A Systems Approach to advanced enzyme technology

1. DINAZYME A Systems Approach to advanced enzyme technology

2. Background History of enzymes Alcoholic fermentation oldest known enzyme reaction Y + G A + CO2 • Phenomena believed to be spontaneous reactions until 1857, when the French chemist Louis Pasteur proved that fermentation occurs only in the presence of living cells. • Subsequently the German chemist Eduard Buchner discovered (1897) that a cell-free extract of yeast can cause alcoholic fermentation. • The ancient puzzle was then solved; the yeast cell produces the enzyme and the enzyme brings about the fermentation • As early as 1783 the Italian biologist Lazzaro Spallanzani had observed that meat could be digested by gastric juices extracted from hawks.

3. Background History of enzymes • Probably first experiment in which a vital reaction was performed outside the living organism • After Buchner's discovery scientists assumed that fermentations and vital reactions in general were caused by enzymes • Nevertheless, all attempts to isolate and identify their chemical nature were unsuccessful • In 1926 the American biochemist James B. Sumner succeeded in isolating and crystallizing urease.

4. Background History of enzymes • Four years later pepsin and trypsin were isolated and crystallized by American biochemist John H. Northrop • Enzymes were found to be proteins, and Northrop proved that the protein was actually the enzyme and not simply a carrier for another compound • Research in enzyme chemistry in recent years has shed new light on some of the most basic functions of life • Ribonuclease, a simple three-dimensional enzyme discovered in 1938 by American bacteriologist René Dubos. • Isolated in 1946 by American chemist Moses unitz • Synthesized by American researchers in 1969

5. RIBONUCLEASE ENZYME

6. Background History of enzymes • The synthesis hooks 124 molecules in a specific sequence to form the macromolecule • Led to identification of those molecular areas that carry out its chemical functions • Opened up the possibility of creating specialized enzymes with new properties • This potential has been greatly expanded in recent years by genetic engineering techniques that have made it possible to produce some enzymes in great quantity

7. How are enzymes manufactured?

8. How are enzymes manufactured?

9. Industry drawbacks • Non-specific reactions may result in poor product yields. • High temperatures and/or pressures needed to drive reactions lead to high energy costs. May require large volumes of cooling water downstream. • Harsh and hazardous processes involving high temperatures, pressures, acidity or alkalinity need high capital investment, and specially designed equipment and control systems. • Unwanted by-products may prove difficult or costly to dispose of. • High chemical and energy consumption, and harmful by-products have a negative impact on the environment.

10. Drawbacks eliminated by enzymes • Reactions carried out under mild conditions • Highly specific • Involve very fast reaction rates • Reactions are carried out by numerous enzymes with different roles. • Industrial enzymes originate from biological systems which contribute to sustainable development through being isolated from microorganisms which are fermented using primarily renewable resources. • Small amounts of enzymes are required to carry out chemical reactions • Reaquires little storage space. • uncomplicated and widely available equipment can be used • Reactions are easily controlled and can be stopped when the desired degree of substrate conversion has been achieved. • Reduce the impact of collateral damage on the environment by reducing the consumption of chemicals and energy, and the subsequent generation of waste. • Developments in genetic and protein engineering have led to improvements in the stability, economy, specificity and overall application potential of industrial enzymes.

11. What are enzymes? An enzyme is a protein which acts as a specific biological catalyst facilitating a given reaction by lowering the amount of required energy. To date, scientists have identified over 1,500 different enzymes.

12. What are enzymes? • Six main classes by type of reaction catalyzed • Classes are split into groups and subclasses • Ex., lactase catalyzes the conversion of milk / sugar to galactose and glucose • Lactase has the systematic name beta-D-galactoside galactohydrolase, and the classification number EC 3.2.1.23.

13. SIX MAIN ENZYME CLASSES

14. What are enzymes? • Globular, water soluble proteins, (few exceptions) • Allows / facilitates chemical reactions to occur such as those that release nutrients from feed during digestion • Without the enzyme catalyst the reaction would either not take place or would happen very slowly • If a reaction is favorable ( ∆G < 0), the activation energy E(act) determines how fast it will go.

15. What are enzymes? • Though an enzymatic catalyst takes part in the chemical reaction it remains unchanged and is available to repeat the task

16. What are the most important enzymes to our industry? • Virtually all enzymes employed in the feed industry are hydrolases. • Some enzymes that are of practical value to the livestock industry: • Xylanases, amylases, phytases, proteases, cellulases, betaglucanases, and pentosanases, are available for use in diet formulations. • These enzymes can be mixed and matched to form an enzyme cocktail to fit any particular diet need.

