Lecture 3

1. Lecture 3 Exploitation of bioprocessing/biotransformation in particularly enzymes (but also other bio-products)

2. Bioprocessing

3. Exploitation of bioprocessing • Bioprocessing is the use of biological materials (organisms, cells, organelles, enzymes) to carry out a process for commercial, medical or scientific reasons. • Some industries have a long tradition of enzyme use: • • In leather tanning, hides are softened and hair removed using the proteasesin faeces. • • In brewing, amylasesin germinating barley are used to convert starch to maltose • Maltoseis then used by yeast for growth and ethanol production. • In cheese-making, the proteins in milk are coagulated using rennin from calf stomachs. Use glucose oxidase to detect glucose

4. Some products of bioprocessing

5. Why use bioprocessing? • They are specific in their action and therefore produce a pure product. • They are extremely efficient, so a little enzyme quickly makes a lot of product. • They are biodegradable and so cause less environmental pollution. • Safer, since any contamination with an enzyme or known microbe is harmless. • They work in mild conditions i.e. low temperatures, neutral pH and normal and normal atmospheric pressure- therefore low energy demand. • Some products (wine, cheese) are virtually impossible to create using chemical alone. • Some foods rely on microbial by-products to create/enhance flavour and so add value.

6. Types of enzymes

7. Enzymes for Downstream processing Extracellular enzymes are better because they are: • More robust and can cope with a wider range of environmental conditions (pH, temperature). • Easier to extract, and so cheaper to buy/use. • Have a longer functional ‘life’ in the commercial application. Steps (extracellular enzyme): • The microbes are removed by filtering or centrifuging. • The filtrate is concentrated (evaporation). • The desired enzyme/product is purified (molecular filtration)- a relatively easy step. • It is dried/packaged for sale. Steps (intracellular enzyme) As above, but with the following additional steps: • The cells are broken open, using ultrasound or other mechanical means. • The protein component is extracted, after removing the cell debris. • The individual enzyme is purified from the hundreds of others – by electrophoresis or chromatography. • This makes them much more expensive and difficult to obtain

8. Extracting intracellular enzymes • Sonication • Use of high frequency sound waves to disrupt cell walls and membranes • Can be used as continuous lysis method. • Better suited to small (lab-scale) operations. • Can damage sensitive proteins. • Pressure cells • Apply high pressure to cells; cells fracture as pressure is abruptly released • Readily adapted to large-scale and continuous operations. • Industry standard (Manton-Gaulin cell disruptor). • Enzymic lysis • Certain enzymes lyse cell walls • Lysozyme for bacteria; chitinase for fungi. • Only useful on small laboratory scale.

9. Industrial exploitation of enzymes • Proteasein biological washing powders: • Helps to break down protein stains such as blood, food and grass • At lower washing temperatures - thus saving energy and are gentler on clothes. • The enzymes are encapsulated in wax and only released during the wash. An extra rinse cycles is introduced to ensure enzymes are all removed before wearing • Pectinasesin food/juice modification. • Pectin forms part of cell walls and holds plant cells together – digested by pectinase. • Used to digest fruit and vegetables in baby food and • to extract fruit/vegetable juices e.g. for cider – it makes the juice clear, not cloudy. • The disadvantage of using free enzymes is that they cannot be re-used and therefore: • Contaminate the end-product and • Are more expensive to use

10. The nature of the downstream processing depends on two considerations: Whether enzyme is intracellular or extracellular. How pure the final product needs to be. Industrial enzymes can be quite crude, but medical enzymes must be extremely pure. The purer the enzyme, the more complex the downstream processing, and the more expensive it is.

11. Advantages of immobilise enzymes Immobilised enzymes are not free in solution – e.g. they can be held in a bead of soft permeable gel or coat the internal surface of a porous solid (see right). • Easier purification of the product as the separation of the enzyme beads is not a problem (thus cheaper). • Easy to recover and recycle the enzymes (thus cheaper). • The enzymes are protected in the beads and so remain functional for longer (thus cheaper).

12. Enzyme (and others natural products) Production/Isolation Methods • The structural complexity of enzymes makes their synthetic preparation a formidable task. They are natural products that are isolated/produced from three principle sources. • Isolation from animal organs (pig insulin- insulin is actually a hormone) • Isolation from plant material (papain- for tenderising meat, is a proteinase) • Microorganism production • Isolation and purification are complicated by the presence of similar proteins and the inherent sensitivity of enzymes to pH, temperature and degradation by other enzymes.

