Download Proteases in the Food Industry

Proteases in the Food Industry
USP Enzyme
Workshop,
July 9, 2009
Rockville, MD
Outline
• Protease definition & basic biochemistry
• Protease sources & classifications
• Commercial protease manufacturing
• Nonfood protease applications
• Protease applications in food processing
• Protease assays – industry rationale
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Protease Definition & Basic Biochemistry
Proteins are polymers composed of linear chains of amino acids linked by
peptide bonds*.
Proteases are enzymes that catalyze the hydrolysis of peptide bonds in
proteins and polypeptides.
PHE
VAL
P2
P1
P1’
P2’
LEU
MET
GLY
LEU
HIS
PHE
LEU
MET
VAL
Protease
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Protease Definition & Basic Biochemistry
•
•
•
There are literally thousands of different protease molecules that have been isolated and
characterized. There are several hundred proteases that have commercial relevance.
There are numerous schemes for classifying proteases…
These classification schemes provide a wealth of relevant information about each
protease.
Example: International Union of Biochemistry & Molecular Biology enzyme nomenclature
system
EC 3.4.22.2 papain
EC 3.4.22.3 ficain
EC 3.4.22.4 now covered by EC 3.4.22.32 and EC 3.4.22.33
EC 3.4.22.5 now EC 3.4.22.33
EC 3.4.22.6 chymopapain
EC 3.4.22.7 asclepain
EC 3.4.22.8 clostripain
EC 3.4.22.9 now EC 3.4.21.48
EC 3.4.22.10 streptopain
EC 3.4.22.11 now EC 3.4.24.56 (supplement 3)
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Protease Classification
By source organism:
Animal: chymosin, trypsin, pepsin
Plant: bromelain, papain, ficin
Bacterial: subtilisin, bacillopeptidases
Fungal: Aspergillopepsin
By proteolytic mechanism:
Serine proteases
Threonine proteases
Cysteine proteases
Crystal structure of trypsin, a serine protease.
Aspartic proteases
Metalloproteases
Glutamic acid proteases
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Protease Mechanism Example: Serine
Protease
.
The serine hydroxyl
(OH) group of the
protease conducts a
nucleophilic attack on
the carbonyl carbon
of the scissile peptide
bond of the substrate.
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Protease Classification (Cont’d)
By active pH range:
Acid proteases
Alkaline proteases
High-alkaline proteases
By peptide bond specificity:
Endopeptidases
Exopeptidases
Carboxypeptidases
120
100
% R e la t iv e A c t iv it y
Neutral proteases
80
Acid
60
Neutral
40
Alkaline
20
High-Alkaline
0
-20 0
2
4
6
8
10
12
14
pH
Aminopeptidases
AA-specific proteases
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Protease Classification (Cont’d)
Example: Subtilisin Carlsberg (EC 3.4.21.14)
Endopeptidase
Bacterial source: Bacillus subtilis
Alkaline pH range (8-9 optimum)
Serine protease
Calcium-dependant (?)
Relatively nonspecific (favor uncharged AA
Crystal structure of Subtilisin Carlsberg
in the P1 position).
Commercial Products:
Alcalase 2.4L (Novozymes)
Protex 6L (Genencor)
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Commercial Protease Manufacturing
Animal & plant-derived proteases are produced primarily by extraction.
Bacterial & fungal proteases are produced primarily by industrial-scale fermentation.
Many bacterial & fungal proteases (and even some animal-derived proteases) have been
genetically modified or engineered. Rationales for genetic modification:
Transfer a gene to a more efficient host.
Produce a multi-copy insert (plasmid).
Better economics
Modify expression and secretion signal sequences.
Delete undesirable genes.
Protein engineering – modify one or more amino acids in the primary protein sequence.
Æ Improved catalytic activity
Æ Modified catalytic specificity
Æ Improved stability (pH, temperature, surfactants, etc.)
Æ Modified pH and temperature profiles
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Industrial Fermentation Processes
Shake
Flask
Pre-seed
Fermentation
Seed
Fermentation
Main
Fermentation
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Industrial Fermentation: Recovery &
Purification
Cell separation
Concentration
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Industrial Fermentation: Recovery &
Purification
Liquid packaging
Liquid formulation
Polish filtration
Granulation &
Packaging
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Nonfood Protease Applications
Medicine
Pharmacology & drug manufacture
Laundry & dishwashing detergents (#1)
Hard surface cleaning formulations
Contact lens cleaning formulations
Waste treatment
Industrial applications
Fermentation (fuel EtOH, etc.)
