December 13, 2016
Finnish Cultural Foundation grant N.: 00140620
Grantee:Anders Filip Mollerup
Title of researchproject:Hiilihydraattioksidaasien muokkausta uusien substraattimieltymysten kehittämiseksi ja suorituskyvyn parantamiseksi käsittelevään
Grant period: September 2014 to September 2015
Short account of the research phases:
According to the submitted work plan the objectives for the supported PhD. project were:
- Improve the performance of GaOx on polysaccharides by constructing fusion proteins that contain selected carbohydrate-binding modules
- Improve production yields and activity of a mutant GaOx oxidizing D-Glucose on polysaccharides
- Construction a mutagenic DNA plasmid library of GOOX through an error-prone PCR based approach
- Site-directed mutagenesis of GOOX to overcome substrate inhibition.
- Assist project partners with production of novel carbohydrate oxidases, identified through genomic mining of existing sequence databases and, by fermentation of recombinant Pichia pastoris strains.
The main part of the research work of objective 1 was completed before the start of the grant period. However, to pass the peer-review for the targeted journals additional experiments on this objective were performed in the grant period. Specifically, the fusion proteins described in the work plan were analyzed for their binding behavior by quartz crystal microbalance with dissipation (QCM-D), isothermal titration calorimetry (ITC) and analytical ultracentrifugation. Results from QCM-D and analytical ultracentrifugation were added to the manuscript to the satisfaction of the reviewers and the manuscripts were accepted during the grant period. Additionally, the manuscript submitted was split into two documents and resulted in a main research article and a supporting data article[1], [2].
The fusion of CBM3to mutGaOx, in Objective 2,increased the stability and activity of GaOx and facilitates immobilization of the enzyme on cellulosic materials. Characterization of the M3-CBM3 (also known as mutGaOx-CBM3) motivated an attempt to further increase the oxidation efficiency on D-glucose through introduction of 3 additional mutations, generating M6-CBM3 from M3-CBM3. The mutations where selected on basis of their ability to increase GaOxwild-type activity on galactose, independently and in conjunction [3]. Whereas M3-CBM3 oxidizes D-glucose (34,6 units*mole-1)1 and D-galactose (228 units*mole-1) at low efficiency, the activity of M6-CBM3 on D-galactose (23.272 units*mole-1) nearly re-gained the activity of GaOx wild-type (26.078 units*mole-1), which was the origin for generating M3-CBM3. However, M6-CBM3 does not oxidize D-glucose suggesting that the mutations gaining enhanced catalysis of D-galactose outweighs the effect of mutations gaining D-glucose activity. These results will be published in the spring of 2017.
Objective 3 and 4 were aborted after recent research insights from the co-research group at University of Toronto [4]. After these research achievements, this research topic is currentlyconcentrated at University of Toronto and my own research topics focused on the CAZy family AA5_2 (galactose oxidase family), as described in objective 1,2,5 and 6.
Objective 5 was successfully completed according to the description in the research plan.Deeper involvement in the characterization of the raffinose oxidase was also agreed with co-authors and consequently I share 1. authorship in the coming publication. A natural galactose oxidase variant, raffinose oxidase, was identified and characterized, which further increased the catalytic diversity ofCAZy family AA5_2. The strict substrate selectivity of raffinose oxidase towards the galactose-containing tri-saccharide raffinose could indicate that theeukaryotic members this family, which is comprised mainly of plant pathogenic fungi,could be involved in inhibition of the raffinosesynthesis during cellular stress response in plants.
Replacing the aborted research projects,Objective 6aimed at selection and characterization of an unknown member ofCAZy family AA5_2.The intelligence collected in Objective 2 and 5 and abioinformatics study of CAZy family AA5_2 advised theprediction of gene sequencesthat would compose of“non-classical galactose oxidase properties” (i.e. having highest catalytic efficiency towards D-galactose). Accordingly, the Pc21g18600 gene(PwiAA5) from Penicilliumrubens(strain ATCC 28089 / DSM 1075 / NRRL 1951 / Wisconsin 54-1255)was successfully cloned and expressed in Pichia pastoris and characterized. Similar to raffinose oxidase from objective 5, PwiAA5 shows higher catalytic efficiency towardsraffinoserelative to galactose. This further substantiate the suggested biological role for this family. Additionally, PwiAA5 shows an inherent, substantial lag-phase during initiation of catalysis. This phenomenon has been observed in GaOx mutants, including M6-CBM3 and partially M3-CBM3, but until now been undiscovered for wild-type members of AA5_2.
1One unit is defined as 1 mole of substrate produced per minute (mole*min-1)
References:
[1]F. Mollerup, K. Parikka, T. V. Vuong, M. Tenkanen, and E. Master, “Influence of a family 29 carbohydrate binding module on the activity of galactose oxidase from Fusarium graminearum,” Biochim. Biophys. Acta - Gen. Subj., vol. 1860, no. 2, pp. 354–362, 2016.
[2]F. Mollerup and E. Master, “Influence of a family 29 carbohydrate binding module on the recombinant production of galactose oxidase in Pichia pastoris,” Data Br., vol. 6, pp. 176–183, 2016.
[3]S. Delgrave et al., “Application of a very high-throughput digital imaging screen to evolve the enzyme galactose oxidase.,” Protein Eng., vol. 14, pp. 261–267, 2001.
[4]T. V Vuong, M. Foumani, B. MacCormick, R. Kwan, and E. R. Master, “Direct comparison of gluco-oligosaccharide oxidase variants and glucose oxidase: substrate range and H2O2 stability.,” Sci. Rep., vol. 6, p. 37356, Nov. 2016.