

"rowing microbes in a fermenter

'iven a suitable nutrient medium and the right conditions !temperature, pH, oxygen levels !many microbes are obligate anaerobes, i.e. are killed by oxygen", it is easy to grow microbes on a laboratory scale in (etri dishes, test tubes and flasks. However, producing substances such as penicillin from

microbes on an industrial scale causes serious problems because massive numbers of organisms have to be grown for commercial use. The microorganisms are grown in very large vessels called fermenters ) as shown in this simplified diagram* The large stainless steel cavity is filled with a sterile nutrient solution, which is then inoculated with a pure culture of the carefully selected fungus or bacterium. (addles rotate the mixture so that the suspension is mixed well. %s the nutrients are used up, more can be added. (robes monitor changes the mixture in pH, and oxygen

concentration and temperature are all computer controlled. % water +acket surrounding the fermenter contains fast flowing cold water to cool the fermenter since fermentation is a heat generating process. ost of the air, including carbon dioxide and other gases produced by cell metabolism, leave the fermenter by an exhaust pipe. There are two main types of culture used in industrial processes* batch cultures and continuous cultures. #atch cultures $ontinuous cultures

cells are grown in a fixed volume of li,uid

medium in a closed vessel

nutrients are added and cells harvested at

a constant rate

$o microorganisms, fluid or nutrients are -sed for producing secondary

added or removed from the culture during the incubation period metabolites, such as penicillin and other antibiotics, which are relatively unstable and not essential for the growth of the culture

/olume of suspension is kept constant Fermenter does not have to be emptied,

cleaned and refilled very often

(roduction is almost continuous 0ontinuous cultivation needs sophisticated

e,uipment to maintain constant conditions. Highly trained staff need to operate the e,uipment. Therefore this

.econdary metabolites can be extracted

economically only when they reach a high concentration in the culture

process can be expensive

Metabolite - organic compound that is a starting material in an intermediate, or an end product of metabolism. Starting materials usually include vitamins and amino acids. They can be used to construct more complex molecules, or they can be broken down into simpler ones. Inrermediatroy metabolites are the most common. they may be synthesised from other metabolites and used to make more complex substances or broken down into simpler compounds

%e&uirements for the production of microbes in fermenters:

anaerobes and oxygen must be excluded

'back to top(

)!ygen is needed for aerobic respiration of !some" micro&organisms ) others are strict a source of $arbohydrate is needed as an energy source for respiration to release energy
needed for growth.

a source of *itrogen is needed need nitrogen for protein synthesis ) Ammonia !$H1" and urea
+!$H2"203" are both widely used as !cheap" sources of useable nitrogen

,solating the Enzyme

-ure enzymes are needed for commercial use4 therefore microbes must be grown in aseptic conditions, free from contaminants & such as unwanted chemicals & and other microbes. 5t is necessary to prevent contamination with other bacteria since*

there may be competition for nutrients4 the re,uired enzyme may not be produced as readily4 the end&product may be contaminated and unsafe.
The re,uired enzyme that is finally produced must also be isolated from the microbial cells. E!tracellular enzymes are present in the culture outside the microbial cells, since they have been secreted. They are often soluble in water, so they can readily be extracted from the culture medium and purified. 6ess common in $ature !though genetic engineering can be used to modify cells to promote this", these enzymes are cheaper to produce and tend to be more stable ) they are therefore the preferred choice, when available.

