The world of proteases-diversity and function. Glitsoe, V. & Pontoppidan. K. Feed Enzymes Seminar presentation, Amsterdam 2014.

1. Amsterdam, May 8th 2014
V Glitsø PhD, Senior Department Manager
K Pontoppidan PhD, Science Manager
Feed Applications
Novozymes R&D
The world of proteases
Diversity and function

2. AGENDA
• The world of proteases
• Proteases in animal feed

3. 1
The world of proteases

4. Protease = Peptidase = Proteinase =
Proteolytic enzyme
An enzyme that degrades protein by
hydrolysis of peptide bonds
DEFINITION OF A PROTEASE
Protease

5. Analysis of complete genomes has shown that about
2% of proteins in all kinds of organisms are proteases
Proteases have many different functions: processing of
proteins, protein turnover, cell division, metabolism, toxins…
OCCURRENCE AND FUNCTIONALITY

6. PROTEASE SUBSTRATES
Protein is a highly diverse substrate
Various types of protein:
Plant proteins
Egg white
Milk
Muscle
Hormones
Enzymes
His
Gly Pro
Ala
20 different
amino acids to
coose from at
each position
Crystal structure of porcine trypsin

7. PROTEASE SPECIFICITY
Specificity is largely determined by the amino acid
residues in the active site of the protease

8. PROTEASE SPECIFICITY
Very specific protease
Trypsin
specific for Arginine and Lysine
Less specific protease
RONOZYME® ProAct
preference for hydrophobic amino acids
Different proteases result in different hydrolysis products
Hydrolysis is limited
(few cuts)
Hydrolysis is aggresive
(many cuts)

9. PROTEASE CLASSIFICATION
Proteases are classified according to:
Functionality (reaction catalyzed)
Molecular structure and sequence homology (MEROPS)

10. FUNCTIONAL CLASSIFICATION
His
Gly Pro
Ala
Endo
Exo

11. MEROPS CLASSIFICATION
Proteases are divided into 7 major protein families and 196
subfamilies based on molecular structure and sequence homology
Catalytic Type
(Major protein families)
MEROPS
families
Serine 45
Cysteine 65
Metallo 59
Aspartic 14
Glutamic 2
Threonine 4
Unknown 7
Total 196
MEROPS Release 9.10 (http://merops.sanger.ac.uk)
Serine residue
involved in the
catalytic action in
the active site

12. Family A1
- Pepsin
Family S1
- Trypsin
- Chymotrypsin
- Elastase
- RONOZYME® ProAct
MICROBIAL PROTEASE DIVERSITY
370,000 SEQUENCES
Family M14
- Carboxypeptidase
A and B

13. PROTEASE ACTIVITY
Protease activity can be measured in many ways using
different substrates and different reaction conditions
There is not one correct way to measure protease activity
An activity number is always dependent on the exact
assay conditions
Therefore, activity units/protease assays cannot be used
to evaluate protease performance – this should be done
under real application conditions

14. PROTEASE ACTIVITY
Protease activity assays are useful to:
Control that our product always contains the same amount of
protease activity (QA/QC)
As a tool in the development process of proteases
Compare the relative (e.g. residual) activity of proteases (e.g.
the stability following a challenge such as pelleting or low pH)

15. 2
Proteases in animal feed

16. KEY CHARACTERISTICS FOR A FEED PROTEASE
Protease activity
Stability in the gut and during processing (pelleting)
Synergy with endogenous proteases
Compatibility with other feed enzymes

17. PROTEASE ACTIVITY
10
25
30
35
50
15
kD
Visualization
e.g. by staining
Solution
with
protein
Heat +
SDS
big proteins
small proteins
Load protein samples here
SDS-page analysis to evaluate protease purity and identification

18. PROTEASE ACTIVITY
10
25
30
35
50
15
10
25
30
35
50
15
10
25
30
35
50
15
kD
kD
kD
Nocardiopsis
Serine protease
(>95% pure)
Bacillus Subtilisin
proteases
>90% estimated to be
wheat protein
Identities confirmed by proteomics analysis (LC-MS/MS)
SDS-page analysis to evaluate protease purity and identification

19. HOW TO EVALUATE IF A PRODUCT
HAS ACTIVE PROTEASE

20. Assay conditions adjusted
according to protocol for
Product A
1448
1
193
105
182
2317
1
770
168
726
0
500
1000
1500
2000
2500
Ronozyme
ProAct
Product A Product B Product C Product D
Relativeproteaseactivity
(ProteaseA=1)
Dosed on equal weight (g product/ml)
Adjusted for 'in feed' recommendations
ELN-13-HALL-0016
PROTEASE ACTIVITY
Quantitive activity assay
Casein-FITC substrate, pH 8.3, 15 min, 37°C, very sensitive assay

