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Enzymes in liquid detergents

Enzymes in liquid detergents. 2009-07-04. CONTENT. Denaturation and degradation of enzymes Factors influencing the enzyme stability in liquid and solutions, and formulation guideline for enzymatic liquid detergents. ENZYME DEGRADATION AND DENATURATION.

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Enzymes in liquid detergents

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  1. Enzymes in liquid detergents 2009-07-04

  2. NOVOZYMES PRESENTATION CONTENT • Denaturation and degradation of enzymes • Factors influencing the enzyme stability in liquid and solutions, and formulation guideline for enzymatic liquid detergents

  3. NOVOZYMES PRESENTATION ENZYME DEGRADATION AND DENATURATION

  4. NOVOZYMES PRESENTATION When the enzyme looses its structure it looses its function The amino acids are located near one another in the active form but not in the denatured from • Denaturation: • High temperature (frying an egg) • Chemical attack • (pH, bleach) Substrate S

  5. Protein inactivation • • Active enzyme in solution Autoproteolysis or proteolysis Denaturation / unfolding over time • • • • • • Chemical modification Inhibition • • • • • • • • • • • • Precipitation

  6. Organogram Use: To illustrate organisation, management, competencies etc. Edit: This slide uses Connectors (Auto Shapes). Lines are connected with boxes. Move the position of the boxes and the lines will still be connected to the original position. Copy/Paste objects and lines for extra elements. Help: For more info search Help (Topmenu) keyword Connectors. NOVOZYMES PRESENTATION guide Factors influencing enzyme stability in a liquid detergent • Water content • pH • Type and amount of enzyme stabilizers • Time • Temperature • Enzyme dose • Major components such as surfactants, builders • Other components in the reaction mix (e.g., preservatives, bleach) These factors interact Each enzyme has a specific range of operating parameters for its optimum performance

  7. NOVOZYMES PRESENTATION Activity - effect of temperature Effect of temperature on the activity of Alcalase Substrate: Denatured haemoglobin Method: Anson pH: 8.5 10 min. time

  8. NOVOZYMES PRESENTATION Activity - effect of pH Activity of Alcalase at different pH-values Substrate: Haemoglobin Temperature: 25 °C 10 min. reaction time

  9. NOVOZYMES PRESENTATION Stability depends on conditions Enzyme stability over time Substrate: Haemoglobin Temperature: 25°C 1 year 1 hour 16 weeks

  10. NOVOZYMES PRESENTATION Enzyme stability Inactive enzyme - does NOT contribute to performance Active enzyme contributes to performance

  11. NOVOZYMES PRESENTATION Enzyme denaturation During storage in liquid detergent, the 3-dimensional structure of the enzyme molecule may be distorted (denatured) through interaction with (mainly): • Surfactants • Builders • Buffers The denaturation may result in loss of enzyme activity as well as increased risk of enzyme degradation.

  12. NOVOZYMES PRESENTATION Enzyme degradation During storage in liquid detergent, the amino-acid chain which forms the ”backbone” of the enzyme molecule may be degradated by hydrolysis (reaction with water). The hydrolysis is accelerated (mainly) by: • Protease • High pH • High water activity

  13. NOVOZYMES PRESENTATION FACTORS INFLUENCING THE ENZYME STABILITY IN LIQUID SOLUTIONS, AND FORMULATION GUIDELINE FOR ENZYMATIC LIQUID DETERGENTS

  14. Organogram Use: To illustrate organisation, management, competencies etc. Edit: This slide uses Connectors (Auto Shapes). Lines are connected with boxes. Move the position of the boxes and the lines will still be connected to the original position. Copy/Paste objects and lines for extra elements. Help: For more info search Help (Topmenu) keyword Connectors. NOVOZYMES PRESENTATION guide Formulation guidelines 1 Water content Preferably : < 50 % Possible : 50-60 % Not Advisable : > 60 % The risk of hydrolytic degradation of the enzyme molecule may be reduced by lowering the water activity of the detergent formulation

