1. Isolation, purification and characterization of a novel glucose oxidase from Penicillium sp. CBS 120262 optimally active at neutral pH Clinton Simpson Researcher – Enzyme Technologies Group

2. © CSIR 2006 www.csir.co.za Hypothesis The Glucose Oxidase (GOX) from CBS 120262 is a novel enzyme that will exhibit different properties to GOX enzymes reported in relevant literature.Aims Cultivate CBS 120262 and determine the intra- and extracellular production levels of GOX, Purify the GOX from CBS 120262 for characterization and activity studies, Determine the kinetic parameters of GOX from CBS 120262, Compare the GOX of CBS 120262 with other GOX enzymes reported in the literature.

3. © CSIR 2006 www.csir.co.za Introduction GOX has been purified from a range of different fungal sources, mainly from the genus Aspergillus and Penicillium, The Penicillium GOX have been shown to exhibit more advantageous kinetics of glucose oxidation over that of Aspergillus GOX, Success - relatively low cost, excellent specificity for β-D-glucose and good stability, GOX finds application in: Glucose biosensor for diabetes monitoring, Biofuel cells, Food and beverage additive, Wine production, Oral hygiene, Gluconic acid.

4. © CSIR 2006 www.csir.co.za Composition of GOX Ascomycetes GOX are dimers consisting of 2 identical polypeptide chain subunits, Covalently linked by disulphide bonds, Each subunit - mol tightly bound but not covalently linked flavine adenine dinucleotide (FAD) as co-factor, GOX’s are glycosylated, Crystal structure of A. niger and P. amagasakiense GOX's are known. Overall structure of GOX from P. amagasakiense model based on 1GPE visualised using YASARA (Wohlfahrt et al., 1999).

5. © CSIR 2006 www.csir.co.za The GOX reaction Reductive - Oxidation of β-D-glucose to D-glucono-δ-lactone which is non-enzymatically hydrolysed to gluconic acid, subsequently FAD reduced to FADH 2, Oxidative - reduced GOX is reoxidised by oxygen to yield hydrogen peroxide. The hydrogen peroxide is cleaved by catalase to produce water and oxygen. GO X H2OH2O Reductive Oxidative

6. © CSIR 2006 www.csir.co.za GOX released by various methods of cell disruption from CBS 120262 Rogalski et al., (1988) medium - 60ml cultivated at 72 hours at 28ºC, (g/L) glucose - 80, peptone - 30, CaCO 3 - 35, (NH 4 ) 2 HPO 4 - 0.388; KH 2 PO 4 - 0.188; MgSO 4.7H 2 O - 0.156.

7. © CSIR 2006 www.csir.co.za Volumetric GOX production The maximum volumetric GOX activity was 18.4 U/ml.

8. © CSIR 2006 www.csir.co.za Biomass specific GOX production The maximum biomass specific GOX activity was shown to be 1.08 U/mg biomass, 70% intracellular and 30% extracellular.

9. © CSIR 2006 www.csir.co.za Comparison of GOX production ND – not determined, N/A – Not Applicable (8% glucose, 3,5% CaCO 3 and 3% peptone).

10. © CSIR 2006 www.csir.co.za Ammonium sulphate precipitation Extracellular – 60% recovery of GOX and 14% recovery of total protein (effective 28U/mg to 122U/mg 4.4-fold purification), Intracellular – 80% recovery of GOX and total protein (ineffective no change in specific activity), GOX glycosylated enzyme.

11. © CSIR 2006 www.csir.co.za Isoelectric Focusing pH4 A BpH7 Zymogram of GOX isoenzymes of the intra- (bottom) and extracellular (top) fractions, Isoelectric focusing – 2 isoenzymes at pH 4.3 (A) and pH 4.67(B), Same isoenzymes intra- and extracellularly.

12. © CSIR 2006 www.csir.co.za Purification table of extracellular GOX

13. © CSIR 2006 www.csir.co.za Molecular weight of final purified GOX Subunit molecular weight of 70kDa, overall molecular weight of 148kDa.

14. © CSIR 2006 www.csir.co.za Temperature activity profile Optimum activity 25 o C, More than 70% of maximum activity between 10 o C and 45 o C.

15. © CSIR 2006 www.csir.co.za pH activity profile Optimally active at pH 7, More than 70% of maximum activity between pH 4.9 and pH 8.9.

16. © CSIR 2006 www.csir.co.za Stability profiles of GOX from CBS 120262 at 25ºC and 37ºC. Residual GOX activity remained relatively unchanged at 25 o C for 10 hours, Half-life of 30 minutes at 37 o C, Immobilisation, polyhydric alcohols.

17. © CSIR 2006 www.csir.co.za Storage stability of lyophilised GOX from CBS 120262 at -20°C Stable for a minimum of 6 months at -20ºC as a freeze-dried preparation without any stabilisers.

18. © CSIR 2006 www.csir.co.za Kinetic parameters of GOX from different sources Values are for free GOX and k cat per mole native enzyme CBS 120262 GOX more advantageous kinetic properties than A. niger GOX, due to higher affinity for β-D-glucose, and higher specificity constant despite the lower turnover number.

19. © CSIR 2006 www.csir.co.za Comparison of different GOX's ND-Not determined by authors

20. © CSIR 2006 www.csir.co.za Final Conclusions Successful isolation, purification and partial characterisation of a novel GOX from CBS 120262, The purified GOX was a novel enzyme based on its distinct characteristics, one of only 2 reported GOX's with neutral optimum pH activity, CBS 120262 GOX displayed more advantageous kinetic characteristics than the A. niger GOX’s it was compared to, Neutral pH optimum, optimum temperature (25- 30ºC), long-term storage stability at -20°C as well as stability at 25°C make this GOX suitable for diagnostic applications.

21. © CSIR 2006 www.csir.co.za Future Work Rhodes University – Application of the GOX from CBS 120262 for biofuel cells, Doctorate – Enhancing the enantioselectivity of Candida antarctica Lipase B with SphereZymes TM Technology.

22. Acknowledgements The research was partially funded by the Innovation Fund, Prof. Chris Whiteley (Supervisor) Rhodes University, Dr. Dusty Gardiner (Co-supervisor) CSIR Biosciences, Dr Justin Jordaan (Academic insight and experimental guidance), The work has been published in Protein Expression and Purification (YPREP 2922).