17. Why are enzymes needed in feed formulations? Trials confirm that enzyme supplementation results in improved animal performance. • Young animals lack many endogenous enzymes or sufficient quantities thereoff. • Sick animals may have a damaged intestinal lumen resulting in limited nutrient absorption. • Animals under stress or at a high level of production may have an impaired digestive system.

18. Why are enzymes needed in feed formulations? Problems in feed ingredients: • Raw materials may contain anti-nutritive factors. Ex. pentosans or betaglucans present in wheat or barley. • Addition of appropriate enzyme aids digestion of the material improving feed value. • Increasing environmental awareness and restrictions on pollutants and contaminants confirm the value of enzymes in the breakdown of such materials. Ex Phytase/ phosphorus

19. How do enzymes work? Specificity • Specific enzymes may be incorporated into specific diets in order to solve specific problems

20. How do enzymes work? • Enzyme catalyzed reactions are often from 100 million to more than 10 billion times faster than the same reaction in the absence of the enzyme. • Most enzymes catalyze the transfer of electrons, atoms or functional groups.

21. Factors influencing enzyme activity • Optimum pH • Optimum Temperature

22. Factors influencing enzyme activity • Optimum Enzyme concentration • OptimumSubstrate concentration

23. Factors influencing enzyme activity • Covalent modification

24. Factors influencing enzyme activity • Inhibitors A competitive inhibitor A non-competitive inhibitor molecule

25. Factors influencing enzyme activity • Allosteric Effectors

26. Factors influencing enzyme activity • Optimum pH:pH at which enzymes operate best. Activity decreases on either side of pH optimum.

27. Factors influencing enzyme activity • Optimum Temperature: • Within a given range, for every 10 degrees the temperature increases, enzyme activity doubles. • Enzymes become denatured at elevated temperatures. • Enzymes have an optimum temperature which varies according to: • Enzyme source. • Salt levels in the medium to which the enzyme is added. (For example, amylases from animal sources are less heat stable than those from fungal sources (Aspergillus) which are in turn less stable than bacterial amylases (Bacillus). • Mineral Content: Certain minerals stabilize enzymes while others cause inactivation. Calcium and magnesium are essential for good starch breakdown (amylases) and increase enzyme stability to temperature. Heavy metals such as iron are typically detrimental to enzymes, and may in some cases be used to inactivate or stop enzyme reactions.

28. Enzyme concentration • Normally enzymes are present in cells in low concentrations. • As enzyme concentration increases the rate of the reaction increases linearly, because there are more enzyme molecules available to catalyse the reaction. • At very high enzyme concentration the substrate concentration may become rate-limiting, so the rate stops increasing.

29. Substrate concentration • As the substrate concentration increases, the rate increases because more substrate molecules can collide with enzyme molecules, so more reactions will take place. • As substrate concentration gets higher the enzyme molecules become saturated so there are few free enzyme molecules. Adding more substrate doesn't make much difference (though it will increase the rate of E-S collisions). • The maximum rate at infinite substrate concentration is called vmax, • The substrate concentration that gives a rate of half the maximum rate vmax is called KM. • The vmax and KM values are useful for characterising an enzyme. • A good enzyme has a high vmax and a low KM. 

30. Substrate concentration

31. Covalent modification • Activity of some enzymes is controlled by others. • These enzymes modify the protein chain by cutting it, or adding a phosphate or methyl group. • Turns inactive enzyme into active (or vice versa). • Used to control many metabolic enzymes and to switch on enzymes in the gut e.g. hydrochloric acid in stomach activates pepsin activates rennin.

32. Inhibitors • Inhibitors inhibit the activity of enzymes, reducing the rate of their reactions. • Found naturally, but are also used artificially as drugs, pesticides and research tools.

33. Inhibitors • There are two kinds of enzymatic inhibitors. Non-competitive Competitive

34. Competitive Inhibitors • Molecule has similar structure to normal substrate molecule. Fits into active site of the enzyme. • Competes with substrate for the active site, so reaction is slower. • Increase KM for enzyme, but no effect on vmax. • The rate can approach a normality if substrate concentration is increased sufficiently. • The sulphonamide anti-bacterial drugs are examples of competitive inhibitors.

35. Non-competitive inhibitors • Inhibitor molecule is different in structure than the substrate molecule • Will not fit into active site. • Binds to another part of the enzyme molecule. • Change enzyme and active site shape so it no longer binds substrate molecules. Result is reduction of active enzyme numbers (just like decreasing the enzyme concentration). Therefore decrease vmax, but have no effect on KM. • Reversible inhibitors - bind weakly and can be washed out. • Irreversible inhibitors - bind tightly and cannot be washed out. • Poisons like cyanide, heavy metal ions and some insecticides are all examples of non-competitive inhibitors.