  15. Organogram Use: To illustrate organisation, management, competencies etc. Edit: This slide uses Connectors (Auto Shapes). Lines are connected with boxes. Move the position of the boxes and the lines will still be connected to the original position. Copy/Paste objects and lines for extra elements. Help: For more info search Help (Topmenu) keyword Connectors. NOVOZYMES PRESENTATION guide Formulation guidelines 2 pH Preferably : 7 - 9 Possible : 9 - 9.5 Not Advisable : 9.5 - 11 High pH increases the risk of enzyme molecule degradation

  16. Organogram Use: To illustrate organisation, management, competencies etc. Edit: This slide uses Connectors (Auto Shapes). Lines are connected with boxes. Move the position of the boxes and the lines will still be connected to the original position. Copy/Paste objects and lines for extra elements. Help: For more info search Help (Topmenu) keyword Connectors. NOVOZYMES PRESENTATION guide Formulation guidelines 3 Anionic surfactants Charged surfactants: Negative influence on enzyme stability AES = None > MES > AOS = soap > LAS alkylethoxysulfatemethylethersulfate Alpha OlefinSulfonate linear alkylbenzenesulphonate Recommendation: Use a combination of non-ionic and anionic surfactants

  17. Organogram Use: To illustrate organisation, management, competencies etc. Edit: This slide uses Connectors (Auto Shapes). Lines are connected with boxes. Move the position of the boxes and the lines will still be connected to the original position. Copy/Paste objects and lines for extra elements. Help: For more info search Help (Topmenu) keyword Connectors. NOVOZYMES PRESENTATION guide Formulation guidelines 4 Nonionic surfactants AEO6 > NP12 > NP4 > None Amides and alkyl-polyglucosides are good Recommendation: Preferably use non-ionic surfactants or combine non-ionic and anionic surfactants Nonylphenyl ether Alkyl polyglycoside Alcohol ethoxylate

  18. Organogram Use: To illustrate organisation, management, competencies etc. Edit: This slide uses Connectors (Auto Shapes). Lines are connected with boxes. Move the position of the boxes and the lines will still be connected to the original position. Copy/Paste objects and lines for extra elements. Help: For more info search Help (Topmenu) keyword Connectors. NOVOZYMES PRESENTATION guide Formulation guidelines 5 Solvent Alcohols/hydroxy groups stabilize proteases and facilitate solubilization of surfactants in water phase Propylene-glycol > Ethanol >> Sorbitol/Glycerol > None Other coupling agents like Na-Xylenesulfonate, Na-cumenesulfonate, phosphate esters, other hydrotropes are also used

  19. Organogram Use: To illustrate organisation, management, competencies etc. Edit: This slide uses Connectors (Auto Shapes). Lines are connected with boxes. Move the position of the boxes and the lines will still be connected to the original position. Copy/Paste objects and lines for extra elements. Help: For more info search Help (Topmenu) keyword Connectors. NOVOZYMES PRESENTATION guide Formulation guidelines 6 Buffer system TEA (Triethanolamine) > DEA > MEA = Urea Keep ethanol amines (MEA, TEA) levels low (below 5%) in case 4-FPBA is used

  20. Organogram Use: To illustrate organisation, management, competencies etc. Edit: This slide uses Connectors (Auto Shapes). Lines are connected with boxes. Move the position of the boxes and the lines will still be connected to the original position. Copy/Paste objects and lines for extra elements. Help: For more info search Help (Topmenu) keyword Connectors. NOVOZYMES PRESENTATION guide Formulation guidelines 7 Builder system Preferably: Citrate, polyacrylates (mild builders) Possible: Zeolite, phosphate Questionable: MGDA (methylglycinediacetic acid) Not Possible : EDTA Most enzymes contain Calcium ions for stabilization of the three-dimensional structure of the enzyme molecule During storage in liquid detergent, strong builder components tend to extract Ca-ions from the enzyme molecules, increasing the risk of enzyme denaturation

  21. Organogram Use: To illustrate organisation, management, competencies etc. Edit: This slide uses Connectors (Auto Shapes). Lines are connected with boxes. Move the position of the boxes and the lines will still be connected to the original position. Copy/Paste objects and lines for extra elements. Help: For more info search Help (Topmenu) keyword Connectors. NOVOZYMES PRESENTATION guide Formulation guidelines 8 • Bleaching system • Preferably: Peroxide, but not very active over a long period of time • May require oxidative stable enzymes like Everlase • Not Possible: Chlorine

