enzymatic tailoring of hydrolysates' properties
TRANSCRIPT
Enzymatic Tailoring of Hydrolysates’ Properties
Outline
• Enzymology– Overview of enzymes
• Enzymatic Hydrolysates of Proteins:– Chemical and Biologiocal Properties– Tailoring Properties: Protein Surgery– Examples
• Opportunities
Classes & Nomenclature of Enzymes
6 subclasses
Enzyme Commission (EC) Designation Numbers*
Group Reaction catalyzed Typical reaction Enzyme example(s) with trivial name
EC 1Oxidoreductases
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 2Transferases
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 3Hydrolases
Formation of two products from a substrate by hydrolysis AB + H2O → AOH + BH Lipase, amylase, peptidase
EC 4Lyases
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 5Isomerases
Intramolecule rearrangement, i.e. isomerization changes within a single molecule
AB → BA Isomerase, mutase
EC 6Ligases
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)
Enzymes• Enzymes:– are catalysts– not spent by the catalyzed reactions– spent by degradation
Enzymes Activity UnitsENZYME 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) FungalSAP (Spectrophotometric acid protease unit); or Leucine aminopeptidase Unit (LAPU)
Pullulunase Bacterial PU (Pullulunase Unit)
Per Food Chemical Codex V & the US Pharmacopia
Enzymes Specificity: Protein Surgery
Practical Impact
Properties of Proteins
DETERMINED BY:
1. SELECTION2. SEQUENCE3. CONDITIONS
DETERMINED BY:
CHEMICAL PROPERTIES
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)
Bioactive Peptides from Food*BIOLOGICAL EFFECT ORIGIN ENCRYPTING PROTEIN NAME AA SEQUENCE
IMMUNOMODULATORY Rice Rice albumin Oryzatensin GYPMYPRLWheat 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 specifiedFish Sardine muscle MY
MilkLactalbumin &Lactoglobulin - MHIRL, YVEEL
HYPOTENSIVE Soy Soy protein - NWGPLV
FishFish muscle protein
(e.g. tuna, squid, sardine) - LKP, IKP, LRPEgg Ovotransferrin & Ovalbumin Ovokinins KVREGTTY, etc.
*Hartmann, R. et al. Food-derived peptides with biological activity: from researchto food applications. Current Opinion in Biotechnology, 18:163-169, 2007
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
Trypsin
Flavo
urzyme
Chymotry
psin
Bromela
in
Alcalas
ePep
sinPap
ain0
10
20
30
40
50
60
70
80
90
100
Degree of Hydrolysis after 24 hrs (%)
DH (%)% DH
PROTEASE
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
Trypsin Flavourzyme Chymotrypsin Bromelain Alcalase Pepsin Papain0
10
20
30
40
50
60
70
80
90
100
DH (%)RSA
Degree of Hydrolysis&
Radical Scavenging Activity (%)
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 ofResulting Protein Hydrolysates. Czech. J. Food Sci., 20:1, 2012
10 30 60 120 240 4800.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
AlcalaseFlavourzymeNovozym
AA/Tyr
Hydrolysis Time (mins)
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 ofResulting Protein Hydrolysates. Czech. J. Food Sci., 20:1, 2012** After 8 hrs
Alcalase Flavorzyme Novozym0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
Degreeof
Hydrolysis(%)**
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 ofResulting Protein Hydrolysates. Czech. J. Food Sci., 20:1, 2012** After 8 hrs of hydrolysis
No Hydrolysis Alcalase Flavourzyme Novozym0
20
40
60
80
100
120
140
160
Foam Stability(mins/100 mm)**
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 ofResulting Protein Hydrolysates. Czech. J. Food Sci., 20:1, 2012** After 8 hrs of hydrolysis
His Ile Leu Lys Met Cys Phe Tyr Thr Trp Val Ala Arg Asp Glu Gly Pro Ser0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
UnhydrolyzedAlcalaseFlavourzymeNovozym
SpecificAA (%)
!!
Sulfur
Nitrogen
Antiox
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 ofResulting 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
Flavourzyme® vs. Alcalase®
Effects on - Color- NVM- Ash- N2
NVM% vs. Protein% in New Proteins Hydrolysates
1 2 3 4 5 6 7 8 90.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
Protein, % (N X 6.25)Non-volatile matter (%)
% NVM
% Protein
Protein %: in Biomass vs. in Hydrolysate
Desiccated Coconut
Chia Pumpkin Isolate Pumpkin Seed Lupin Chlorella Cocoa0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
Protein Contentin Biomass (%)Protein Content in Hydrolysate (%)
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
