Expression and characterization of fifteen Rhizopus oryzae 99-880 polygalacturonase enzymes in Pichia pastoris

Authors: Mertens, Jeffrey; Bowman, Michael

Submitted to: Current Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/1/2010
Publication Date: 4/1/2011
Citation: Mertens, J.A., Bowman, M.J. 2011. Expression and characterization of fifteen Rhizopus oryzae 99-880 polygalacturonase enzymes in Pichia pastoris. Current Microbiology. 62(4):1173-1178.

Interpretive Summary: Utilization of agricultural biomass will be required to meet future fuel and specialty chemical needs. Biomass typically needs to be broken down by chemical pre-treatments and enzymes into simple sugars in order to be utilized. The fungus Rhizopus oryzae produces enzymes called polygalacturonases that are very effective in the conversion of pectin, a major component of plant cell walls, into simple sugars. Industrial interest in this enzyme is strong due to the use of this enzyme in the clarification of fruit juices, retting of flax as well as its potential in the conversion of biomass to simple sugars. In this work we have determined the biochemical properties of a large family of previously uncharacterized Rhizopus polygalacturonase enzymes. This work leads to additional knowledge of polygalacturonase enzymes and identifies useful enzyme species that will ultimately lead to a decrease in the cost of using agricultural crops for the production of high value products.

Technical Abstract: Polygalacturonase enzymes hydrolyze the long polygalacturonic acid chains found in the smooth regions of pectin. Interest in this enzyme class continues due to their ability to macerate tissues of economically important crops and their use in a number of industrial processes. Rhizopus oryzae has a large polygalacturonase gene family with 15 of 18 genes encoding unique active enzymes. The polygalacturonase enzymes, 12 endo-polygalacturonases and 3 exo-galacturonases, were expressed in Pichia pastoris and purified enabling biochemical characterization to gain insight into the maintenance of this large gene family within the Rhizopus genome. The 15 polygalacturonase enzymes have a pH optima ranging from 4.0 to 5.0. Temperature optima of the 15 polygalacturonase enzymes vary from 30 deg C to 45 deg C. While the pH and temperature optima do little to separate the enzymes, the specific activity of the enzymes is highly variable ranging from over 200 umols/min/mg to less than 1 umol/min/mg. A general pattern related to the groupings found in the phylogentic tree was visible with the group containing the exo-polygalacturonase enzymes demonstrating the lowest specific activity. Finally, the progress curves of the polygalacturonase enzymes, contained within the phylogenetic group that includes the exo-polygalacturonase enzymes, acting on trigalacturonic acid lends additional support to the idea that the ancestral form of polygacturonase in Rhizopus is endolytic and exolytic function evolved later.