Enzymatic tailoring of hydrolysates' properties
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This document discusses enzymatic tailoring of hydrolysate properties. It provides an overview of enzymes, including enzyme classes and nomenclature. It describes how enzymatic hydrolysis can be used to customize the chemical and biological properties of protein hydrolysates, such as solubility, molecular weight distribution, and amino acid profile. Several examples are given of how different proteases can be used to tailor the degree of hydrolysis, antioxidant activity, foam stability, and amino acid composition of soy flour hydrolysates. The document concludes that enzymatic tailoring provides opportunities to engineer product specifications, improve yields and recoveries, customize protein properties, and develop new active ingredients and product paradigms from plant sources.
![Enzyme Commission (EC)
Designation Numbers*
Group Reaction catalyzed Typical reaction
Enzyme example(s)
with trivial name
EC 1
Oxidoreductases
To catalyze oxidation/reduction
reactions; transfer of H and O atoms or
electrons from one substance to
another
AH + B → A + BH
(reduced)
A + O → AO (oxidized)
Dehydrogenase, oxidase
EC 2
Transferases
Transfer of a functional group from one
substance to another. The group may
be methyl-, acyl-, amino- or phosphate
group
AB + C → A + BC Transaminase, kinase
EC 3
Hydrolases
Formation of two products from a
substrate by hydrolysis
AB + H2O → AOH + BH Lipase, amylase, peptidase
EC 4
Lyases
Non-hydrolytic addition or removal of
groups from substrates. C-C, C-N, C-O
or C-S bonds may be cleaved
RCOCOOH → RCOH + CO2
or [X-A-B-Y] → [A=B + X-Y]
Decarboxylase
EC 5
Isomerases
Intramolecule rearrangement, i.e.
isomerization changes within a single
molecule
AB → BA Isomerase, mutase
EC 6
Ligases
Join together two molecules by
synthesis of new C-O, C-S, C-N or C-C
bonds with simultaneous breakdown of
ATP
X + Y+ ATP → XY + ADP +
Pi
Synthetase
*Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB)](https://image.slidesharecdn.com/enzymatictailoringofhydrolysatespropertiesdmitri-160330142845/85/Enzymatic-tailoring-of-hydrolysates-properties-4-320.jpg)
Enzymatic tailoring of hydrolysates' properties
- 1. Enzymatic Tailoring of Hydrolysates’ Properties
- 2. Outline • Enzymology – Overview of enzymes • Enzymatic Hydrolysates of Proteins: – Chemical and Biologiocal Properties – Tailoring Properties: Protein Surgery – Examples • Opportunities
- 3. Classes & Nomenclature of Enzymes 6 subclasses
- 4. Enzyme Commission (EC) Designation Numbers* Group Reaction catalyzed Typical reaction Enzyme example(s) with trivial name EC 1 Oxidoreductases To catalyze oxidation/reduction reactions; transfer of H and O atoms or electrons from one substance to another AH + B → A + BH (reduced) A + O → AO (oxidized) Dehydrogenase, oxidase EC 2 Transferases Transfer of a functional group from one substance to another. The group may be methyl-, acyl-, amino- or phosphate group AB + C → A + BC Transaminase, kinase EC 3 Hydrolases Formation of two products from a substrate by hydrolysis AB + H2O → AOH + BH Lipase, amylase, peptidase EC 4 Lyases Non-hydrolytic addition or removal of groups from substrates. C-C, C-N, C-O or C-S bonds may be cleaved RCOCOOH → RCOH + CO2 or [X-A-B-Y] → [A=B + X-Y] Decarboxylase EC 5 Isomerases Intramolecule rearrangement, i.e. isomerization changes within a single molecule AB → BA Isomerase, mutase EC 6 Ligases Join together two molecules by synthesis of new C-O, C-S, C-N or C-C bonds with simultaneous breakdown of ATP X + Y+ ATP → XY + ADP + Pi Synthetase *Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB)