13. Immobilised enzymes • When purified enzymes are used to make large quantites of another product, downstream processing can be difficult and expensive. • Immobilising enzymes are cheaper. • Enzyme molecules are attached to a support matrix rather than free in solution. They still function properly but can be kept separate from the reactants and the products. • Immobilised enzymes are usually used in continuous flow-through re-actors, which have a low volume.

14. Advantages of Immobilization: • The production becomes a continuous process • Biocatalysts (enzymes) are not lostthroughout the reaction • Allows easy separation of the products • Provides increased resistance to changes in pH and temperature Other Examples of Immobilization Applications: • Transformation (modification) of steroids byimmobilized enzymes • Detoxification of pesticidepolluted water by immobilized bacteria

15. Range of enzymes and applications Dairy: Calf rennet (chymosin) is used in the coagulation of milk protein for cheese production, without loss of sensitive components. Lactase hydrolyses the principal carbohydrate of milk, lactose. This processes a cheese byproduct and relieves lactose intolerance. Detergents: Protein stain removal is facilitated by the hydrolysis of proteins into oligopeptides. Enzyme stability with respect to storage, pH, temperature and bleach are key concerns. Leather Production: Proteasesare widely used for the soaking and de-wooling stages of hide processing in which selective protein degradation results in a softer produce without substantial loss of strength. Food and Feed: Starch conversion to high-fructose corn syrup is an important process to the beverage industry.

16. Starch Conversion: High Fructose Corn Syrup • a-amylase degrades amylose to D-glucose, but a second enzyme, glucoamylase is needed to breakdown oligosaccharides derived from amylopectin. • This product can be used as substrate for yeast fermentation to produce ethanol as an alternate fuel source. Much of the glucose produced by starch degradation is isomerized to fructose for use as a low-cost (relative to sucrose) natural sweetener in soft drinks

17. Industry Focus: Food and beverage Fermentation Products • cheese • soy products • yoghurt • wine, beer • bread Enzymes • adjust food flavour • adjust food texture • improve nutritional quality • high fructose corn syrup

18. Fermentation- benefit to the organism • A form of anaerobic respiration occurring in certain microorganisms (ex. yeasts) • Alcoholic fermentation is a series of biochemical reactions by which pyruvate is converted to ethanol and CO2.

19. Examples of someimportantfermentations • Fermentation of sugarbyyeasts (basisforwineandbeerproductionandleavening of bread): C6H12O6 (sugar) + yeast 2C2H5OH (ethanol)+ 2CO2 • Fermentation of alcoholbybacteria (basisforvinegar, aceticacid, production): C2H5OH + O2 + bacteria CH3COOH (aceticacid) + H2O • Fermentation of milksugar, lactose, bybacteriaproduceslacticacidwhichcausesprecipitation of thecurd in themilk (basisforcheeseproduction).

20. The maximum enzyme production is usually in stationary phase of microbe growth, so a batch or fed-batch process are usually used. The medium must be chosen to stimulate the microbe into synthesising the correct enzyme. For example to stimulate a microbe to synthesise amylase enzymes, a medium with starch but no sugars is used. Optimising enzyme production

21. Winemaking fermenter

22. Must is fresh fruit juice Preparation of ‘must’ by stemmingand crushing of grapes (or other fruit) Addition of starter cultureof yeast and bacteria Fermentationof must(crushedfruit) or ofjuice aloneinto wine Figure 25.2 The wine-making process Clarificationof wine Aging of wine Bottling of wine

23. Barley is moistened and germinated,producing enzymes that convertstarch into sugars. Barley is then driedto halt germination, and crushed toproduce malt. Mashing malt andadjuncts with warmwater allowsenzymatic activityto generate moresugars. Solids areremoved toproduce wort. Figure 25.3 The beer-brewing process Mashing kettle Addition of hops for flavoring Cooking of wort halts enzymaticactivity, extracts flavor from hops,and kills the microorganisms present. Removalof hops Addition of yeast culture Wort fermentsinto beer. Aging, filteringor pasteurization,and bottlingfinish theprocess.

24. Beer making • 2. Malting • The appropriate variety of barley (some are more suitable to the production of malt whiskey or food rather than beer), • are allowed to soak in water for about 40 hours, with draining and new water added every 8 hours. • Once the barley grains reach 40-45% moisture the barely is allowed to germinate around 15.5˚ C. • Germination of the grain allows for plant enzymes to convert carbohydrates into more simple sugars like glucose. • Once the grain shoot forms, the grains are dried with a gradual rise in temperature.