  22. Organogram Use: To illustrate organisation, management, competencies etc. Edit: This slide uses Connectors (Auto Shapes). Lines are connected with boxes. Move the position of the boxes and the lines will still be connected to the original position. Copy/Paste objects and lines for extra elements. Help: For more info search Help (Topmenu) keyword Connectors. NOVOZYMES PRESENTATION guide Formulation guidelines 9 • Preservative • Almost all the preservatives compatible with enzymes: • Kathon 886 MW, • Bronopol , • Proxel CRL or GXL • Nipasol M, • Irgasan DP300, • Dowicide A • Aldehydes are generally not advisable • Not possible: formaldehyde

  23. Organogram Use: To illustrate organisation, management, competencies etc. Edit: This slide uses Connectors (Auto Shapes). Lines are connected with boxes. Move the position of the boxes and the lines will still be connected to the original position. Copy/Paste objects and lines for extra elements. Help: For more info search Help (Topmenu) keyword Connectors. NOVOZYMES PRESENTATION guide Formulation guidelines 10 Enzyme stabilization system Calcium (CaCl2) and formate (HCOONa) important for stability of proteases and amylases Dosage: 0.02 - 0.1 % (w/w) CaCl2 (~100 ppm free Calcium)

  24. NOVOZYMES PRESENTATION Calcium binding sites Lipex with Ca-binding site

  25. Organogram Use: To illustrate organisation, management, competencies etc. Edit: This slide uses Connectors (Auto Shapes). Lines are connected with boxes. Move the position of the boxes and the lines will still be connected to the original position. Copy/Paste objects and lines for extra elements. Help: For more info search Help (Topmenu) keyword Connectors. NOVOZYMES PRESENTATION guide Formulation guidelines 11 Enzyme stability Acetamide, glycine, maltodextrin may be added to improve enzymatic stability Dosage: 0 - 5 % (w/w) Not compatible: Glutamic acid (Phase sep.)

  26. Organogram Use: To illustrate organisation, management, competencies etc. Edit: This slide uses Connectors (Auto Shapes). Lines are connected with boxes. Move the position of the boxes and the lines will still be connected to the original position. Copy/Paste objects and lines for extra elements. Help: For more info search Help (Topmenu) keyword Connectors. NOVOZYMES PRESENTATION guide Formulation guidelines 12 • Enzyme stability • Boricacid and Borax (1-3 wt%), use at pH>8 • Sodiumformate (1-3 wt%), use at pH<8 • 4-FPBA (0.005-0.02 wt%), included in ”Ultra” proteases • Propyleneglycol (5-10%) • Since enzymes are proteins, protease molecules in a liquid detergent will tend to decompose (hydrolyse) other enzyme molecules present in the formulation

  27. NOVOZYMES PRESENTATION Example 1

  28. NOVOZYMES PRESENTATION Example 2

  29. NOVOZYMES PRESENTATION Example 3

  30. Process Illustration 2 Use: For illustration of processes. NOVOZYMES PRESENTATION H H-O C=O B H-O guide 4-FPBA - highly effective protease inhibitor • Highly efficient protease inhibitor compared to borax/propylene glycol combination. • Results in good stability of protease and other enzymes in the presence of protease • 4-FPBA can replace borax and propylene glycol with minor formulation adjustments including lowering of pH. Molecular structure 4-formylphenyl boronic acid

  31. Process Illustration 2 Use: For illustration of processes. NOVOZYMES PRESENTATION H H-O H-O C=O B B O-H H-O H-O guide 4-FPBA is a reversible protease inhibitorthat stabilizes proteases in liquid detergents Boricacid 4-formyl phenylboronic acid (4-FPBA) Borax = di-Sodium- tetraborate, 10.H2O Ki = 10 mM 4-FPBA requireslessornopropyleneglycol for stabilization Ki= 0.03 - 0.1 mM

