Industrielle Herstellung von Enzymen für Lebensmittel

Industrielle Herstellung von
Enzymen für Lebensmittel
AB Enzymes
Dr. Patrick Lorenz
11. FEI-Kooperationsforum 2012
Bonn, 17. April 2012
MUTTERKONZERN
Associated British Foods
Incorporated
Employees
1935
> 97,000 in 44 countries
Revenues
> £11.1 billion (€12.2 billion) in 2011
Activities
International food, ingredients and retail
Selected Leading Positions
World´s second largest sugar producer
Leading global speciality tea brand
World´s second largest yeast business
World leader in speciality enzymes
2
UNTERNEHMEN
AB Enzymes
Established
1907
Ownership
100% owned by Associated British Foods (ABF)
Employees
> 200
Revenues
Growth Rate
Focus
R&D Investment
Patents
> €100 million
> 10% p.a. since 1999
Feed, Food & Specialities, Technical, Biomass Ethanol
> 10% of revenues
> 450 (owned and licensed)
3
FIRMENGESCHICHTE
Ein internationales Traditionsunternehmen
1907
Röhm and Haas founded. Invention of first industrial enzyme for the leather industry – Oropon
1934
1954
1991
Invention of the first enzyme for the food industry: PECTINOL® now ROHAPECT®
Alko (now Altia), starts producing microbial enzymes for captive use in grain processing
1958
First enzyme for the milling and baking industry launched: VERON®.
1973
Invention of first industrial Xylanase
1985
First patent application for Trichoderma cellulase enzymes by Alko
Roal JV founded by Röhm and Alko for enzyme manufacturing for both companies
1996
Röhm Enzyme GmbH, is incorporated
1997
Röhm Enzyme buys the non-Trichoderma enzyme business of Alko
1999
ABF acquires Röhm Enzyme
2001
Röhm Enzyme renamed AB Enzymes
2002
2003
Acquisition of Gamma Chemie, a food enzymes distributor
Integration of AB Enzymes into the newly formed division, ABF Ingredients
2007
Global distribution arrangement for feed with ABF sister company, AB Vista
2008
Acquisition of Quantum Phytase business and technology
4
FORSCHUNG & ENTWICKLUNG
We have the full R&D skill set
Isolation
Screening
Enzyme
Gene
Cloning
Mutagenesis
Evolution
Expression
NH2
COOH
NH2
COOH
Strain
construction
Fermentation
DSP
Production
strain
Bulk
production
Product
Cp(cal/°C)
Bioprospecting
Temperature (°C)
Nature
Microorganisms
Wild-type
enzymes
Analytical
amounts for
primary
characterisation
Optimised
enzymes
Formulation
5
PRODUKTION
Proud to partner with Roal Oy
Location
Ownership
Activities
Technology
Facility
Capacity
Rajamäki, Finland
Roal Oy, a 50/50 joint venture between ABF and Altia,
a Finnish alcoholic beverages company
Manufacturing, research, process and product
development, pilot plant
Patented production technology based on
Trichoderma, Bacillus and Aspergillus production strains
Modern fermentation facility with downstream,
drying and mixing capabilities
Major capacity expansion in 2009 to support strong
business growth
6
MARKT UND WETTBEWERBER
Global Market 3.4 billion USD
2.6 billion €
Big Players:
Novozymes
DuPont (Danisco/Genencor)
DSM
BASF
Others:
AB Enzymes
Ajionomoto
Amano
Henkel
Kerry
Biocatalyst
Lyven
Chr. Hansen
7
ANWENDUNGSFELDER ENZYME
Food accounts for 30% of enzyme usage
Enzyme Sales
Industrial enzyme application can
be divided in
- Food
- Feed
- Technical Applications
- Biofuel
Biofuel
15%
Technical
39%
Food
29%
Feed
17%
8
HERKUNFT UND PRODUKTIONSPLATTFORMEN
Microbial dominance
Origin of industrial Enzymes
265 enzymes are known to be
industrially produced
Animals
5%
Plants
3%
The majority originate from fungal
and bacterial sources*
Fungal and bacterial production
strains produce similar tonnage of
active enzyme protein.
Bacteria
28%
Fungi
64%
*Source: AMFEP list of enzymes (www.amfep.org)
9
EXPRESSIONSWIRT ≠ PRODUKTIONS PLATTFORM
Wenige starke Plattformen in „Bulk“-Liga
Expression Host
An „organism“ (virus, bacterium, fungus, yeast, insect, cultured
cells, transgenic animals and plants - any living thing that can
be manipulated) used to produce a product of interest (a
protein/enzyme, a chemical compound, a polysaccharide, an
antibody, etc.)
