What are enzymes?

2006-02093-01

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Agenda

• Enzymes in general• Discovery of enzymes• Production of enzymes• Enzymes for detergents

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What are enzymes and what do they do

• Enzymes are biological catalysts in the form of proteins

• Enzymes are present in all living cells, where they drive chemical reactions and vital metabolic processes

• Enzymes reduce resource usage and increase the efficiency of the process

• Enzymes are not consumed in the process• Enzymes operate under gentle conditions

• Enzymes are fully biodegradable, and can replace chemicals in industrial production

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Enzymes as proteins

Ascorbate oxidase

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Enzymes as proteins

Bacterial alpha amylase

Active site residues

Calcium ions

Calcium ions

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Enzymes as proteins

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Binding SpecificityEven when different substrate molecules are present, only those that have the specific shape complementary to the active site are

able to bind with the enzyme's active site.

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Enzymes as catalysts

Enzymes catalyse reactions by lowering the activation energy necessary for a reaction to occur.

E + S ES E + P

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Enzymes in general - conclusions

•Chemical reactions that take place under mild conditions

•Highly specific action•Very fast reaction rates•Numerous enzymes for different tasks

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Agenda

•Enzymes in generalEnzymes in general•Discovery of enzymes•Production of enzymesProduction of enzymes•Enzymes for detergentsEnzymes for detergents

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Enzyme discovery and screening

Found in nature

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Enzyme discovery and screeningGrown in Laboratory

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Screening in many ways

Growth conditions inducing enzyme production

Growth on selective media

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Screening in many ways

Detection of enzyme activity

High through put

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Screening process to identify relevant enzymes

Relevant substance (stain) is defined by customer

Thousands of enzymes are applied to the substance to identify relevant/efficient enzyme

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Identification of final enzyme

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Enzyme fermentation

• Fermentation to produce industrial enzymes starts with a vial of dried or frozen microorganisms called a production strain.

• This production strain is selected to produce large amounts of the enzyme(s) of interest.

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Enzyme fermentation

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Agenda

•Enzymes in generalEnzymes in general•Discovery of enzymesDiscovery of enzymes•Production of enzymes•Enzymes for detergentsEnzymes for detergents

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Fermentation of enzymes

Compressor

Mixing of nutrient medium

Water

Inoculation flask

Seed fermenter

Air Sterile filter Fermented broth forrecovery of enzyme

Cooling water

Exhaust

Sterilisation

Raw materials

Ground grain/cornStarchGlucoseSugar

Soy bean mealGluten

Corn steep liquorCasein

PhosphatesSulphatesAmmonium salts

Carbohydrates:

Proteins:

Salts:

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Recovery of enzymesFermenter

Culturebroth

Cooling

Ultrafiltration Stabilisation

Bacterial filtration

Bacterial filtration

A

B

Drum filtration Filtration of enzyme crystals

for granulation

Liquid concentrate

Liquidproduct

Pretreatment

Filter aidis added

Preservativesare added

Crystallisation

Enzyme crystals for granulation

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Granulation of enzymes

Raw materials

Enzyme crystals or liquid

concentrate from the recovery plant

Granulation mixer

Fluid-bed dryer Sifter

Sifter

Hopper

Fibre drums

Coating mixer

Fluid-bed cooler

Granulated final product

Processing aidsare added

Big bag

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Produced in large quantities

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Commercial enzyme formulations (stabilized and standardized)

• Liquids• Non dusty granulates (incapsulated enzymes)• Immobilized dry products

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Savinase T

Coated core, rounded beads, irregular shape.

Note the very smooth coating without holes and the wide variation in size.

0,8 mm

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Novozymes granulation technology

• Enzyme composition• Granulate Stability• Dust• Enzyme release• Color• Particle size distribution

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PEGTiO2Kaolin/CaCO3

Na2SO4Cellulose fibersEnzymeDextrinKaolin/CaCO3

Sugar/starchPVA

EnzymePVA

MgSO4

PVA TiO2Contrast colourSurfactant

T-granulate Competitor product

Comparison of granulate structure

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Microscope picture

T-granulate Competitor product

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SEM Pictures

T-granulate Competitor product

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Set-up for mechanical stability test of granules

Microscope and camera

Weight

Glass plateGranule

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Competitor granulate

Photographs of granulateupon increased pressure

0 g

200 g

400 g

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T-granulate

Photographs of granulateupon increased pressure

400 g

1000 g

0 g

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Agenda

•Enzymes in generalEnzymes in general•Discovery of enzymesDiscovery of enzymes•Production of enzymesProduction of enzymes•Enzymes for detergents

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6 different enzyme classes are used in detergents world wide

Protease

Lipase

Amylase

Cellulase

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Proteases are basic ingredients