25. Beer making • 3. Mashing • The barley is cracked open so that water can get inside and activate the enzymes. • These enzymes called diastases, become most active around 69 ºC. They convert the starches from the barley into simple sugars. • After the solids are strained out the dark, sweet liquid is called "wort." • The wort must be boiled for 30-90 minutes depending on the recipe.

26. Beer making • 4. Hops are added at different times during the boiling phase. • Hops have tiny oil glands that contain oils and resin that contribute to the aromatic flavor and bouquet of the beer. • Hops contribute to the bitterness of the beer are added early in the boil so the resins have time to dissolve into the wort. • Hops that are added for their aromatic flavouring are added within the last few minutes of the boil. Otherwise the quickly dissolved oils get steamed out of the wort.

27. Beer making • 5. The wort is cooled, so the yeasts to be added next don't die. • 6. The yeast is added either as a freeze-dried powder or as an actively growing liquid. Each has its advantages. • 7. The yeast is allowed to ferment the wort for up to 10 days, depending on the type of beer. During this phase, the alcohol is made and the carbonation is allowed to escape through the fermentation lock.

28. 7. Beer making • Respiration--the yeast converts simple sugars to carbon dioxide and water. • Fermentation--the conversion of sugar to alcohol and carbon dioxide. It is the longest of the three phases. At its peak, which is also the start of sedimentation, the yeast has a density of 50 million cells per milliliter. • Sedimentation--the yeast cells settle to the bottom of the fermentation vessel because most of the sugars have been converted and utilized for respiration, and the begin to prepare for dormancy. Sedimentation last for 2-3 days. At the time the beer appears clear, the yeast's density is less than 1 million cells per milliliter.

29. Making Yoghurt • Starter cultures contain bacteria that make lactic acid from the sugar (lactose) in the milk. lactose lactic acid (sugar in milk) (thickens and gives taste) • Lactic acid thickens the milk and gives the yoghurt its taste. • Lactobacillus and Streptococcus

30. Pasteurization killsunwanted microorganisms Addition of starterbacterial culture Figure 25.1 The cheese-making process Coagulation of milkproteins (curd formation) Production ofunprocessedcheeses Disposal ofliquid whey aswaste product Cutting of curds Production of processedcheeses through pressing,addition of secondarymicrobial cultures, andaging (ripening)

31. Industry Focus: Textiles

32. Indigo dye adheres to denim surface Cellulase enzyme removes some of the dye by partially hydrolyzing the cotton surface Denim is faded by abrasive action of pumice stones Stone washing denim traditional method new method • lower costs • shorter treatment times • less solid waste • weakens the fabric

33. Detergents • Detergent industry is the largest single market for enzymes at 25 - 30% of total sales • Dirt comes in many forms and includes proteins, starches and lipids (fats and oils) • proteases, amylases, lipases are enzymes used in detergents • enzymes allows lower temperatures and less agitation for washing Inner core of enzyme Protective waxy coat that disperses in the wash

34. Industrial and medical applications of immobilised enzymes

35. Glucose Biosensor • Use in diabetes monitoring • Glucose + O2 → glucose oxidase →gluconic acid + H2O2

36. Exploitation of immobilised enzymes • Applications in food industry: • Lactose removal in milk • Milk contains 5% lactose which must be removed before feeding babies deficient in β-galactosidase (lactose hydrolyzing enzyme). A column packed with β-galactosidase immobilized in polyacrylamide gel allows continuous lactose removal. • Hydrogen peroxide removal in milk after sterilization • Immobilized catalase by covalent binding with carboxychloride resin • Bitterness removal in orange juice • Naringinase (produced by Bacillus) immobilized on copolymer, used in both batch and column systems

37. Exploitation of immobilised enzymes • Broad in waste treatment and medicine: • Reduction of phenol content in wastewater using immobilized polyphenol oxidase. • Removal of cyanide: • Improve use of microorganisms to decompose cyanide by entrapping enzyme in polyacrylamide gel and packed into column. • Wound dressings: Immobilization of proteolytic enzymes (trypsin- hydrolyses protein) onto textiles used as wound dressings to accelerate cleaning and healing of infected wounds and reduces amount of enzyme used as compared with use of enzyme solutions.