Download The world of proteases Diversity and function

The world of proteases
Diversity and function
Amsterdam, May 8th 2014
V Glitsø PhD, Senior Department Manager
K Pontoppidan PhD, Science Manager
Feed Applications
Novozymes R&D
AGENDA
• The world of proteases
• Proteases in animal feed
1
The world of proteases
DEFINITION OF A PROTEASE
Protease = Peptidase = Proteinase =
Proteolytic enzyme
An enzyme that degrades protein by
hydrolysis of peptide bonds
Protease
OCCURRENCE AND FUNCTIONALITY
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…
PROTEASE SUBSTRATES
Protein is a highly diverse substrate
20 different
amino acids to
coose from at
each position
Various types of protein:
His
Plant proteins
Egg white
Milk
Muscle
Hormones
Enzymes
Crystal structure of porcine trypsin
Gly Pro
Ala
PROTEASE SPECIFICITY
Specificity is largely determined by the amino acid
residues in the active site of the protease
PROTEASE SPECIFICITY
Different proteases result in different hydrolysis products
Very specific protease
Less specific protease
Trypsin
specific for Arginine and Lysine
RONOZYME® ProAct
preference for hydrophobic amino acids
Hydrolysis is limited
(few cuts)
Hydrolysis is aggresive
(many cuts)
PROTEASE CLASSIFICATION
Proteases are classified according to:
Functionality (reaction catalyzed)
Molecular structure and sequence homology (MEROPS)
FUNCTIONAL CLASSIFICATION
Endo
His
Gly Pro
Ala
Exo
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
Metallo
Aspartic
Serine residue
involved in the
catalytic action in
the active site
65
59
14
Glutamic
2
Threonine
4
Unknown
7
Total
196
MEROPS Release 9.10 (http://merops.sanger.ac.uk)
MICROBIAL PROTEASE DIVERSITY
370,000 SEQUENCES
Family A1
- Pepsin
Family M14
- Carboxypeptidase
A and B
Family S1
- Trypsin
- Chymotrypsin
- Elastase
- RONOZYME® ProAct
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
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)
2
Proteases in animal feed
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
PROTEASE ACTIVITY
SDS-page analysis to evaluate protease purity and identification
Load protein samples here
Solution
with
protein
kD
Heat +
SDS
50
big proteins
35
Visualization
e.g. by staining
30
25
small proteins
15
10
PROTEASE ACTIVITY
SDS-page analysis to evaluate protease purity and identification
kD
kD
kD
50
50
35
30
25
15
Nocardiopsis
Serine protease
(>95% pure)
50
35
35
30
30
25
25
15
15
10
10
10
Bacillus Subtilisin
proteases
>90% estimated to be
wheat protein
Identities confirmed by proteomics analysis (LC-MS/MS)
HOW TO EVALUATE IF A PRODUCT
HAS ACTIVE PROTEASE
PROTEASE ACTIVITY
Quantitive activity assay
Relative protease activity
(Protease A=1)
2500
2317
Dosed on equal weight (g product/ml)
Adjusted for 'in feed' recommendations
2000
1500
Assay conditions adjusted
according to protocol for
Product A
1448
1000
770
726
500
193
1
0
Ronozyme
ProAct
1
Product A
Product B
105 168
Product C
182
Product D
Casein-FITC substrate, pH 8.3, 15 min, 37°C, very sensitive assay
ELN-13-HALL-0016
QUANTITATIVE ACTIVITY ASSAY
Protease activity determined using a highly sensitive assay
(Casein-FITC substrate, pH 8.3, 15 min, 37°C)
Relative protease activity
(Protease A=1)
2500
Dosed on equal weight (g product/ml)
2317
Adjusted for 'in feed' recommendations
2000
1500
Assay conditions adjusted
according to 3x
protocol for
Product A
1448
1000
7.5x
770
726
500
193
1
0
Ronozyme
ProAct
1
Product
Product A
A
Product B
B
Product
105
168
Products
C
Product C
182
Product DD
Product
ELN-13-HALL-0016
PROTEASE ACTIVITY
Qualitative activity assay for fast indicative answers
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 assay for activity and stability
Skimmed milk plates for a simple test of activity and acid stability
Acid
instability
Acid
stability
A
ProAct
A
C
B
C
B
Dilution in pH 7
buffer
Spot on plate
Extraction of solid products in pH 6
buffer (1 hour, 100 rpm, 26°C)
recovery of liquid fraction
Incubation
½ h at pH 3
dilution in pH 7
buffer
Incubation of plate (2½ hour, pH 6,
37°C). All extracts diluted 25x in total.
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)
ELN-13-KPON-0002
SYNERGY WITH ENDOGENOUS PROTEASES
In vitro digestion model –
a useful tool when screening for complimentary effects
Test enzyme
Analyse soluble
Feed
protein
Analyse degree of
hydrolysis (DH)
The results reflect at the same time survival and action in the digestion model
SYNERGY WITH ENDOGENOUS PROTEASES
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
Improvement relative to control (%)
In vitro conditions:
- SBM-maize (30:70)
- 40°C
- pH 3/pepsin: 1hour
- pH 7/pancreatin: 4 hours
- Protease: 10 x rec. dosage
120
Improvement relative to control (%)
Protein solubilization
110
*
115
*
*
110
105
100
95
90
85
80
105
*
*
Degree of hydrolysis
100
95
90
85
* Significant increase (P<0.05, all-pairwise Tukey-kramer HSD)
SYNERGY WITH ENDOGENOUS PROTEASES
Effect of ProAct on protein sol. of different raw materials in vitro
Control
100
100 mg EP/kg
Sol protein of total (%)
90
80
70
60
50
40
SBM, Brazil Full fat SBM, Sorghum, Maize, Brazil
SBM
Full
fat Sorghum
Maize
1
Brazil
Brazil
SBM
MLRA080001 & HALL100003
MBM, US
MBM
Feather Wheat Midds Corn DDGS
Feather
Wheat
Corn
meal,
Brazil
(Dakota
meal
midds
DDGS
Gold)
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
Using ProAct, same
effect is obtained
with ~½ amount of
PTN
ELN-13-HALL-0001
COMPATIBILITY WITH OTHER FEED ENZYMES
In vitro incubations with enzyme combinations
Phytate degradation by phytase
and phytase + ProAct (20x)
Corn-sbm diet: pH 6/30 min -> pH 3/pepsin/10 min
Phytase @ recom. dose, ProAct @ 20x rec. dose
Xylan solubilisation by xylanase
and xylanase + ProAct (10x)
Wheat bran: pH 7/3 hours
Xylanase @ recom. dose, ProAct @ 10x rec. dose
ELN-11-KPON-0001 & ELN-11-CAAO-0005
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
Thank you for your attention
MICROBIAL PROTEASE DIVERSITY
partate
As
Serine
4,169
reonine
Th
132,946
Metallo
ate
m
uta
674
10,833
ss
ncla ifie
140,155
d
U
8,778
Gl
Peptidases
divided on basis
of catalytic
mechanism
Cysteine
59,135
Major protease
families
bubble areas are
proportional to
sequence counts
370.000 in total