DINATEC A Systems Approach to advanced enzyme technology.

DINATECDINATEC

A Systems Approach to advanced enzyme technology

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Background History of Background History of enzymesenzymes

Phenomena believed to be spontaneous reactions until 1857, Phenomena believed to be spontaneous reactions until 1857, when the French chemist Louis Pasteur proved that when the French chemist Louis Pasteur proved that fermentation occurs only in the presence of living cells.fermentation occurs only in the presence of living cells.

Subsequently the German chemist Eduard Buchner Subsequently the German chemist Eduard Buchner discovered (1897) that a cell-free extract of yeast can discovered (1897) that a cell-free extract of yeast can cause alcoholic fermentation. cause alcoholic fermentation.

The ancient puzzle was then solved; the yeast cell produces The ancient puzzle was then solved; the yeast cell produces the enzyme and the enzyme brings about the the enzyme and the enzyme brings about the fermentation fermentation

As early as 1783 the Italian biologist Lazzaro Spallanzani had As early as 1783 the Italian biologist Lazzaro Spallanzani had observed that meat could be digested by gastric juices observed that meat could be digested by gastric juices extracted from hawks. extracted from hawks.

Alcoholic fermentation oldest known enzyme reaction Alcoholic fermentation oldest known enzyme reaction Y + G A + COY + G A + CO22

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Probably first experiment in which a vital reaction was Probably first experiment in which a vital reaction was performed outside the living organismperformed outside the living organism

After Buchner's discovery scientists assumed that After Buchner's discovery scientists assumed that fermentations and vital reactions in general were caused fermentations and vital reactions in general were caused by enzymesby enzymes

Nevertheless, all attempts to isolate and identify their Nevertheless, all attempts to isolate and identify their chemical nature were unsuccessfulchemical nature were unsuccessful

In 1926 the American biochemist James B. Sumner In 1926 the American biochemist James B. Sumner succeeded in isolating and crystallizing urease. succeeded in isolating and crystallizing urease.

Background History of Background History of enzymesenzymes

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Background History of Background History of enzymesenzymes

Four years later pepsin and trypsin were isolated and Four years later pepsin and trypsin were isolated and crystallized by American biochemist John H. Northrop crystallized by American biochemist John H. Northrop

Enzymes were found to be proteins, and Northrop Enzymes were found to be proteins, and Northrop proved that the protein was actually the enzyme and proved that the protein was actually the enzyme and not simply a carrier for another compoundnot simply a carrier for another compound

Research in enzyme chemistry in recent years has shed Research in enzyme chemistry in recent years has shed new light on some of the most basic functions of life new light on some of the most basic functions of life

Ribonuclease, a simple three-dimensional enzyme Ribonuclease, a simple three-dimensional enzyme discovered in 1938 by American bacteriologist discovered in 1938 by American bacteriologist René Dubos. René Dubos.

Isolated in 1946 by American chemist Moses unitzIsolated in 1946 by American chemist Moses unitz

Synthesized by American researchers in 1969 Synthesized by American researchers in 1969

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RIBONUCLEASE RIBONUCLEASE ENZYMEENZYME

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The synthesis hooks 124 molecules in a The synthesis hooks 124 molecules in a specific sequence to form the macromolecule specific sequence to form the macromolecule

Led to identification of those molecular areas Led to identification of those molecular areas that carry out its chemical functionsthat carry out its chemical functions

Opened up the possibility of creating Opened up the possibility of creating specialized enzymes with new properties specialized enzymes with new properties

This potential has been greatly expanded in This potential has been greatly expanded in recent years by genetic engineering recent years by genetic engineering techniques that have made it possible to techniques that have made it possible to produce some enzymes in great quantity produce some enzymes in great quantity

Background History of Background History of enzymesenzymes

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How are enzymes How are enzymes manufactured?manufactured?

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How are enzymes How are enzymes manufactured?manufactured?

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Industry drawbacksIndustry drawbacks Non-specific reactions may result in poor product yields.Non-specific reactions may result in poor product yields.