Protease

Lipase

Amylase

Cellulase

• used in 75 % of all detergents• used since 1963 • removes stains• basic ingredient in ADW• cannot be substituted by any other ingredients

Cocoa

Grass

Protease no yes

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Wash Performance of Savinase 6.0T25 °C, TOM, Regular powder, 10 °dH, 20 min, EMPA 117 swatch

05

1015202530

0 0,2 0,4 0,6 0,8 1

Enzyme Concentration (%)

D R

emis

sio

n

UnwashedUnwashed

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Concrete examples of test/wash results

Protease effect on blood spots

not washed washed 3 times, no protease

washed 3 times, with protease

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Concrete examples of test/wash results

No protease

With Protease

Re-soiling

Re-soiling

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Protease Action

Particulate stain

Protein

Textile fiber

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Particulate stain

Protein

Textile fiber

Surfactant

Protease Action

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Particulate stain

Textile fiber

Surfactant Enzyme

Protease Action

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Particulate stain

Textile fiber

Surfactant

Protease Action

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Amylases make white whiter

• available already in the sixties• often used in South America (40 % penetration)• basic ingredient in ADW• removes stains• reduces redeposition

Protease

Lipase

Amylase

Cellulase

Tomatosauce

Chocolatepudding

Amylase no yes

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Amylases• Amylases hydrolyse -1,4-glucose-

linkages randomly forming soluble dextrins and oligosaccharides

• The viscosity of the gelatinized starch is reduced and the solubility enhanced

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Savinase® & Termamyl®on Cocoa Stain (EMPA 112)

38

40

42

44

46

48

50

52

54

Rem

issi

on

40 °C 60 °C

Without Enzyme

1 % Savinase 6.0T

1 % Termamyl 60T

0.5 % Savinase 6.0T& 0.5 % Termamyl60T

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Removal of soils containing starch

• Gelatinization and swelling are needed to make the starch susceptible to enzymatic breakdown

• For most starchy foods, various degrees of gelatinization result from cooking

• Amylases boost the removal of starch e.g. from pasta, potato, gravy, chocolate, and baby food

• Amylases prevent starch from adhering to the surface of laundry and dishes and act as a glue for particulate soils

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Lipases give higher in-depth cleaning than surfactants

Protease

Lipase

Amylase

Cellulase

• remove fatty matter at low temperature• give in-depth cleaning• reduces redeposition• can replace surfactants• first protein engineered enzyme introduced in 1988

+ Lipase

- Lipase

Lipstick

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Lipex™

without Lipex with Lipex at different dosages

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Electron microscopically analysisLongitudinal specimen of yarns soiled with olive oil

Olive oil

UnwashedOlive oil

Washed withdetergent

Olive oil

Washed withdetergent &lipase

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Electron microscopically analysisCross sectional specimen of yarns soiled with olive oil

Olive oil

UnwashedOlive oil

Washed withdetergent

Olive oil

Washed withdetergent &lipase

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Cellulases have multiple effects

• available since 1985 • gives multiple effects (colour care, soil removal, whitening, softening)• in top brands and special products

- cellulase+ cellulase

EMPA101

Protease

Lipase

Amylase

Cellulase

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SEM-picture of FibersCotton, 20x wash, European HDP 5 g/l, 40 °C, 18 °dH, WITHOUT Carezyme 900 T

Cellulase effect

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Cellulase effectSEM-picture of FibersCotton, 20x wash, European HDP 5 g/l, 40 °C, 18 °dH, WITH 1 % Carezyme 900 T

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Removal of Fuzz

Clearer Colors

Slide No. 55

Novozymes Protease Products

• Alcalase: Low-medium alkalinity / ”wool proof”• Esperase: High pH & temperature tolerance• Savinase: Medium-high alkalinity • Everlase: Bleach tolerance• Polarzyme: Low-temp. & cost efficacy

• Savinase Ultra • Alcalase Ultra With build-in stabiliser for Liquid detergents

Slide No. 56

Novozymes Amylase Products

• Termamyl: High pH & temperature tolerance• Duramyl: Bleach tolerance• Termamyl Ultra: Builder tolerance• Stainzyme: Effective on “hydrophobic” and “complex”

starch

Slide No. 57

Novozymes Lipase Products

• Lipolase: Enhanced removal of stubborn fat stains

• Lipex: Effect in 1st. wash

Slide No. 58

Novozymes Cellulase Products

• Celluzyme: Reduces re-deposition and greying

• Carezyme: Color Care efficacy and anti-pilling

• Endolase: Anti-redeposition effect and improved soil removal

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The End

• Enzymes in general• Discovery of enzymes• Production of enzymes• Enzymes for detergents

Thank you !