 To obtain an intracellular enzyme, the microbe cells are harvested (by filtration or
centrifugation) from the culture and are then broken up. The mixture is next centrifuged to remove large cell fragments and the enzymes !all of them7" are precipitated from solution by a salt or alcohol. The re&uired enzyme must then be purified by techni,ues such as electrophoresis or column chromatography. This last process is complicated and expensive, so these enzymes are only used when no other alternative is available. 8y their very nature, they tend to be more sensitive to their operating conditions, which makes their commercial use less easy. 3n the other hand, they are much more common in $ature7

$omparing intra- and e!tra- cellular enzymes

,ntracellular enzymes ore difficult to isolate E!tracellular enzymes Easier to isolate $o need to break cells ) secreted in large amounts into medium surrounding cells 3ften secreted on their own or with a few other enzymes ore stable (urification9downstreaming processing is easier9cheaper /icroorganisms such as bacteria and fungi are saprobionts i.e. they feed saprophytically0 secreting enzymes onto their food ) making them a good source of extracellular enzymes. For example, the fungus Aspergillus niger produces an enzyme called pectinase, which breaks down pectin, a substance found in the cell walls of plant cells. The fruit +uice industry uses pectin widely, since when fruit is crushed to extract the +uice, pectin prevents some being released and also makes the +uice cloudy. The stainless steel fermenter with complicated control systems is not actually the most expensive part of the process. %lmost :;< of the cost is accounted for by downstream processing: the isolation, extraction, and purification of the product at the end of the culture in the fermenter. Downstreaming uses a variety of techni&ues. 5n the first stage cells need to be separated from the

0ells have to be broken apart to release them

Have to be separated out from cell debris and a mixture of many enzymes and other chemicals 3ften stable only in environment inside intact cell (urification9downstreaming processing is difficult9expensive

li,uid part of the suspension. This can be done by sedimentation, centrifugation or filtration. 5f the cells themselves are the desired product !e.g. for single&cell protein production for animal feed" then they need to be sterilised, washed, dried and packaged. 5f the desired product is a chemical within the cells, the cells have to be broken apart to release the chemical and the cellular components removed. The desired chemical is then extracted and purified by a number of techni,ues such as precipitation and chromatography. Finally, the purified chemical has to be dried and packaged in a suitable form. 5n the case of the enzymes in biological washing powders, this means coating the granules with wax to ensure that they remain dry until used ) otherwise the enzymes would digest themselves7

/edical uses of enzymes

Diagnostic:
=eagent strips have been designed to perform rapid and semi&,uantitative analysis for glucose. They are easy to use and re,uire no addition laboratory e,uipment or reagents. % $linisti! contains molecules of two enzymes fixed onto the end of a plastic strip. >hen this is dipped into a sample, the first, glucose o!idase0 converts any glucose molecules, by reaction with atmospheric oxygen, into gluconic acid and hydrogen peroxide. The second enzyme, pero!idase0 then enables the hydrogen peroxide to react with an indicator to give a purple colour. % colour chart on the strip will match the shade of purple to the glucose concentration. The idea of fixing an enzyme to a plastic support is the basic principle of biosensors & mobile, cheap and accurate sensors which can monitor a number of biochemicals in blood, urine and also in food and soil. 3ver the next few years, the use of biosensors is likely to increase dramatically. .tep ?
Glucose oxidase

'lucose &&&&&&&&&&&&&&&&&&&&&&&&&&&&@ 'luconic %cid A Hydrogen (eroxide .tep 2

Peroxidase

Hydrogen peroxide A !reduced" orthotolidine &&&&&&&&&&&&&&&&&&&&&&&&&&&&@ >ater A !oxidized" orhotolidine

Brown Blue

Treatment:

The approved treatments for strokes are i.v. prescription drugs -rokinase, .treptokinase and t&(% !tissue& plasminogen&activator". %ll three are available in the form of intravenous infusion only. To work best, they must be given within three hours from the onset of the attack. % floating time bomb, an embolism could be trapped and block any artery.

Fibrin filaments wrap around and entrap a single red blood cell.

The beginning of a blood clot* (latelet and =ed 8lood 0ells are trapped in a network of fibrin cables.