High temperatures and/or pressures needed to drive reactions High temperatures and/or pressures needed to drive reactions lead to high energy costs. May require large volumes of cooling lead to high energy costs. May require large volumes of cooling water downstream. water downstream.

Harsh and hazardous processes involving high temperatures, Harsh and hazardous processes involving high temperatures, pressures, acidity or alkalinity need high capital investment, pressures, acidity or alkalinity need high capital investment, and specially designed equipment and control systems. and specially designed equipment and control systems.

Unwanted by-products may prove difficult or costly to dispose Unwanted by-products may prove difficult or costly to dispose of. of.

High chemical and energy consumption, and harmful by-High chemical and energy consumption, and harmful by-products have a negative impact on the environment.products have a negative impact on the environment.

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Drawbacks eliminated by Drawbacks eliminated by

enzymesenzymes Reactions carried out under mild conditions Reactions carried out under mild conditions Highly specific Highly specific Involve very fast reaction rates Involve very fast reaction rates Reactions are carried out by numerous enzymes with different roles. Reactions are carried out by numerous enzymes with different roles. Industrial enzymes originate from biological systems which contribute to Industrial enzymes originate from biological systems which contribute to

sustainable development through being isolated from microorganisms sustainable development through being isolated from microorganisms which are fermented using primarily renewable resources.which are fermented using primarily renewable resources.

Small amounts of enzymes are required to carry out chemical reactions Small amounts of enzymes are required to carry out chemical reactions Reaquires little storage space. Reaquires little storage space. uncomplicated and widely available equipment can be used uncomplicated and widely available equipment can be used Reactions are easily controlled and can be stopped when the desired Reactions are easily controlled and can be stopped when the desired

degree of substrate conversion has been achieved. degree of substrate conversion has been achieved. Reduce the impact of collateral damage on the environment by reducing Reduce the impact of collateral damage on the environment by reducing

the consumption of chemicals and energy, and the subsequent the consumption of chemicals and energy, and the subsequent generation of waste. generation of waste.

Developments in genetic and protein engineering have led to Developments in genetic and protein engineering have led to improvements in the stability, economy, specificity and overall improvements in the stability, economy, specificity and overall application potential of industrial enzymes.application potential of industrial enzymes.

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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.

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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 Ex., lactase catalyzes the conversion of milk / sugar to galactose and glucose galactose and glucose

Lactase has the systematic name beta-D-galactoside Lactase has the systematic name beta-D-galactoside galactohydrolase, and the classification number EC galactohydrolase, and the classification number EC 3.2.1.23. 3.2.1.23.

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SIX MAIN ENZYME CLASSES

CLASS REACTION PROFILE

1: Oxidoreductases Involves movement of electrons from one molecule to another. In biological systems we usually see the removal of hydrogen from the substrate. Enzymes in this class are called dehydrogenases. Ex., alcohol dehydrogen-ase catalyzes reactions of the type R-CH2OH + A → R-CHO + H2A, where A is an acceptor molecule. If A is oxygen, the relevant enzymes are called oxidases; if A is hydrogen peroxide, the relevant enzymes are called peroxidases.

2: Transferases This class of enzymes catalyzes the transfer of groups of atoms (radicals) from one molecu-le to another. Aminotransferases or transaminases promote the transfer of an amino group from one amino acid to an alpha-keto-acid.

3: Hydrolases Hydrolases catalyze reactions between a substrate and water, and bind water to certain molecules. In this way larger molecules are broken up into smaller units. This class of enzymes catalyzes the cleavage of peptide bonds in proteins, glucosidic bonds in carbohydrates, and ester bonds in lipids.

4: Lyases Lyases catalyze the addition of groups to double bonds or the formation of double bonds through the removal of groups. Thus bonds are cleaved using a different principle to hydrolysis. Pectate lyases, for example, split the glycosidic linkages by beta-elimination.

