Selective Recovery From the Interstitial Spaces of Leaves

In planta production of apoplast-targeted cellulose degrading enzymes may be a valuable approach for efficient production of concentrated enzyme preparations that can be used for saccharification of cellulose in pretreated feedstocks. A vacuum infiltration-centrifugation method has been developed for recovery of transiently-produced cellulase enzymes in the apoplast wash fluid (AWF) of tobacco leaves.  A single AWF recovery at the time of peak production following transient agroinfiltration of harvested tobacco leaves removes up to 67% of the Acidothermus cellulolyticus thermostable endo-1,4-β-glucanasecatalytic domain (E1cd) activity.  However, recovering AWF daily from agroinfiltrated leaves more than tripled the amount of the enzyme activity recovered in the AWF and doubled the amount of active E1cd produced overall.  Although the mechanism for the yield increase is not yet understood, rinsing the apoplast of agroinfiltrated leaves during the production/incubation period has potential advantages of improved protein stability, secretion kinetics, and leaf health.  The AWF collected also had higher purity and enzyme concentration, up to 124- and 19-fold improvements respectively compared to recovery from homogenate extracts, facilitating its use in the saccharification process.