Production host (Cell factory)
It is an expression host with sufficient efficiency for industrial
production.
10
PLATTFORMEN - ZELLFABRIKEN
Strains
Enzyme Example
Trichoderma reesei
Cellulases, Hemicellulases,
Bacillus spec.
B. subtilis
B. amyloliquefaciens
B. licheniformis
Amylases, Proteases
Aspergillus spec.
Lipases, Amylase,
Pichia spec.
Phytase, Lipase
11
MIKROBIELLE ZELLFABRIKEN
3 Plattformen
2 Kategorien
GMO
recombinant
DNA
Trichoderma
Aspergillus
Bacillus
GMO
GMO
GMO
UV
CMO
classical
Microbe
DNA
CMO
CMO
CMO
Enzyme
gene
12
Trichoderma reesei Produktionsplattform
•T. reesei QM6a was isolated in Solomon islands 1944
•Naturally efficient cellulase producer
•High cellulase producing mutants secrete up to 40-100 g/l protein
•Techniques and tools for genetic engineering developed in the
1980’s
•Strong cbh1 promoter used for production
•Non-pathogenic to man
•Used for over 40 years in large scale manufacturing of enzymes
•Whole genome has been published
•Key patents of Trichoderma technology are exploitable by ROAL
13
DIMENSIONEN - BULK
Trichoderma 120 m3 Fermentation
FERMENTATION - Feed ca.
C-Quelle Dextrine
N-Quelle komplex
(NH4)2 SO4
Anti-foam
KH2PO4
H2SO4
NaOH
NH3-WATER
H2O
Air
Energy (Electricity plus heated steam)
37 000 kg
4 000 kg
600 kg
30 kg
400 kg
90 kg
400 kg
7 000 kg
32 000 kg
70-80 m3/min
100 MWh
DOWNSTREAM PROCESS
FILTER AIDS
FLOCCULANTS
FILTER SHEETS
CELL WASTE
Protein (harvest, BCA)
850 kg
540 kg
300
17 000 kg
16 000 kg
14
TECHNOLOGISCHER FORTSCHRITT
Chance und Bürden des tech. Fortschritts
1970
Today
• Microbiology
• Fermentation Technology
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Microbiology
High throughput Screening
Enzyme Engineering Technologies
(Gene shuffling, Saturation
Mutagenesis, Recombination,
Creating Diversity, Directed Evolution )
Genome Sequencing (ultra fast )
Bioinformatics
Genome mining
Transcriptome analysis
Proteom analysis
Metabolome analysis
Metabolic flux analysis
Fermentation technology
Simulation / modeling
Down stream processing technology /
Separation technology
Granulation technology
15
EIGENSCHAFTEN PLATTFORMEN
Sicherheit, Nebenprodukte,
Trophismen
1
1
• Biological Safety
o
o
o
No toxins, strain not pathogenic
No mobilizing elements
No spore formation
• No production of unwanted side products (clean background)
o
o
o
o
Low in secreted proteases
Slime (polysaccharide, poly amino acid, protein)
Malodor (fatty acids, sulfur compounds)
Pigments /coloured metabolites
• Inexpensive growth media / conditions
• Physiological Information available
o
o
o
o
o
Active metabolic pathways
Metabolic potential
No auxotrophy
No overflow metabolites
No metabolisation of target product
16
EIGENSCHAFTEN PLATTFORMEN
2
Produktivität/Sekretion, Manipulierbarkeit
• Own Intellectual Property or no IP – freedom to operate
• Capable of producing large amounts of the desired product in a short time
(high yield) - excellent secretion performance for extracellular enzymes
• Accessability to molecular biology
o
o
o
o
o
Easy to manipulate
Sufficient transformation rates
Knock out mutants possible
No Restriction/Modification systems
Genome available (DNA Arrays)
• Genetic Stability
o
Absence of phages, prophages, transposons
• Long term experience in technical processes
• Experience with various fermentation conditions
o
Acceptance of various of nitrogen sources
17
ENZYM & PLATTFORM & INFRASTRUKTUR
Enzymspezifische Plattform und Prozess
o Enzyme, production organism
and cultivation conditions are
linked Influence on yield
o Single amino acid exchanges
may reduce productivity by
99%
o Design of fermenters
determines yields by fixing
aeration and cooling options
18
Rohalase® PL-XTRA
Enzymatische Ölentschleimung
ÖL RAFFINATION
Pflanzliche Öle müssen gereinigt werden
4,3
3,2
3,0
Oilseed cell
5,0
31,0
Soy
10,2
Sunflower
Disturbing substances after pressing/extraction
Rapeseed
Phospholipids
Free fatty acids (FFA)
Pigments (chlorophyll, carotinoides)
S-compounds
Disodourous and flavouring substances
Trace metals (Ca, Mg, Fe etc.)