5: Isomerases Isomerases catalyze the transfer of groups from one position to another on the same molecule. These enzymes change the structure of a substrate by rearranging its atoms.

6: Ligases Ligases join molecules together with covalent bonds. These enzymes participate in biosynthetic reactions where new groups of bonds are formed. Such reactions require the input of energy in the form of co-factors such as ATP.

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Globular, water soluble proteins, (few exceptions)

What are enzymes?

Allows / facilitates chemical reactions to occur such as those that release nutrients from feed during digestion

If a reaction is favorable ( ∆G < 0), the activation energy E(act) determines how fast it will go.

Without the enzyme catalyst the reaction would either not take place or would happen very slowly

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What are enzymes?

Though an enzymatic catalyst takes part in the chemical reaction it remains unchanged and is available to repeat the task

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Some enzymes that are of practical value to the livestock industry:

These enzymes can be mixed and matched to form an enzyme cocktail to fit any particular diet need.

Xylanases, amylases, phytases, proteases, cellulases, betaglucanases, and pentosanases, are available for use in diet formulations.

What are the most important enzymes to our industry?

Virtually all enzymes employed in the feed industry are hydrolases.

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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.

Young animals lack many endogenous enzymes or sufficient quantities thereoff.

Why are enzymes needed in feed formulations?

Trials confirm that enzyme supplementation results in improved animal performance.

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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

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.

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Specific enzymes may be incorporated into specific diets in order to solve specific problems

How do enzymes work?

Specificity

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How do enzymes work?

Enzyme catalyzed reactions are often Enzyme catalyzed reactions are often from 100 million to more than 10 from 100 million to more than 10 billion times faster than the same billion times faster than the same reaction in the absence of the enzyme. reaction in the absence of the enzyme.

Most enzymes catalyze the transfer Most enzymes catalyze the transfer of of electrons, atoms or functional electrons, atoms or functional groups.groups.

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Factors influencing enzyme Factors influencing enzyme activityactivity Optimum pHOptimum pH

Optimum TemperatureOptimum Temperature

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Optimum Enzyme concentrationOptimum Enzyme concentration

OptimumOptimum Substrate concentrationSubstrate concentration

Factors influencing enzyme Factors influencing enzyme activityactivity

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Covalent modificationCovalent modification

Factors influencing enzyme Factors influencing enzyme activityactivity

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InhibitorsInhibitors

A competitive inhibitor A non-competitive inhibitor molecule

Factors influencing enzyme Factors influencing enzyme activityactivity

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Factors influencing enzyme Factors influencing enzyme activityactivity

Allosteric EffectorsAllosteric Effectors

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Factors influencing enzyme Factors influencing enzyme activityactivity

Optimum pH:Optimum pH: pH at which enzymes pH at which enzymes operate best. Activity decreases on operate best. Activity decreases on either side of pH optimum.either side of pH optimum.

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Optimum Temperature:Optimum Temperature:

Within a given range, for every 10 degrees the temperature increases, enzyme Within a given range, for every 10 degrees the temperature increases, enzyme activity doubles. activity doubles.

Enzymes become denatured at elevated temperatures. Enzymes become denatured at elevated temperatures.

Enzymes have an optimum temperature which varies according to:Enzymes have an optimum temperature which varies according to:

Enzyme source.Enzyme source.

Salt levelsSalt levels in the medium to which the enzyme is added. (For example, in the medium to which the enzyme is added. (For example, amylases from animal sources are less heat stable than those from fungal amylases from animal sources are less heat stable than those from fungal sources (Aspergillus) which are in turn less stable than bacterial amylases sources (Aspergillus) which are in turn less stable than bacterial amylases (Bacillus).(Bacillus).

Mineral Content:Mineral Content: Certain minerals stabilize enzymes while others cause Certain minerals stabilize enzymes while others cause inactivation. Calcium and magnesium are essential for good starch breakdown inactivation. Calcium and magnesium are essential for good starch breakdown (amylases) and increase enzyme stability to temperature. Heavy metals such as (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 iron are typically detrimental to enzymes, and may in some cases be used to inactivate or stop enzyme reactions.inactivate or stop enzyme reactions.

