What next potential improvements in phytases? Author: Jari Vehmaanperä Company: Roal - AB Enzymes IPS2: Dec 13 th, 2012 / Rome, Italy - PDF

What next ptential imprvements in phytases? Authr: Jari Vehmaanperä Cmpany: Ral - AB Enzymes IPS2: Dec 13 th, 2012 / Rme, Italy Intrductin 2 QUICK FACTS Assciated British Fds Incrprated 1935 Emplyees 106,000

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What next ptential imprvements in phytases? Authr: Jari Vehmaanperä Cmpany: Ral - AB Enzymes IPS2: Dec 13 th, 2012 / Rme, Italy Intrductin 2 QUICK FACTS Assciated British Fds Incrprated 1935 Emplyees 106,000 in 47 cuntries Revenues 12.3 billin ( 15.2 billin) in 2012 Activities Selected Leading Psitins Internatinal fd, ingredients and retail Wrld s secnd largest sugar prducer Leading glbal speciality tea brand Wrld s secnd largest yeast business Wrld leader in speciality enzymes 3 QUICK FACTS AB Enzymes Established 1907 Ownership 100% wned by Assciated British Fds (ABF) Emplyees 200 Revenues 100 millin Grwth Rate 10% p.a. since 1999 Fcus R&D Investment Patents Feed, Fd & Specialities, Technical, Bimass Ethanl 10% f revenues 450 (wned and licensed) 4 AB ENZYMES - HISTORY 1907 Röhm and Haas funded. Inventin f OrpnTM fr the leather industry First enzyme fr the milling and baking industry launched: VERON 2005 AB Enzymes restructures t achieve a glbal leadership psitin 2007 Glbal distributin arrangement fr feed with ABF sister cmpany AB Vista 2009 Capacity expansin at manufacturing site in Finland 1934 Inventin f the first enzyme fr the fd industry: ROHAPECT 1999 ABF acquires Röhm Enzyme which is renamed t AB Enzymes 2006 Glbal marketing and sales netwrk established 2008 Cmpletin f the VERON, ECOSTONE, ROHAPECT and ROHALASE prduct ranges 2010 Further expansin f the BIOTOUCH prduct range, and launch f Vern xtender 5 AB ENZYMES - COMMERCIAL Industry Segments Baking Beverages Specialities Textiles Detergents Feed Bread Fruit Juice Prtein mdificatin Bi-finishing Laundry Pultry Biscuits / Crackers Wine Oil prcessing Pre-treatment Swine Other Brewing Starch prcessing Denim Ruminants Distilling Pulp & Paper Other 6 Phytases CLASSIFICATION OF PHYTASES Class I: Histidine acid phsphatases (HAPhy) 3-phytases (E.C ) Aspergillus 6-phytases (E.C ) (4-phytase based n 1D-numbering) Escherichia cli Class II: Beta-prpeller (BPPhy) 3-phytases (E.C ) Bacillus subtilis Class III: Cysteine phytases (Cphy) Class IV: Purple acid phytases (PAPhy) 8 CLASSIFICATION OF PHYTASES Class I Histidine acid phytases Class II Alkaline phytases A.niger A.awamrii A.terreus T.thermphilus A.fumigatus B.Subtilis VTT E B.subtilis B.amylliquefaciens DS11 B.amylliquefaciens FZB45 B.subtilis TS B.subtilis 168 B.lichenifrmis E.nidulans M.thermphilia A.niger S.cerevisiae S.pmbe Human prstate Rat prstate Human lyssme Muse lyssme E.cli Grup I, 3-phytase ph 2.5, 5.0 Grup II, 3-phytase ph 2.5 Grup III, 6-phytase ph Grup IV, 3-phytase ph Adapted frm: Oh B.-C. et al.