  32. Process Illustration 3 Use: For illustration of processes. NOVOZYMES PRESENTATION guide Benefits of stabilizer - the Savinase protease product containing a stabilizer Formulationoptimization Costsavings Reduced level of boric compounds Simplermanufacturingprocesses Due to removal or reduction in level of traditional stabilizers Due to removal or reduction in level of traditional stabilizers 4-FPBA represents a boric acid substitution option: 0.1% of 4-FPBA can replace 1-3% boric acid normally used EU have re-classified boric acid as reprotoxic substance above 5.5% Decrease in number of ingredients Avoid dissolving step for Borax

  33. NOVOZYMES PRESENTATION How do borate stabilizers work? Acidification of borax in a liquid detergent yields boric acid and borates. The borate species react with propane-diol to form 1:1 and 1:2 borate-diol ester complexes. The 1:1 complex weakly associates with the protease at the active site and provides competitive inhibition.

  34. NOVOZYMES PRESENTATION Mechanism of boric acid • Boric acid is a subtilisin inhibitor (alkaline serine protease inhibitor). • Dissolution of Borax in a liquid detergent yields boric acid and borates. Boric acid associates with the protease at the active site and provides competitive inhibition. • The borate species react with propane-diol to form 1:1 and 1:2 borate-diol ester complexes. The 1:1 complex may also provide inhibition.

  35. This is what 4-FPBA does? 35 10/30/2014 NOVOZYMES PRESENTATION 4-FPBA has the same effect as borate, but in much smaller amounts. 4-FPBA weakly associates with the protease at the active site and provides competitive inhibition.

  36. Mechanism of 4-FPBA 36 10/30/2014 4-FPBA is a subtilisin inhibitor as boric acid. 4-FPBA fits well in the binding pocket of the enzyme and is effective in much smaller amounts than boric acid. 4-FPBA requires less or no propylene glycol for stabilisation. NOVOZYMES PRESENTATION

  37. Process Illustration 2 Use: For illustration of processes. 37 10/30/2014 NOVOZYMES PRESENTATION guide 4-FPBA in US type model detergentwith MPG, without Borax, pH 7, 30°C • Improved protease stability • Lipolase stable in basis formulation without protease • 4 FPBA significantly reduces proteolysis of Lipolase

  38. Process Illustration 2 Use: For illustration of processes. 38 10/30/2014 NOVOZYMES PRESENTATION guide 4-FPBA effect in EU structured, 40°C • Savinase stability improved • Lipolase activity from poor to very good • 4-FPBA is 1000 X more efficient than Boric Acid based on weight

  39. Organogram Use: To illustrate organisation, management, competencies etc. Edit: This slide uses Connectors (Auto Shapes). Lines are connected with boxes. Move the position of the boxes and the lines will still be connected to the original position. Copy/Paste objects and lines for extra elements. Help: For more info search Help (Topmenu) keyword Connectors. NOVOZYMES PRESENTATION guide Formulation guidelines 13 Physical stability Sorbitol or glycerol (glycerine) may be added to improve physical stability. Dosage: 0 - 5 % (w/w)

  40. NOVOZYMES PRESENTATION Formulation guidelines- Improve the physical stability in the detergent • Add glycerol ~2% • Lower CaCl2·2H2O to ~0.06 % • Lower amount of surfactant • Add hydrotrope, e.g. SXS ~2% • Increase amount of ethanol or MPG • Lower amount of carboxylic acids: citrate, borate or formate • Change ratio between anionic and non-ionic surfactants • Increase pH

  41. NOVOZYMES PRESENTATION Formulation guidelines- Improve the enzymatic stability in the detergent • Add CaCl2·2H2O to ~0.06 % • Add borate + MPG • Add formate • Lower the water content to ~40 % • Lower the anionic surfactant content, especially LAS (AS) • Examine "strange" detergent components • Lower pH

  42. Process Illustration 2 Use: For illustration of processes. NOVOZYMES PRESENTATION Stability depends largely on the formulation of the liquid detergent guide Formulation guidelines - detergent influence And only to a very limited extend on the formulation of the enzyme product

  43. Slide Divider Use: Drawing attention to different chapters or themes in the presentation. Animation: Auto Supergraphic light animation. Notice Changes automatically to next slide after 10 seconds. Edit: Change/Set the auto Slide Transition in top menu: > Slideshow > Slide Transition NOVOZYMES PRESENTATION THANK YOU guide

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