Factors influencing enzyme Factors influencing enzyme activityactivity

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Enzyme Enzyme concentrationconcentration Normally enzymes are present in cells in low Normally enzymes are present in cells in low

concentrations.concentrations.

As enzyme concentration increases the rate of As enzyme concentration increases the rate of the reaction increases linearly, because there the reaction increases linearly, because there are more enzyme molecules available to are more enzyme molecules available to catalyse the reaction.catalyse the reaction.

At very high enzyme concentration the At very high enzyme concentration the substrate concentration may become rate-substrate concentration may become rate-limiting, so the rate stops increasing. limiting, so the rate stops increasing.

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Substrate Substrate concentrationconcentration As the substrate concentration increases, the rate increases As the substrate concentration increases, the rate increases

because more substrate molecules can collide with enzyme because more substrate molecules can collide with enzyme molecules, so more reactions will take place. molecules, so more reactions will take place.

As substrate concentration gets higher the enzyme molecules As substrate concentration gets higher the enzyme molecules become become saturatedsaturated so there are few free enzyme molecules. Adding so there are few free enzyme molecules. Adding more substrate doesn't make much difference (though it will more substrate doesn't make much difference (though it will increase the rate of E-S collisions).increase the rate of E-S collisions).

The maximum rate at infinite substrate concentration is called The maximum rate at infinite substrate concentration is called

vvmax, max, The substrate concentration that gives a rate of half the maximum The substrate concentration that gives a rate of half the maximum

rate rate v vmax is called max is called KKM. M.

TheThe v vmax and max and KKM values are useful for characterising an M values are useful for characterising an enzyme. enzyme.

A good enzyme has a high A good enzyme has a high vvmax and a low max and a low KKM. M. 

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Substrate Substrate concentrationconcentration

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Covalent Covalent modificationmodification Activity of some enzymes is controlled by others. Activity of some enzymes is controlled by others.

These enzymes modify the protein chain by cutting it, These enzymes modify the protein chain by cutting it, or adding a phosphate or methyl group. or adding a phosphate or methyl group.

Turns inactive enzyme into active (or vice versa).Turns inactive enzyme into active (or vice versa).

Used to control many metabolic enzymes and to switch Used to control many metabolic enzymes and to switch on enzymes in the gut e.g. hydrochloric acid in stomach on enzymes in the gut e.g. hydrochloric acid in stomach activates pepsin activates rennin. activates pepsin activates rennin.

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InhibitorsInhibitors

Inhibitors inhibit the activity of enzymes, Inhibitors inhibit the activity of enzymes, reducing the rate of their reactions. reducing the rate of their reactions.

Found naturally, but are also used Found naturally, but are also used artificially as drugs, pesticides and artificially as drugs, pesticides and research tools. research tools.

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There are two kinds of enzymatic inhibitors. There are two kinds of enzymatic inhibitors.

Competitive

Non-competitive

InhibitorsInhibitors

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Competitive Competitive InhibitorsInhibitors

Molecule has similar structure to normal Molecule has similar structure to normal substrate molecule. Fits into active site of the substrate molecule. Fits into active site of the enzyme. enzyme.

CompetesCompetes with substrate for the active site, with substrate for the active site, so reaction is slower. so reaction is slower.

Increase Increase KKM for enzyme, but no effect on M for enzyme, but no effect on vvmax. max.

The rate can approach a normality if substrate The rate can approach a normality if substrate concentration is increased sufficiently. concentration is increased sufficiently.

The sulphonamide anti-bacterial drugs are The sulphonamide anti-bacterial drugs are examples of competitive inhibitors.examples of competitive inhibitors.

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Non-competitive Non-competitive inhibitors inhibitors

Inhibitor molecule is different in structure than the substrate Inhibitor molecule is different in structure than the substrate moleculemolecule

Will not fit into active site.Will not fit into active site.