(2004) Appl. Micrbil. Bitechnl 63: COMMERCIAL PHYTASES Dnr rganism Prductin rganism Phytase class Cmpany Reference Aspergillus ficuum Aspergillus niger 3 phytase BASF (DSM) EFSA Jurnal (2010) Aspergillus niger Trichderma reesei 3 phytase Ral / AB Vista Zyla et al Penicillium funiculsum Penicillium funiculsum 3 phytase Adisse EFSA Jurnal (2007) Peniphra lycii Aspergillus ryzae 6 phytase Nvzymes / DSM EFSA Jurnal (2010) Escherichia cli Schizsaccharmyces pmbe 6 phytase DuPnt (DAN) EFSA Jurnal (2009) Pichia pastris 6 phytase AB Enzymes /AB Vista EFSA Jurnal (2010) Trichderma reesei 6 phytase Ral / AB Vista EFSA Jurnal (2011) Buttiauxella sp. Trichderma reesei 6 phytase DuPnt (DAN) 2012 PSA Annual meeting, US GA Citrbacter braakii Aspergillus ryzae 6 phytase Nvzymes / DSM Guggenbuhl et al All phytases, including the bacterial phytases, are prduced by eukarytic hsts. Thus these mlecules are glycsylated, which they are nt in their native hsts. 10 IDENTITY PROFILE OF BACTERIAL 6-PHYTASES Amin acid level, signal peptides remved BIOCHEMICAL PROPERTIES E.cli QPT2 Aspergillus niger Buttiauxella Citrbacter braakii Yersinia mllaretii Active enzyme prtein equivalent t 500 phytase units, [mg] ~0.22 ~ 0.45 ~ 0.48 ph ptimum / Melting pint, Tm 60 C 89 C 63 C 63 C K m [mm] (ph 5.0) * 0.1% phytate = 1.52 mm *ph 4.5 Lehmann et al Kim et al Shivange et al. 2012 ALIGNMENT OF 6-PHYTASES FROM ENTEROBACTERIA active centre Imprvement f heat stability WO2008/ Steer et al. E. cli WO2007/ De Maria et al. Citrbacter braakii WO2009/ Kensch et al. Buttiauxella sp ACTIVE CENTRE D/L-Ins(1,2,3,4,5,6)P 6 His 17 . A phylgenetic tree f the Enterbacteriaceae Bacteria; Prtebacteria; Gammaprtebacteria; Enterbacteriales; Enterbacteriaceae; Yersinia Klebsiella Escherichia Citrbacter Paradis et al Buttiauxella Hafnia 15 DIFFERENCIAL SCANNING CALORIMETRY (DCS) E.cli phytase Quantum XT Quantum Blue Reference 60.1 C 74.9 C 88.7 C 82.7 C Differential Scanning Calrimetry Sample cell Cp Reference cell Cp(cal/ C) measuring the unflding f the phytase prtein Temperature ( C) Next generatin phytases? 17 TYPICAL FLOW OF A MOLECULE DISCOVERY PROJECT Biprspecting Islatin Screening Enzyme Gene Clning Expressin Mutagenesis Evlutin Strain cnstructin Fermentatin DSP Prduct NH2 COOH NH2 COOH Cp(cal/ C) Temperature ( C) Nature Micrrganisms Wild-type enzymes Analytical amunts fr primary characterisatin Optimised enzymes Prductin strain Bulk prductin Frmulatin 18 ENABLING TECHNOLOGIES New mlecule discvery r directed evlutin? Screen the bidiversity Current cmmercial phytases are frm saprhytic fungi, plant pathgens r frm the gut flra Screen based n phytase activity Pssibility t find nvel backbnes Expressin f phytases may require special culture cnditins Genme mining f published genmes Use degenerate primers fr clning N expressin needed Limited t knwn mlecules Evlve ne f the existing mlecule backbnes further Expect incremental imprvements One characteristic at the time Pssible t evlve features nt relevant in nature Appraches: Directed evlutin» Randm mutagenesis» Shuffling f dmains Ratinal site-directed mutagenesis Cnsensus apprach Cp(cal/ C) NH2 COOH NH2 COOH Temperature ( C) What yu screen is what yu get Need sanity checks in the applicatin at regular intervals 19 ENZYME PRODUCER VIEW Bundary cnditins fr the enzyme supplier High specific activity Influences the cst f enzyme prtein Influences the capacity usage at the manfacturing Next generatin: expect same level r higher than current mlecules Gd expressin in a micrbial prductin hst Current hsts prefer enzymes frm micrbial surces Enzymes frm metagenmic libraries, archaebacteria r higher eukarytes d nt usually express well Preferably a secreted phytase Cst-effective recvery during prductin Secreted enzymes inherently mre stable Intracellular enzymes