An Al(porphyrin) functionalized with a large axial ligand was incorporated into a porous organic polymer (POP) using a cobalt-catalyzed acetylene trimerization strategy. Removal of the axial ligand afforded a microporous POP that is catalytically active in the methanolysis of a nerve agent simulant. Supercritical CO2 processing of the POP dramatically increased the pore size and volume, allowing for significantly higher catalytic activities.

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Microbes engineered to display heterologous proteins could be useful biotechnological tools for protein engineering, lignocellulose degradation, biocatalysis, bioremediation and biosensing. Bacillus subtilis is a promising host to display proteins, as this model Gram-positive bacterium is genetically tractable and already used industrially to produce enzymes. To gain insight into the factors that affect displayed protein stability and copy-number, we systematically compared the ability of different protease-deficient B. subtilis strains (WB800, BRB07, BRB08 and BRB14) to display a Cel8A-LysM reporter protein in which the Clostridium thermocellum Cel8A endoglucanase is fused to LysM cell wall binding modules. Whole-cell cellulase measurements and fractionation experiments demonstrate that genetically eliminating extracytoplasmic bacterial proteases improves Cel8A-LysM displays levels. However, upon entering stationary phase, for all protease-deficient strains, the amount of displayed reporter dramatically decreases, presumably as a result of cellular autolysis. This problem can be partially overcome by adding chemical protease inhibitors, which significantly increase protein display levels. We conclude that strain BRB08 is well-suited for stably displaying our reporter protein, as genetic removal of its extracellular and cell wall-associated proteases leads to the highest levels of surface accumulated Cel8A-LysM without causing secretion stress or impairing growth. A two-step procedure is presented that enables the construction of enzyme coated vegetative B. subtilis cells that retain stable cell-associated enzyme activity for nearly 3 days. The results of this work could aid the development of whole cell display systems that have useful biotechnological applications.