Binds to another part of the enzyme molecule. Binds to another part of the enzyme molecule.

Change enzyme and active site shape so it no longer binds Change enzyme and active site shape so it no longer binds substrate molecules. Result is reduction of active enzyme substrate molecules. Result is reduction of active enzyme numbers (just like decreasing the enzyme concentration). numbers (just like decreasing the enzyme concentration). Therefore decrease vmax, but have no effect on KM. Therefore decrease vmax, but have no effect on KM.

RReversible inhibitorseversible inhibitors - bind weakly and can be washed out. - bind weakly and can be washed out.

Irreversible inhibitors -Irreversible inhibitors - bind tightly and cannot be washed out. bind tightly and cannot be washed out.

Poisons like cyanide, heavy metal ions and some insecticides are Poisons like cyanide, heavy metal ions and some insecticides are all examples of non-competitive inhibitors. all examples of non-competitive inhibitors.

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RATE EQUATION FOR PRODUCT RATE EQUATION FOR PRODUCT

INHIBITIONINHIBITION

Michaelis-Menten equation RATE EQUATION

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Allosteric Allosteric Effectors Effectors

Allosteric site is distinct from the active site Allosteric site is distinct from the active site Different molecules can inhibit or activate the Different molecules can inhibit or activate the

enzyme, allowing sophisticated control of the reaction enzyme, allowing sophisticated control of the reaction raterate

Few enzymes can do this. They are often at the start Few enzymes can do this. They are often at the start of long biochemical pathways of long biochemical pathways

Generally activated by the substrate of the pathway Generally activated by the substrate of the pathway and inhibited by the product of the pathway, thus only and inhibited by the product of the pathway, thus only turning the pathway on when it is neededturning the pathway on when it is needed

Activity of some enzymes is Activity of some enzymes is controlled by certain controlled by certain

molecules molecules binding to a specific binding to a specific regulatory or regulatory or allostericallosteric site site on the enzyme.on the enzyme.

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Allosteric Allosteric EffectorsEffectors

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Economic Economic benefitsbenefits

Increases daily weight gain Increases daily weight gain Increases egg productionIncreases egg production Lowers feed conversionLowers feed conversion More uniform weights / increased nutrient More uniform weights / increased nutrient

absorptionabsorption Lower incidence of digestive problems caused by Lower incidence of digestive problems caused by

unassimilated fiber which also improves litter quality unassimilated fiber which also improves litter quality Reduces fecal volume and nitrogen excretion levelsReduces fecal volume and nitrogen excretion levels Cleaner eggs and better egg yolk colorCleaner eggs and better egg yolk color Use of lower cost ingredientsUse of lower cost ingredients Maintains and improves performance levelsMaintains and improves performance levels Increases ratio of lean to fat tissueIncreases ratio of lean to fat tissue Can "inactivate" mycotoxins in feedsCan "inactivate" mycotoxins in feeds

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What is Dinazyme?What is Dinazyme?

Dinazyme B/W Dry and Liquid Dinazyme B/W Dry and Liquid

Dinazyme C/S PBM Dry and Liquid Dinazyme C/S PBM Dry and Liquid

Dinazyme PSE, (Phytase) DryDinazyme PSE, (Phytase) Dry

Several types of enzyme technologies are offered as Dinazyme

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What is Dinazyme C/S What is Dinazyme C/S PMBPMB

Supplement for corn soy based poultry and pig diets containing high glucan barley levels.

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A diet supplement which enhances nitrogen utilization and A diet supplement which enhances nitrogen utilization and increases protein digestibility with the active ingredient increases protein digestibility with the active ingredient protease, resulting in increased absorption of amino acids and protease, resulting in increased absorption of amino acids and peptides. peptides.

DINAZYME C/S® also contains amylase-breaks down starch DINAZYME C/S® also contains amylase-breaks down starch content and content and Xylanase, Xylanase, aa complex hydrolytic enzyme complex hydrolytic enzyme preparation which has preparation which has anan effect on hemicellulose substrates effect on hemicellulose substrates containing xylan, manan containing xylan, manan and and glucan. glucan.