prbably have a ph ptimum at neutral range 20 Trichderma reesei is Our Key Prductin Platfrm T. reesei QM6a was islated in Slmn island 1944 naturally efficient cellulase prducer high cellulase prducing mutants secrete up t g prtein / l techniques and tls fr genetic engineering develped in the 1980 s the strng cbh1 prmter used fr prductin nn-pathgenic t man grws nly at r near the equatr used fr ver 40 years in large scale manufacturing f enzymes the whle genme has been recently published the key patents f Trichderma technlgy are explitable by Ral 21 PRODUCTION PROCESS Nutrient additin PRODUCTION PROCESS Slurry line Washing line Acids / alkali Sterilisatin: Exterminatin f ther micrbes Seed fermenter Carbhydrate surce: - starch, sugars Nitrgen surce: - Yeast extract, spent grain, CSP Salts: -MgSO 4, KH 2 PO 4, (NH 4 ) 2 SO 4 Sampling valve Jacket cling water circulatin Parameters: -ph - temperature - aeratin - feed flw Aeratin line Prcessing Prduct recvery Filtering Ultrafiltering Stabilizing Drying POSSIBLE CHARACTERISTICS TO DEVELOP Thermstability T survive the highest temperatures in pelleting High specific activity Reducing csts at the manufacturing High turnver t release phsphrus during the time windw in the stmach ph prfile Phytate degradatin at ph Lw Km High affinity t IP6 at stmach ph High anti-nutrient activity Fast phsphrus release frm IP6 t lw IPx Pepsin resistance Often linked t better thermstability Other unmet needs? LPS-dephsphrylatin? 23 QUANTUM BLUE THERMO-TOLERANCE Quantum Blue Therm-tlerance 120 With Quantum Blue we are almst there. Further aim: Imprved recvery at 95 C? % Recvery against mash diet meal Cnditining Temperature ( C) Surce: AB Vista 24 HIGH SPECIFIC ACTIVITY Flexibility f the active site lp essential fr the high specific activity? Clsed Open Shivange et al ph PROFILE Activity related t ph 5.5, % Stability and pepsin resistance at ph are relevant, nt the ph-ptimum per se (?) Quantum Blue E. cli WT Fungal 6 phytase Fungal 3 phytase Redrawn frm data f AB Vista ph 600 Spes act, U/mg prt Quantum Blue E. cli WT Fungal 6 phytase Fungal 3 phytase ph Specific activity ph curve 26 AFFINITY TOWARDS PHYTATE (Km) Availability f substrate in stmach limits phytase actin Hwever, ur knwledge f affinities f different phytases is limited, e.g., at the stmach ph, between different IP-frms, under viscus cnditins, with ther salts Sluble and insluble phytate in gizzard f brilers fed with r withut 500FTU Phytase. Phytate(IP6) cncentratin % N phytase Phytase Sluble Phytate Unsluble Phytate Leslie, % phytate = 1.52 mm 27 SUMMARY OF POSSIBLE DIRECTIONS Thermstability Fr nvel mesphilic mlecules this is a pririty (cating is always a cst) Easy t screen, prven track recrd Patent landscape?, real need with mlecules like Quantum Blue? High specific activity Recent understanding f phytase active site dynamics culd give insight fr targeted mutagenesis Lking utside the family Enterbacteriaceae? ph ptimum twards Can this be cmpensated by high specific activity and stability? Affinity Culd be a limiting factr, but mre data wuld prbably help Pepsin resistance Likely t crrelate with better thermstability 28 ACKNOWLEDGEMENTS Dr. Dietrich Löbel, AB Enzymes Dr. Imke Kühn, AB Vista / AB Enzymes + the Darmstadt R&D team Dr. Mike Bedfrd, AB Vista 29 THANK YOU FOR YOUR ATTENTION! 30
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