The industrial use of enzymes +using the whole microbe1

Historically, three examples of the industrial use of microbes !and their enzymes" are*

#rewing* in which yeast !Saccharo yces cere!isiae) reacts with the sugars in fruit or malted barley to
produce ethanol and carbon dioxide. 5n $ature, the yeast is competing with bacteria for the available sugar in the wild fruit. 5ts response is a form of Bchemical warfareC, since the ethanol it makes it poisonous to many bacteria and, indeed, ethanol can be used as a disinfectant !though it stings a lot7". The process of fermentation takes several days or weeks and results in a product with a maximum alcohol content of about ?2< & above which the yeast is itself killed. ore alcoholic beverages can only be made by distilling the raw brew. From wine we get brandy, cider gives 0alvados, ale gives whisky. .urplus yeast could then be used to mix with flour and water to make !leavened" bread. Hence brewing and baking are closely related. 5n baking, the carbon dioxide is the important product, since it makes the

dough rise. the ethanol evaporates off in the baking, so you cannot get drunk by eating bread7 The reason why Bin&store bakeriesC are so popular in supermarkets is that the smell of baking bread !and the ethanol" in the air circulates throughout the store and this stimulates our Bhunger centreC and so we buy more & ,uite true7 These days the surplus yeast is heated and processed to make armite.

2inegar production:
6ouis (asteur was employed by Emperor $apoleon 555 in ?:DE to research why !sometimes" wine went BoffC or turned to vinegar. (asteur soon showed that the historic Bspontaneous generationC theory was wrong ) substances did not spontaneously go BbadC4 instead he formulated the modern Bgerm theoryC. This states that it is the existence of microbes which makes food rot. The secret to keeping wine was thus to keep the microbes out i.e. bottle it, rather than storing it in open casks. To make vinegar, wine is slowly pored over oak chips in a tall tower, open to the air. 8acteria !Acetobacter) on the wood oxidise the ethanol in the wine and turn it into ethanoic acid or vinegar, giving out a great deal of heat as well. 5f the vinegar is made from fermented raisins and stored in oak vats !similar to the solera syste making sherry" then the sweet, highly&prized Balsamic vinegar is made ) mainly around >orld7 used for odena in

5taly. $ote how different forms of Bsweet and sourC dishes are a part of local cuisine from all over the

3oghurt production:
ilk goes sour within a few hours in the hot conditions common in the iddle East. 5f stored in a leather

bag and mixed with a suitable starter culture, however, it rapidly turns into yoghurt, which will keep for several days. This happy accident led to the development of the modern industry, which thus has its roots in 8iblical times !%braham was said owe his longevity to drinking yoghurt". 5n =ussia,"efir is a similar ancient product with a fascinating modern commercial history, beginning in ?F;: with the attempted seduction of a (rince 8archarov, the kidnapping of a beautiful maiden !5rina" and a court case with a fine of Bthe (rophetCs 'rainsC, which were the sacred starter culture for Gefir. 5n ?FH1 the for Food in the -..= wrote to 5rena thanking her for bringing Gefir to the .oviet people7 inister

The industrial use of enzymes +not using the whole microbe1

4eather:

The earliest example of enzymes in industry is a colourful one7 To make leather soft, it has to be bated0 which means that some of the protein fibres are removed. 3therwise, the leather will be hard & perfect for the soles of shoes but of little use for anything else. The =oman writer (liny reported the use of pigeon droppings for this process over 2;;; years ago. 6ater, leather was bated by smearing it with dog excrement7 (eople used to go around the streets collecting dog turds and then rubbed them into the skins by hand, paddle or by trampling it in by foot. 8y the early ?F;;Cs it was known that the excrement was rich in bacteria which produced p roteases, which degraded part of the leather. 5t was a highly skilled +ob to prevent the enzymes damaging the leather, which is largely made up of protein. 8ut thanks to the The 'erman scientist =Ihm, developed a standardized bate in ?F;:, based on an extract from the pancreases of slaughtered animals. This contained trypsin & one of a mixture of enzymes found in the digestive system. .ince then, all bates have been based on enzyme preparations, though now bacterial and fungal enzymes are used instead.