A combination of amylase, Xylanase and protease boosts the A combination of amylase, Xylanase and protease boosts the digestibility of typical corn and soybean meal-based diets, digestibility of typical corn and soybean meal-based diets, resulting in more nutrients available for growth. resulting in more nutrients available for growth.

Inclusion of DINAZYME C/S® in diet supplements provides Inclusion of DINAZYME C/S® in diet supplements provides endogenous enzymes animals lack or produce in low endogenous enzymes animals lack or produce in low amounts.amounts.  

What is Dinazyme C/S What is Dinazyme C/S PMBPMB

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WHAT MAKES DINAZYME MORE EFFECTIVE THAN OTHER WHAT MAKES DINAZYME MORE EFFECTIVE THAN OTHER ENZYMECOMBINATIONSENZYMECOMBINATIONS

EEffective action ffective action ddue to presence of other ue to presence of other important hydrolytic enzymes, which decompose important hydrolytic enzymes, which decompose cellulose, lichenin, araban cellulose, lichenin, araban and and pectin. pectin.

Dinatec makes use of important technical Dinatec makes use of important technical concepts such as:concepts such as:

Covalence modification,Covalence modification,

The use of specific allosteric substances and The use of specific allosteric substances and enzyme co-factors that are conducive to higher enzyme co-factors that are conducive to higher enzymatic efficacyenzymatic efficacy

Enzyme concentrationEnzyme concentration

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What can Dinazyme C/S PMB do for What can Dinazyme C/S PMB do for you?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.

* Non Starch Polysaccharide

Xylanase reduces digesta viscosity; decreases anti-nutritional effects of NSP; reduces soluble NSP in digesta hence increased absorption of amino acids and peptides.

Pectinase, more complete hydrolysis (digestion) of pectins in wheat and corn based diets

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Why should you use Why should you use Dinazyme?Dinazyme?

Undigested feed promotes "nutritional scours" and provides substrate for the growth of diarrhea-causing pathogens.

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.

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Why should you use Why should you use Dinazyme?Dinazyme?

To enable use of normally undigestible alternate lower To enable use of normally undigestible alternate lower cost ingredientscost ingredients

The Solution…The Solution… Dinazyme C/S-PMBDinazyme C/S-PMB

To supplement the immature endogenous enzyme To supplement the immature endogenous enzyme system.system. To maximize performance, even with limited digestive To maximize performance, even with limited digestive capacity, capacity, e.g. case of young animals or rapid diet e.g. case of young animals or rapid diet changes.changes.

To optimize nutrient utilization of high energy feedstuffs.To optimize nutrient utilization of high energy feedstuffs.

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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.

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Specifications for Dinazyme C/S Specifications for Dinazyme C/S PBMPBM

EFFICACY Effective over a wide pH and temperature range.

PACKAGING Available in 5, 10 and 20 kg pails or 55 lb. bags.

STORAGE In dry location. Do not exceed 26°C.

SHELF LIFE One year in original sealed container. Three months in stored premixes and feedstuffs.

USE & DOSE For any corn/soy based feed. Layer/Breeder feeds. 200 grms/metric ton, Broiler feeds. 250 grms/metric ton. Pig diets. 300 grms/metric ton.

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ENZYME TYPE CONTENT U/GRAM

Amylase ECC IUB # 3.2.1.1 Protease ECC IUB # 3.4.24.28 Xylanase ECC IUB # 3. 2.1.8 Pectinase ECC IUB # 3. 2.1.8 Betaglucanase ECC IUB # 3. 2.1.8

1600   

16000   

1200  

120   

600

GUARANTEED GUARANTEED ANAlYSISANAlYSIS

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The End…….…for now…….

Diversified Nutri-Agri Technologies Diversified Nutri-Agri Technologies Inc.,Inc.,

“a Dynamic Approach to Nutri-Agri Product Research and Technology Development”