Purification of coniferyl alcohol oxidase from lignifying xylem of sitka spruce using immobilised metal affinity chromatography

摘要

Extracts of lignifying xylem of sitka spruce (Picea sitchensis), obtained by a combined extraction/affinity method that selects for cell-wall-associated glycoproteins, were enriched in oxidase and peroxidase activity but peroxidase activity was approximately 80 times more abundant than oxidase activity. Previous attempts to purify the oxidase using ion-exchange, hydrophobic interaction and gel filtration chromatographic methods have been unsuccessful and it has proved particularly difficult to separate completely oxidase activity from peroxidase activity. All of the oxidase activity in the xylem cell wall extracts bound to an affinity matrix loaded with immobilised copper (Cu2+) ions and was fractionated into two main peaks by eluting the bound proteins with a gradient of histidine. The second oxidase peak, which represents a subset of oxidase that has higher affinity for the bound metal ions, contained a third of the total oxidase activity at a 2-fold increase in specific activity. In addition,this fraction had greatly reduced although still detectable peroxidase activity. Oxidase activity was split into a bound and an unbound fraction when extracts were applied to a matrix of immobilised cobalt (Co2+) ions. In this case, a larger proportion of the applied oxidase activity than peroxidase activity bound to the Co2+-loaded matrix and the specific activity was higher than that in the unbound fraction. This suggested that this bound fraction of the oxidase population had a higher affinity for metal ions and could therefore be isolated from peroxidase. When applied to a Zn2+-loaded matrix, the oxidase activity also split into bound and unbound portions but effectively all of the peroxidase activity was eluted in the unbound fraction yielding a bound fraction enriched in oxidase activity with negligible peroxidase contamination. The nature of this "high affinity fraction" of oxidase and the heterogeneity in the oxidase population is discussed. Gel permeation on Superose-6 partially resolved oxidase from the residual peroxidase activity in the Zn-bound fraction and gave an apparent Mr of 62 kDa for the oxidase activity. However, an examination of the protein profile of fractions enriched in oxidase activity by SDS-PAGE strongly suggests that a protein band of apparent Mr 80 kDa is responsible for the oxidase activity. This apparent disparity is discussed. It is concluded that immobilised metal affinity chromatography on Zn-loaded matrix provides a rapid method to separate oxidase from peroxidase activities enabling studies of the substrate specificity and the physical properties of the oxidase to be carried out.