36. Michaelis-Menten equation RATE EQUATION RATE EQUATION FOR PRODUCT INHIBITION

37. Allosteric Effectors • Allosteric site is distinct from the active site • Different molecules can inhibit or activate the enzyme, allowing sophisticated control of the reaction rate • Few enzymes can do this. They are often at the start of long biochemical pathways • Generally activated by the substrate of the pathway and inhibited by the product of the pathway, thus only turning the pathway on when it is needed • Activity of some enzymes is controlled by certain molecules binding to a specific regulatory or allosteric site on the enzyme.

38. Allosteric Effectors

39. Economic benefits • Increases daily weight gain • Increases egg production • Lowers feed conversion • More uniform weights / increased nutrient absorption • Lower incidence of digestive problems caused by unassimilated fiber which also improves litter quality • Reduces fecal volume and nitrogen excretion levels • Cleaner eggs and better egg yolk color • Use of lower cost ingredients • Maintains and improves performance levels • Increases ratio of lean to fat tissue • Can "inactivate" mycotoxins in feeds

40. What is Dinazyme? Several types of enzyme technologies are offered as Dinazyme • Dinazyme B/W Dry and Liquid • Dinazyme C/S PBM Dry and Liquid • Dinazyme PSE, (Phytase) Dry

41. What is Dinazyme C/S PMB • Supplement for corn soy based poultry and pig diets containing high glucan barley levels.

42. What is Dinazyme C/S PMB • A diet supplement which enhances nitrogen utilization and increases protein digestibility with the active ingredient protease, resulting in increased absorption of amino acids and peptides. • DINAZYME C/S® also contains amylase-breaks down starch content and Xylanase, a complex hydrolytic enzyme preparation which has an effect on hemicellulose substrates containing xylan, manan and glucan. • A combination of amylase, Xylanase and protease boosts the digestibility of typical corn and soybean meal-based diets, resulting in more nutrients available for growth. • Inclusion of DINAZYME C/S® in diet supplements provides endogenous enzymes animals lack or produce in low amounts.

43. WHAT MAKES DINAZYME MORE EFFECTIVE THAN OTHER ENZYMECOMBINATIONS • Effective action due to presence of other important hydrolytic enzymes, which decompose cellulose, lichenin, araban and pectin. • Dinatec makes use of important technical concepts such as: • Covalence modification, • The use of specific allosteric substances and enzyme co-factors that are conducive to higher enzymatic efficacy • Enzyme concentration

44. What can Dinazyme C/S PMB do for you? • Contents / effects: • Protease, enhanced nitrogen utilization and increased protein digestibility • Amylase, increased digestibility of starch in pig and poultry diets • Betaglucanase, reduced digesta viscosity in poultry diets; decreases anti- nutritional effects of NSP*; reduces soluble NSP in disgesta. • Pectinase, more complete hydrolysis (digestion) of pectins in wheat and corn based diets • Xylanase reduces digesta viscosity; decreases anti-nutritional effects of NSP; reduces soluble NSP in digesta hence increased absorption of amino acids and peptides. * Non Starch Polysaccharide

45. Why should you use Dinazyme? High energy diets high on starch and protein content are desirable at an early age for the monogastric. Young animal's endogenous enzyme system not fully developed. Unable to adapt quickly enough for demands of current feed management programs. Immature pancreas needs time to adapt to new diet and produce necessary amounts and types of digestive enzymes. Result - Undigested feed is wasted. Undigested feed promotes "nutritional scours" and provides substrate for the growth of diarrhea-causing pathogens.

46. Why should you use Dinazyme? The Solution…Dinazyme C/S-PMB • To enable use of normally undigestible alternate lower cost ingredients • To supplement the immature endogenous enzyme system. • To maximize performance, even with limited digestive capacity, e.g. case of young animals or rapid diet changes. • To optimize nutrient utilization of high energy feedstuffs.

47. ECONOMICS Data suggest that an average improvement in nutrient utilization of 3 – 5% can be obtained Leeson and Summers (1976) reported that high moisture content corn harvest necessitated high temperature drying & time retention conditions, reduced ME value of corn by as much as 3% compared to the expected value.

48. Specifications for Dinazyme C/S PBM

49. GUARANTEED ANAlYSIS

50. Diversified Nutri-Agri Technologies Inc., The End …….…for now……. “a Dynamic Approach to Nutri-Agri Product Research and Technology Development”