Basidiomycetous enzymes. versatile tools in industrial biotechnology

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1 Basidiomycetous enzymes versatile tools in industrial biotechnology Holger Zorn Justus Liebig University Giessen Institute of Food Chemistry and Food Biotechnology

2 Recycling of renewable resources - Secretome of Pleurotus sapidus Enzymatic degradation of carotenoids Nootkatone a biotechnological challenge Conclusions

3 Growth of P. sapidus on rape straw

4 Secretome of P. sapidus Bouws et al. Appl Microbiol Biotechnol 80:

5 Secretome of P. sapidus Overlay of the secretomes of the 6 th (orange) and 12 th (blue) culture day

6 Secretome of P. sapidus

7 Secretome of P. sapidus

8 Recycling of renewable resources - Secretome of Pleurotus sapidus Enzymatic degradation of carotenoids Nootkatone a biotechnological challenge Conclusions

9 Co-Oxidation Plant enzymes Glycosidases Fungi? Enzymatic degradation of carotenoids

10 Culture supernatant + -carotene incubation extraction analysis (GC-MS / LC-MS)

11 Cleavage of -carotene to flavour compounds

12 MsP2 IEF electrophoresis MsP1 + MsP2 from M. scorodonius Zorn et al. Eur Pat Appl EP

13 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

14 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 MsP 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

15 M (calculated) ~ 49 kda ~ 70 kda Characteristics of MsP1 Scheibner et al. Appl. Microbiol. Biotechnol. 77:

16 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:

17 random coil structure α-helices loops and turns intra-molecular β-sheets inter-molecular β-sheets Relative activity [%] Pressure stability of MsP1

18 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

19 Bleaching of food by 3-enzyme-system

20 b-value t [h] Bleaching of whey by 3-enzyme-system

21 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

22 Abs 280 nm [mau] blank Versatile Peroxidase MsP1 t [min] modified after Liers et al Conversion of adlerol

23 B 10 9 reducing sugars as xylose[%] incubation time [h] + 2 U MsP1 - MsP1 Saccharification of MsP1-pretreated corn stover with Cellulase Zorn et al. Eur. Pat. Appl /EP/P0

24 Recycling of renewable resources - Secretome of Pleurotus sapidus Enzymatic degradation of carotenoids Nootkatone a biotechnological challenge Conclusions O (Z) (R) (S) (R)

25 odour threshold: mg L -1 water natural occurrence in trace amounts price of natural compound: ~ 5,000 $ kg -1 Nootkatone

26 Dosage L -1 Costs L -1 Medium [ ] Molassis 100 ml 0.10 NH 4 NO g 0.01 Yeast extract 3.0 g 0.10 KH 2 PO g 0.06 MgSO g 0.06 Trace element solution 1 ml 0.33 Medium optimization

27 Culture up-scaling

28 Ultra Turrax (630x) High pressure homogenization (630x) Bead mill (630x) Lyophilisate (630x) Mycelium disruption

29 Pleurotus sapidus lyophilisate 20 mm Tris-HCl buffer ph 7.5 T = 24 C 150 rpm O Biotransformation of (+)-valencene to (+)-nootkatone

30 1, [mg L -1 ] 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

31 661 AA M = 74.2 kda P. sapidus oxygenase Fraatz et al. J Mol Catal B Enzym 61:

32 Recycling of renewable resources - Secretome of Pleurotus sapidus Enzymatic degradation of carotenoids Nootkatone a biotechnological challenge Conclusions

33 Oxygenases Oxidases PsP Endo- Glucanases Cutinases / Lipases

34 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

35 LCB 2011 Many thanks!