Industrielle Herstellung von Enzymen für Lebensmittel
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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]