5ashing powders:
=Ihm was ,uite a genius & he was the first to examine the chemical composition of dirt on laundry and he came up with the idea of using the pancreatic extract to wash clothes. His wife tested trypsin at home on their dirty underwear & and found it was excellent7 >hen soaked overnight, their clothes became clean and the water became dirty. .o, he patented his idea and in ?F?E, developed the first enzymatic washing agent. 5t was so effective that only a small ,uantity was re,uired* it was sold as a spot remover. -nfortunately 'erman housewives were used to bulky washing powders that produced lots of lather so they regarded it with suspicion. 5n ?F?J, some people even thought it was a hoax. The product was investigated by scientists who found that it really did work ) indeed, it was about J; years ahead of its time* it wasnCt until the ?FD;s that enzymatic detergents gained widespread acceptance. The mass&production of an alkaline protease suitable for wash conditions began in ?FD2. -nlike trypsin, this wasnCt an animal extract but a product of microbial fermentation. This new enzyme was initially shunned by detergent manufacturers but there were exceptions. 5n ?FD1, it was incorporated into Biotex, which took the market by storm. 5ndustry began to take notice of enzymes and by ?FDH their widespread use in domestic detergents was commonplace.

Enzymes used: These are produced from Bacillus lichenifor is. They are usable at high pH and temperatures up to D;o 0 and are all relatively non&specific proteases. They attack the 0&terminal of carboxyl amino acids producing small peptides which can be readily dissolved by the detergent. There is currently considerable

interest in developing better proteases for washing powders through protein engineering, particularly in engineering oxidation&resistance into the proteases. #ngineered Subtilisin for i pro!ed wash perfor ance *ot 6ust proteases .ince the ?FF;Cs, amylases have also been added to detergents to remove stains from spaghetti, sauces, oatmeal and baby foods. 5n ?F:: the first detergent lipase was released & the first commercial enzyme to be produced from a genetically&modified organism !' 3". Today more than F;< of detergent enzymes are made from ' 3s. The detergent industry has been the largest market for industrial enzymes for over 2J years, accounting for 1H< of world sales of enzymes. %part from laundry detergents, many automatic dishwashing detergents now also contain enzymes. To maximise the effectiveness and to be as economical as possible in the production process the enzyme molecules must be brought into maximum contact with the substrate molecules. This can be achieved by mixing the solutions of enzyme and substrate in suitable concentrations. in cheese manufacture* owever this means that the enzyme is BlostC with each batch of product made and that the end&product will be contaminated too ) as

$heese making: >arm milk is mixed !about 2;;;*?" with the enzyme rennin +rennet) !formerly
extracted from !dead" calvesC stomachs, but now produced from bacteria" and allowed to react for several hours. The caesinogen in the milk is uncoiled and clots to casein. This turns the milk solid. The curds !solid" are then cut with a knife and the whey !li,uid" drained away and fed to animals !remember iss TuffetK". The chopped up curd is then salted and placed in a mould before s,ueezing to remove any trapped air !a process known as BcheddaringC ) hence 0heddar cheese". .ometimes the cheese is then dipped in brine or a solution of fungal spores to inoculate it and produce a surface BrindC. The cheese is then left at a constant, low, temperature !in the old days, in a cave, hence many cheeses are associated with cave&rich districts" to mature. this may take up to a year or more, so cheese&making was an important way of preserving a valuable food through the winter in the days before refrigeration. The BblueC in cheeses such as .tilton, is added by pushing spore&covered wires ! Penicilliu notatu & the

same fungus that gave us penicillin" into the partially ripened cheese. This fungus needs oxygen to make the blue pigment, so holes have to be made in the cheese ) the more holes, the faster the blue veins develop. The role of the rennin in young mammals is to clot the motherCs milk in babyCs stomach. This then BtricksC the stomach into keeping the contents there for several hours, thus allowing protein digestion and the

mother to get some !much&needed" rest7 5n most mammals the rennin is only made until the animal is weaned, but in 0aucasian people, milk was !historically" drunk throughout life and so the enzyme continues to be made, even in adults. %lso made is the enzyme lactase, which breaks down the milk sugar and stops the bacteria from fermenting it in the colon, with subse,uent large volumes of gas produced and embarrassing side)effects7

