Basidiomycetous enzymes versatile tools in industrial biotechnology Holger Zorn Justus Liebig University Giessen Institute of Food Chemistry and Food Biotechnology
Recycling of renewable resources - Secretome of Pleurotus sapidus Enzymatic degradation of carotenoids Nootkatone a biotechnological challenge Conclusions
Growth of P. sapidus on rape straw
Secretome of P. sapidus Bouws et al. Appl Microbiol Biotechnol 80:381-388
Secretome of P. sapidus Overlay of the secretomes of the 6 th (orange) and 12 th (blue) culture day
Secretome of P. sapidus
Secretome of P. sapidus
Recycling of renewable resources - Secretome of Pleurotus sapidus Enzymatic degradation of carotenoids Nootkatone a biotechnological challenge Conclusions
Co-Oxidation Plant enzymes Glycosidases Fungi? Enzymatic degradation of carotenoids
Culture supernatant + -carotene incubation extraction analysis (GC-MS / LC-MS)
Cleavage of -carotene to flavour compounds
MsP2 IEF electrophoresis MsP1 + MsP2 from M. scorodonius Zorn et al. Eur Pat Appl EP 64132 20060712
MsP1, M ~ 70 kda, pi ~ 3.7: Edman-sequence ASF?AGL?LTDIQGDIL?TMKKNKELFF ESI-MS/MS ( L = L or I ) GGEYFFSPSLSALR / QLPYGPEVTDAEK / NSGTDAPNTPR MsP2, M ~ 50 kda, pi ~ 3.5: Edman-sequence: AP?L?LTDIQGDILIGMKKNKE?FF ESI-MS/MS ( L = L or I ) DGSFLAFR / PQNTNNHLLR / LVLNTDFVSSR / FLADNAALTQGNADLL / SGAPVDLAPTADDVDLANDPQR
MsP2.1 Ile 428 Leu, Asn 431 Ala, Ile 448 Val, Asn 455 Val, Ser 465 Gly, Thr 479 Lys, Leu 483 Ile, Val 489 Ser MsP2 510 AA Homology >98% MsP2.4 Ile 428 Leu, Asn 431 Ala, Ile 448 Val, Asn 455 Val, Ser 465 Gly, Thr 479 Lys, Leu 483 Ile, Val 489 Ser, Thr 106 Ala MsP1.2 59 AA differing, 5 AA less Homology 87% MsP1.1 Asn 35 Ser, Asp 139 Gly, Leu 192 Pro, Ala 212 Glu, Lys 402 Arg, Val 450 Ala MsP1 513 AA Homology >98% MsP1.3 Ala 125 Thr, Leu 199 Ser, Ala 212 Glu, Ala 288 Val, Asp 353 Val Iso-enzymes of MsP1 and MsP2
M (calculated) ~ 49 kda ~ 70 kda Characteristics of MsP1 Scheibner et al. Appl. Microbiol. Biotechnol. 77:1241-1250
random coil structure α-helices loops and turns intra-molecular β-sheets inter-molecular β-sheets Activity (U/mL) Temperature and ph dependence of MsP1 activity Puehse et al. BBA Proteins Proteom 1794:1091-1098
random coil structure α-helices loops and turns intra-molecular β-sheets inter-molecular β-sheets Relative activity [%] Pressure stability of MsP1
O O O carotene -ionone -ionone-5,6-epoxide O O O O OH dihydroactinidiolide -cyclocitral 2-hydroxy-2,6,6- trimethyl cyclohexanone zeaxanthin O O HO HO 3-hydroxy- -ionone 3-hydroxy- -cyclocitral lutein O O HO HO 3-hydroxy- -ionone 3-hydroxy- -ionone neoxanthin / violaxanthin HO O O 3-hydroxy- -ionone-5,6-epoxide lycopene O O 6-methyl-5-hepten-2-one geranial Release of volatiles from carotenes and xanthophylls by MsP1 catalysis
Bleaching of food by 3-enzyme-system
b-value 60 58 56 54 52 50 48 46 44 42 40 38 36 34 32 30 28 26 24 22 20 18 16 14 12 10 8 6 4 2 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 t [h] Bleaching of whey by 3-enzyme-system
1 2 3 4 5 Bleaching of whey + -carotene by 3-enzyme-system (ph 4.6; 90 min, RT) 1 4: 5 whey + -carotene; 5: control (whey); 2: + MsP1 + Lactase + GOX; 3: + MsP1 heat inactiv. + Lactase + GOX; 4: + H 2 O + Lactase + GOX Bleaching of whey by 3-enzyme-system
Abs 280 nm [mau] blank Versatile Peroxidase MsP1 t [min] modified after Liers et al. 2010 Conversion of adlerol
B 10 9 reducing sugars as xylose[%] 8 7 6 5 4 3 2 1 0 6 12 24 incubation time [h] + 2 U MsP1 - MsP1 Saccharification of MsP1-pretreated corn stover with Cellulase Zorn et al. Eur. Pat. Appl. 26485/EP/P0
Recycling of renewable resources - Secretome of Pleurotus sapidus Enzymatic degradation of carotenoids Nootkatone a biotechnological challenge Conclusions O (Z) (R) (S) (R)
odour threshold: 0.001 mg L -1 water natural occurrence in trace amounts price of natural compound: ~ 5,000 $ kg -1 Nootkatone
Dosage L -1 Costs L -1 Medium [ ] Molassis 100 ml 0.10 NH 4 NO 3 2.4 g 0.01 Yeast extract 3.0 g 0.10 KH 2 PO 4 1.5 g 0.06 MgSO 4 0.5 g 0.06 Trace element solution 1 ml 0.33 Medium optimization
Culture up-scaling
Ultra Turrax (630x) High pressure homogenization (630x) Bead mill (630x) Lyophilisate (630x) Mycelium disruption
Pleurotus sapidus lyophilisate 20 mm Tris-HCl buffer ph 7.5 T = 24 C 150 rpm O Biotransformation of (+)-valencene to (+)-nootkatone
1,000 16 14 750 12 [mg L -1 ] 500 250 10 08 06 04 valencene [µl] -nootkatol nootkatol (+)-nootkatone (+)-valencene (dosage) 02 0 (1.4 µl, 0 h) + 2 µl after 4, 8, 12, 16, and 20 h (5.0 µl, 0 h) + 2 µl after 4, 8, 12, 16, and 20 h Production of nootkatone in fed batch culture Fraatz et al. EP 08.1711483
661 AA M = 74.2 kda P. sapidus oxygenase Fraatz et al. J Mol Catal B Enzym 61:202-207
Recycling of renewable resources - Secretome of Pleurotus sapidus Enzymatic degradation of carotenoids Nootkatone a biotechnological challenge Conclusions
Oxygenases Oxidases PsP Endo- Glucanases Cutinases / Lipases
Basidiomycetes unique catalytic properties food grade, non-toxic (edible fungi) unlimited availability high enzyme stability simple enzyme recovery (often extra-cellular) potential applications in biorefinery concepts Conclusions
LCB 2011 Many thanks!