21. QUANTITATIVE ACTIVITY ASSAY
Protease activity determined using a highly sensitive assay
(Casein-FITC substrate, pH 8.3, 15 min, 37°C)
1448
1
193
105
182
2317
1
770
168
726
0
500
1000
1500
2000
2500
Ronozyme
ProAct
Product A Product B Product C Product D
Relativeproteaseactivity
(ProteaseA=1)
Dosed on equal weight (g product/ml)
Adjusted for 'in feed' recommendations
Product A Product B Products C Product D
ELN-13-HALL-0016
7.5x
3x
Assay conditions adjusted
according to protocol for
Product A

22. Protease activity on agarose plates with 1% AZCL-casein (pH 7, 22°C)
pH 5 buffer extracts of protease products were used for the spot test
Release of blue color indicate protein hydrolysis
t = 0 t = 30 min t = 60 min t = 120 min
ELN-14-BERA-0003
PROTEASE ACTIVITY
Qualitative activity assay for fast indicative answers

23. Skimmed milk plates for a simple test of activity and acid stability
Of the tested products RONOZYME® ProAct showed the highest protease
activity and it was the only product that was stable at pH 3 (30 min)
Extraction of solid products in pH 6
buffer (1 hour, 100 rpm, 26°C)
recovery of liquid fraction
Spot on plate
Incubation
½ h at pH 3
dilution in pH 7
buffer
Dilution in pH 7
buffer
Incubation of plate (2½ hour, pH 6,
37°C). All extracts diluted 25x in total.
ProAct A C B
ELN-13-KPON-0002
BC
Acid
instability
Acid
stability
A
PROTEASE ACTIVITY
Qualitative assay for activity and stability

24. In vitro digestion model –
a useful tool when screening for complimentary effects
SYNERGY WITH ENDOGENOUS PROTEASES
The results reflect at the same time survival and action in the digestion model
Test enzyme
Feed
Analyse degree of
hydrolysis (DH)
Analyse soluble
protein

25. 80
85
90
95
100
105
110
115
120
Improvementrelativetocontrol(%)
*
*
* *
In vitro conditions:
- SBM-maize (30:70)
- 40°C
- pH 3/pepsin: 1hour
- pH 7/pancreatin: 4 hours
- Protease: 10 x rec. dosage
Products A, B, C and D
represent products claiming
protease activity as main
activity or as side activity
ELN-09-HALL-0008 & ELN-10-LNBR-0065
Protein solubilization
85
90
95
100
105
110
Improvementrelativetocontrol(%)
Degree of hydrolysis*
* Significant increase (P<0.05, all-pairwise Tukey-kramer HSD)
SYNERGY WITH ENDOGENOUS PROTEASES

26. 40
50
60
70
80
90
100
SBM, Brazil
1
Full fat SBM,
Brazil
Sorghum,
Brazil
Maize, Brazil MBM, US Feather
meal, Brazil
Wheat Midds Corn DDGS
(Dakota
Gold)
Solproteinoftotal(%)
Control 100 mg EP/kg
SorghumSBM Maize MBM Feather
meal
Wheat
midds
Corn
DDGS
Full fat
SBM
MLRA080001 & HALL100003
SYNERGY WITH ENDOGENOUS PROTEASES
Effect of ProAct on protein sol. of different raw materials in vitro

27. SYNERGY WITH PANCREATIC PROTEASES
Substrate: Commercially toasted SBM
Incubation: 3 hours, pH 7, 40°C
Enzymes: Pancreatic Trypsin Novo (PTN), RONOZYME® ProAct
Analysis: Colorimetric analysis of cleaved peptide bonds with OPA reagent
ELN-13-HALL-0001
Using ProAct, same
effect is obtained
with ~½ amount of
PTN

28. COMPATIBILITY WITH OTHER FEED ENZYMES
ELN-11-KPON-0001 & ELN-11-CAAO-0005
In vitro incubations with enzyme combinations
Phytate degradation by phytase
and phytase + ProAct (20x)
Xylan solubilisation by xylanase
and xylanase + ProAct (10x)
Corn-sbm diet: pH 6/30 min -> pH 3/pepsin/10 min
Phytase @ recom. dose, ProAct @ 20x rec. dose
Wheat bran: pH 7/3 hours
Xylanase @ recom. dose, ProAct @ 10x rec. dose

29. CONCLUDING REMARKS
Large protease diversity exists:
• A protease is not just a protease
• Also there are large differences between the products
claiming protease activity
For feed protease it is important to ensure that the key
characteristics are in place:
• Detectable protease activity – ability to hydrolyse protein
• Stability in gut and during processing
• Synergy with endogenous enzymes
• Compatibility with other feed enzymes

30. Thank you for your attention

31. MICROBIAL PROTEASE DIVERSITY
Aspartate
Gl
utam
ate
Threonine
U
nclassifie
d
Metallo
Serine
Cysteine
132,946
4,169
8,778
140,155
59,135
674
10,833
Major protease
families
bubble areas are
proportional to
sequence counts
370.000 in total
Peptidases
divided on basis
of catalytic
mechanism