- 5. Enzymes • Enzymes: – are catalysts – not spent by the catalyzed reactions – spent by degradation
- 6. Enzymes Activity Units ENZYME NAME SOURCE POTENCY UNITS alpha-Amylase Bacterial DU (dextrin unit) alpha-Amylase Fungal BAU (bacterial amylase unit) Cellulase unspecified CU (cellulase unit) alpha-Galactosidase Fungal GaIU (galactosidase unit) beta-Glucanase Bacterial BGU (beta-glucanase unit) Glucoamylase (amyloglucosidase) Fungal GU or AGU units Hemicellulase Fungal HCU Invertase (sucrase) Fungal INVU or Sumner Unit (SU) Lactase (beta-galactosidase) Fungal Acid Lactase Unit (ALU) Lipase Fungal FIP (Fungi Lipase-Int'l) or LU Lysozyme Animal or micro Lysozyme unit Phytase unspecified FTU (phytase or "f"ytase Unit) Protease Plant PU (papain unit) Protease (alkaline, Alcalase) Bacterial Anson Units (AU); HUT (Hemoglobin Unit on Tyrosine Basis) Protease (e.g. Flavourzyme) Fungal SAP (Spectrophotometric acid protease unit); or Leucine aminopeptidase Unit (LAPU) Pullulunase Bacterial PU (Pullulunase Unit) Per Food Chemical Codex V & the US Pharmacopia
- 7. Enzymes Specificity: Protein Surgery
- 9. Properties of Proteins DETERMINED BY: 1. SELECTION 2. SEQUENCE 3. CONDITIONS DETERMINED BY: CHEMICAL PROPERTIES
- 10. Properties of ProteinsChemical Properties of Proteins Customizable via Enzymology: - Protein/nitrogen recovery, yield - Degree of Hydrolysis - Aesthetics & stability: color, odor, formation of precipitates - NVM content - Nature of organic NVM present - Solubility (water or oil) - Substantivity (to surfaces) - Interactions/reactivity (covalent, electrostatic) with other chemicals (e.g. dyes, vitamins) - Permeation (through biomembranes and from defined vehicles/formulas into skin/hair) - Molecular Weight Distribution - Free AA profile - Terminal Amino Acids - Emulsification capacity - Water retention/humectancy - Film formation - Foaming - Digestability (or metabolic behavior when applied to living tissues, e.g. skin, hair follicle)
- 11. Bioactive Peptides from Food* BIOLOGICAL EFFECT ORIGIN ENCRYPTING PROTEIN NAME AA SEQUENCE IMMUNOMODULATORY Rice Rice albumin Oryzatensin GYPMYPRL Wheat Wheat gluten Immunopeptides multiple OPIOID AGONISTS Wheat Wheat gluten Gluten-exorphins GYYPT, etc. OPIOID ANTAGONISTS Milk Lactoferrin Lactoferroxins multiple CHOLESTEROL LOWERING Soy Glycinin - LPYPR ANTIOXIDANT Wheat Wheat germ protein Peptides not specified Fish Sardine muscle MY Milk Lactalbumin & Lactoglobulin - MHIRL, YVEEL HYPOTENSIVE Soy Soy protein - NWGPLV Fish Fish muscle protein (e.g. tuna, squid, sardine) - LKP, IKP, LRP Egg Ovotransferrin & Ovalbumin Ovokinins KVREGTTY, etc. *Hartmann, R. et al. Food-derived peptides with biological activity: from research to food applications. Current Opinion in Biotechnology, 18:163-169, 2007
- 12. Palm Kernel Cake Hydrolysis* *Zarei, M. et al. Production of Defatted Palm Kernel Cake Protein Hydrolysate as a Valuable Source of Natural Antioxidants. Intl. J. Mol. Sci., 13:8097-8111, 2012 0 10 20 30 40 50 60 70 80 90 100 Degree of Hydrolysis after 24 hrs (%) DH (%) % DH PROTEASE