-roducing a pure product more cheaply 7 re-using the enzyme

5t would obviously be more economic to retain the enzyme and so be able to re&use it for several batches of product. 5n addition, the product would be pure and uncontaminated by any enzyme !though, since this is a natural product and a simple protein, it is unlikely to do any harm to the end&user". However, the enzyme needs to be in contact with the substrate in order to react. The solution to this dilemma is to use Bimmobilised enzymes8:

8y attaching the enzyme molecules to an inert surface !such as plastic beads" and then bringing
the surface into contact with a solution of the substrate. This method has the advantage of enabling the enzyme molecules to be used over and over again, with the result that a lot of product can be made from a relatively small amount of enzyme. %n example of continuous production using an immobilised enzyme is*

Fructose syrup production from glucose using glucose isomerase

,mproving the Enzyme: ,mmobilsation

%s enzymes are catalytic molecules they are not directly used up by the process in which they are used. However due to denaturation, they do loose activity with time. Therefore they should be stabilised against denaturation. >hen the enzymes are used in a soluble form they can contaminate the product, and its removal may involve extra purification costs. 5n order to eliminate wastage and improve productivity the enzyme and product can be separated during the reaction. The enzyme can be imprisoned allowing it to be reused but also preventing contamination of the product ) this is known as immobilisation. -nstable enzymes may be immobilised by being attached to or located within an insoluble support, therefore the enzyme is not free in solution. 3nce attached, an enzymeCs stability is increased, possibly because its ability to change shape is reduced. There are four main methods available for immobilising enzymes:

a b. c. d.

%dsorption in glass or alginate beads ) enzyme is attached to the outside of an inert material 0ross&linkage to another chemical e.g. cellulose or glyceraldehydes. Entrapment in a silica gel ) enzyme is held in a mesh or capsule of an inert material. embrane confinement

$ompared with free enzymes0 immobilised enzymes have several other advantages: Advantages of immobilisation ?. 2. 1. Easier to separate enzyme and products %llows catalysis in unfavourable media 5ncreases stability and can be manipulated Disadvantages of immobilisation ?. 2. 5mmobilisation may alter shape of enzyme ay alter catalytic ability

easily E. %llows continuous production9enzyme used

1.

Enzyme may become detached

for longer J. D. Enzyme can be recovered and reused Enzyme does not contaminate product9no

E.

Expensive

purification re,uired

,mproving the Enzyme: 9tability

The stability of an enzyme refers to its ability to retain its tertiary structure so that it continues to be effective under a wide range of conditions. environments. ost enzymes are relatively unstable and work only within narrow ranges of temperature and p . They ,uickly become denatured when sub+ected to unnatural any industrial processes re,uire enzymes to work in the presence of chemicals such as organic solvents, at high temperatures and extremes of pH ) conditions with cause most enzymes to lose their shape and become inactive. 5t is possible to overcome this problem by taking advantage of microbes that live naturally in harsh environments. 3rganisms evolve to produce enzymes that are adapted to their environmental conditions. .ome bacteria are adapted to extreme environments e.g. some bacteria may live in hot volcanic springs. They will produce thermostable enzymes that do not denature at high temperatures ) they work effectively in the temperature range DJ&HJL0. They will also remain effect at lower temperatures for much longer than BnormalC enzymes and so are the preferred choice for industry. These enzymes are also resistant to organic solvents and tolerate a wide range of pH. The gene for a thermostable enzyme can be isolated from the natural bacteria and transferred to a microbe BhostC that can be used in the industrial process. This enables a thermostable version of the desired enzyme to be produced.