Chondroitin & heparin production
Animal feed additives
Digestive supplements
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Protease Applications in Food Processing
Proteases are also used in a wide range of foods & food processing
applications.
Dairy: milk coagulation, flavor development
Baking: gluten development
Fish & seafood processing: fishmeals, enhanced oil recovery, aquaculture
Animal protein processing: improved digestibility, reduced allergenicity,
improved flavor, meat tenderization
Plant protein processing: improved functionality & processing, generation of
bio-active peptides.
Yeast hydrolysis: flavor compounds.
Production of Value-Added Food
Ingredients
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Protease Applications in Food Processing
Basic rationale: Proteases are a powerful tool for modifying the properties of
food proteins.
Improved digestibility
Improved solubility
Modified functional properties: emulsification, fat-binding, water-binding,
foaming properties, gel strength, whipping properties, etc.
Improved flavor & palatability
Improved processing: viscosity reduction, improved drying, etc.
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An Example: Soy Protein Modification
. Soybeans
Solvent
Extraction
Defatted
Soymeal
Soy Oil
Aqueous
Extraction
The protein in the soy
isolate has been
subjected to several
harsh denaturing
processes. As a result it
has very poor solubility
and functionality
compared to a “native”
soy protein.
Isoelectric
Precipitation
CHO residue
“Whey”
Soy Protein
Isolate (90%)
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An Example: Soy Protein Modification
SPI has very poor solubility & limited digestibility. It has poor functionality for many
food applications.
By treating the SPI with protease it is possible to restore much of the protein’s original
solubility and functionality.
Treatment of SPI with an alkaline endopeptidase (DH = 4%) significantly improves its
solubility:
Protein Solubility (% )
120
100
80
Native SP
60
SP Isolate
40
SP Hydrolysate
Improved solubility Æ
improved digestibility, better
performance in low-pH
beverages.
20
0
0
2
4
6
8
10
12
pH
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General Enzyme QA & QC
Objectives: Assure that enzyme products are safe and effective and meet the
regulatory requirements for their intended applications.
Food-grade vs. technical-grade specifications
GM vs. non-GM
Shelf-life requirements & storage recommendations
Formulations requirements
Kosher, Chometz, Halal, and organic certifications
Allergen statements
Non-BSE and non-TSE statements
Physical specifications – pH, specific gravity, color, etc.
Microbiological specifications
Activity specifications
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Protease Activity Assays
Just as there are hundred’s of commercially relevant proteases, there are very
many analytical assay methods used to measure protease activity.
FCC methods
USP methods
Industry methods (HUT assay, TNBS casein assay, etc.)
Proprietary methods developed by enzyme producers & users
At Genencor we have >35 different protease assays. Why so many?
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Protease Assays: Producer’s Perspective
From an industrial enzyme producer’s perspective, we have certain “demands”
that we place on an enzyme assay method:
Must be extremely repeatable & reproducible.
Must be valid for all stages of production from main fermentation to final
product. Wide range of activities.
Should only use highly pure defined substrates and reagents. (We use a lot
of synthetic peptide substrates.)
Should be at temperature & pH that are close to enzyme’s optima.
Should be readily adaptable to automation – we test many 100’s of samples
every day.
Does not necessarily need to reflect the performance of an enzyme in its
intended applications. (A single product might have >20 different
applications.)
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Protease Assays: End-User’s Perspective
“How is this protease product going to perform in my application?” Every
application is unique….
For example, in a laundry detergent formulation proteases are combined with
surfactants, oxidizers, and other ingredients and expected to remain stable
for many months at room temperature. And they are also expected to have
good efficacy in soil removal…
In a food application the same protease molecule might be expected to
hydrolyze whey protein to produce a specific bioactive peptide. Under
defined parameters (substrate, pH, time, temperature, dosage, etc.) the
protease should produce a defined concentration of the desired bioactive
peptide.
In an animal feed application… Well, you get the picture.
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Protease Assays
It is virtually impossible to develop a single assay method that meets the
enzyme producer’s needs and all of the potential end-users’ needs.
Enzyme producers work very diligently with our customers to identify the
correct protease & usage conditions for their unique application.
Then our QA release assay method and our specifications provide the
necessary assurance that every batch of our protease product will give
acceptable performance in our customer’s application.
This is not an absolutely perfect system… For example, sometimes an
unmeasured side-activity might be critical in an application. If this sideactivity varies significantly from batch-to-batch Æ problems.
This is why we ask our customers a lot of questions and typically perform labscale studies to determine the best product and optimum usage parameters.
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Thank You!
Thank you for your kind attention. Questions?
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