- 13. Palm Kernel Cake Hydrolysis* *Zarei, M. et al. Production of Defatted Palm Kernel Cake Protein Hydrolysate as a Valuable Source of Natural Antioxidants. Intl. J. Mol. Sci., 13:8097-8111, 2012 PROTEASE 0 10 20 30 40 50 60 70 80 90 100 DH (%) RSA Degree of Hydrolysis & Radical Scavenging Activity (%)
- 14. Hydrolysis of Soy Flour: Tyr* *Hrckova, M. et al. Enzymatic Hydrolysis of Defatted Soy Flour by Three Different Proteases and their Effect on the Functional Properties of Resulting Protein Hydrolysates. Czech. J. Food Sci., 20:1, 2012 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10 30 60 120 240 480 Alcalase Flavourzyme Novozym AA/Tyr Hydrolysis Time (mins)
- 15. Hydrolysis of Soy Flour:DH* *Hrckova, M. et al. Enzymatic Hydrolysis of Defatted Soy Flour by Three Different Proteases and their Effect on the Functional Properties of Resulting Protein Hydrolysates. Czech. J. Food Sci., 20:1, 2012 ** After 8 hrs 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0 Alcalase Flavorzyme Novozym Degree of Hydrolysis (%)**
- 16. Hydrolysis of Soy Flour: Foam* *Hrckova, M. et al. Enzymatic Hydrolysis of Defatted Soy Flour by Three Different Proteases and their Effect on the Functional Properties of Resulting Protein Hydrolysates. Czech. J. Food Sci., 20:1, 2012 ** After 8 hrs of hydrolysis 0 20 40 60 80 100 120 140 160 No Hydrolysis Alcalase Flavourzyme Novozym Foam Stability (mins/100 mm)**
- 17. Hydrolysis of Soy Flour: AA* *Hrckova, M. et al. Enzymatic Hydrolysis of Defatted Soy Flour by Three Different Proteases and their Effect on the Functional Properties of Resulting Protein Hydrolysates. Czech. J. Food Sci., 20:1, 2012 ** After 8 hrs of hydrolysis 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 His Ile Leu Lys Met Cys Phe Tyr Thr Trp Val Ala Arg Asp Glu Gly Pro Ser Unhydrolyzed Alcalase Flavourzyme Novozym Specific AA (%) !! Sulfur Nitrogen Antiox
- 18. Hydrolysis of Soy Flour: MWD* *Hrckova, M. et al. Enzymatic Hydrolysis of Defatted Soy Flour by Three Different Proteases and their Effect on the Functional Properties of Resulting Protein Hydrolysates. Czech. J. Food Sci., 20:1, 2012 Soy Flour (before hydrolysis) Reference Ladder NOVOZYM FLAVOURZYME ALCALASE After 8 hrs of hydrolysis Molecular Weight Distribution
- 19. Flavourzyme® vs. Alcalase® Effects on - Color - NVM - Ash - N2
- 20. NVM% vs. Protein% in New Proteins Hydrolysates 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 1 2 3 4 5 6 7 8 9 Protein, % (N X 6.25) Non-volatile matter (%) % NVM % Protein
- 21. Protein %: in Biomass vs. in Hydrolysate 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Desiccated Coconut Chia Pumpkin Isolate Pumpkin Seed Lupin Chlorella Cocoa Protein Content in Biomass (%) Protein Content in Hydrolysate (%)
- 22. OPPORTUNITIES- ENGINEER PRODUCT SPECS (COLOR, PRECIPITATION, NVM, ASH) - HIGHER YIELD AND PROTEIN/NITROGEN RECOVERY; LOWER COSTS - NEW ACTIVES FROM PLANT MATTER - CUSTOMIZING PROTEIN PROPERTIES – FROM SAME SOURCE - BUILDING UNIQUE KNOW-HOW & IP AS BARRIERS TO ENTRY - BUILDING TECHNICAL CREDIBILITY WITH CUSTOMERS - COMMANDING HIGHER PRICE POINTS - NEW PRODUCT PARADIGMS, A UNIQUE TAKE ON PROTEINS: - USING TRANSFERASES TO SYNTHESIZE LMW COMPOUNDS - USING LIPASES IN BOTH PROTEIN AND LIPID SYNTHESYS
- 23. Спасибо! TRI-K Industries, Inc. | info@tri-k.com | www.tri-k.com | 973.298.8850