3D imaging of enzymes working in situ.

Today, development of slowly digestible food with positive health impact and production of biofuels is a matter of intense research. The latter is achieved via enzymatic hydrolysis of starch or biomass such as lignocellulose. Free label imaging, using UV autofluorescence, provides a great tool to follow one single enzyme when acting on a non-UV-fluorescent substrate. In this article, we report synchrotron DUV fluorescence in 3-dimensional imaging to visualize in situ the diffusion of enzymes on solid substrate. The degradation pathway of single starch granules by two amylases optimized for biofuel production and industrial starch hydrolysis was followed by tryptophan autofluorescence (excitation at 280 nm, emission filter at 350 nm). The new setup has been specially designed and developed for a 3D representation of the enzyme-substrate interaction during hydrolysis. Thus, this tool is particularly effective for improving knowledge and understanding of enzymatic hydrolysis of solid substrates such as starch and lignocellulosic biomass. It could open up the way to new routes in the field of green chemistry and sustainable development, that is, in biotechnology, biorefining, or biofuels.