contaminants (pesticides, herbicides)
Palm
15,6
27,7
World Oil consumption [%]
Soy, Rapeseed, Sunflower: 65 Mio tons/a
Impacting further processing, taste and storage stability
20
KONKURRIERENDE VERFAHREN
Ölentschleimung - Degumming
CHEMICAL REFINING
PHYSICAL REFINING
ENZYMATIC DEGUMMING
Crude Oil
NaOH treatment
Water degumming
Hydratable
P-lipids
3 x neutralisation
washing,
centrifugation
Acid degumming
Non
Hydratable
P-lipids
FFA +
P-lipids as
Soapstock
Enzym. degumming
Centrifugation
Centrifugation
Bleaching
Bleaching
Bleaching
Deodorization
Steam stripping
Deodorization
FFA
Deodorization
RBD Oil
21
WIRKPRINZIP VON PHOSPHOLIPASEN
Verringerung der Emulsionsstabilität
Free Fatty Acid
Phospholipid
1-Acyl-Lysophospholipid
Water
PLA Hydrolysis
Oil
The cleavage of a fatty acid makes the phospholipid more hydrophilic
and therefore easy to hydrate and to remove with the water phase.
Relevant enzyme classes: PLA and PLC
22
ROHALASE PL-XTRA PROZESS
Kürzere Prozessdauer, niedriger pH
pH : 3,8 – 4,5 / Temperature: 50 – 57 °C / Dosage: min. 25g/MT / Reaction time: >1h (up to 6h)
CRUDE OIL / WATER DEGUMMED OIL
Reaction tank
batch for 1-6h !
Cool to
Heat to 70 °C
50 - 57 °C
Rohalase
PL-XTRA
Centrifuge
mixing
Gums
Citric acid
chelate metals
shift pH
NaOH
high
shear
mixing
adjust pH between
3,8 - 4,5 (<5 !)
DEGUMMED
OIL (RBD)
heat to 70 °C
23
EIN UMWELTFREUNDLICHERER PROZEß
Weniger Energie, weniger Ressourcen
Resource
Caustic soda*
Chemical
degumming
5,3 kg
Enzymatic
degumming
0,43 kg
Phosphoric acid* 2,0 kg
-
Sulphuric acid*
5,3 kg
-
Citric acid*
-
1,0 kg
Water*
127,8 kg
10,76 kg
Cooling water*
1,5 m3
-
Energy*
7,7 kWh
7,0 kWh
Steam*
75 kWh
22 kWh
Enzyme*
-
0,014 kg
*per MT oil
Report German Federal Environmental Agency 2005
24
ROHALASE® PL-XTRA
Technical Performance
Aspergillus fumigatus PLA
110
100
90
80
70
60
50
40
30
20
10
0
Activity [%]
•
•
•
•
•
1,5
2
2,5
3
3,5
4 4,5
pH
5
5,5
6
Treatment
6,5
IP filed WO2011051322A1
Acid phospholipase, 20 kDa
Optimally active at pH4: less fouling
Superior efficiency per unit enzyme
Process time drastically reduced
7
Units
/100g oil
Time (h)
Phosphorous
(ppm)
Untreated Soybean oil, Serbia
157
Citric acid
143
Lecitase Ultra 55°C, pH5
50
4
6
15.5
11.1
Rohalase XTRA 55°C, pH4
25
4
6
13.9
8.8 (<10ppm)
25
ROHALASE® PL-XTRA
Technical Performance
Degumming of Soybean Oil
Units
Degumming of soybean
oil
Time
(min)
Phosp
(ppm)
60
173.8
15
60
129.0
25
60
83.0
50
60
22.4
100
60
120
18.0
6.0
200
60
120
5.8
3.4
100 g oil
Citric Acid
Rohalase
PL Xtra
57°C, pH 4.0, 2% water, target <10ppm phosphorous
A novel phospholipase operating at pH 4 to avoid Me-citrate fouling
An activity profile allowing the reduction of enzymatic batch processing time to 1 hour
26
Vielen Dank für Ihre
Aufmerksamkeit
[email protected]