Cellulose synthase is an interesting enzyme because its product is an assembly of many polymer molecules – microfibril. Then this enzyme has molecular assembling mechanism in addition to glycosyltransferase activity to polymerize glucose residues. Our group has attempted to produce cellulose microfibril with crystallographic cellulose I polymorph in order to clarify the molecular assembling mechanism of cellulose synthase. However, cellulose II aggregation is the product of our in vitro cellulose synthesis with bacterial enzyme, while several reports have recently demonstrated cellulose microfibril formation in vitro with plant cellulose synthase. In this study, we aimed to understand cellulose II aggregation process by in situ measurement of in vitro cellulose synthesis using time-resolved small angle X-ray scattering (SAXS). Crude enzyme of cellulose synthase was extracted from Komagataeibacter xylinus ATCC53524 (formerly Acetobacter xylinus) by solubilizing membrane fraction with detergent, decyl-β-d-maltoside or dodecyl-β-d-maltoside. In vitro synthesis of cellulose with the obtained enzyme preparation was conducted in a liquid cell with quartz plate window (light path = 3 mm) for in situ SAXS measurement. The time-resolved SAXS measurement was made with a wavelength of 0.1 nm every 4 – 5 min at 28°C, at BL40B2 in SPring-8, Japan. Two-dimensional scattering pattern was azimuthally integrated to have 1-D scattering profile with absorbance correction. An increase of intensity was observed specifically for the reaction with c-di-GMP, the activator of bacterial cellulose synthase. Given that the purified cellulose product showed a significant scattering in the corresponding q-range, the gradual increase of scattering is due to accumulation of cellulose molecules during the in vitro reaction. However, a scattering in a lower q-region was slightly increased even in the absence of c-di-GMP. This increase was partly suppressed in the reaction with 6% glycerol, which has been reported to inhibit the protein aggregation by X-ray radiation damage. Then the increased scattering in this lower q could be partly derived from protein aggregates in addition to cellulose accumulation. Compiling the data, the increase of scattering intensity during the reaction of our in vitro system appears to be monotonic in a q-range measured in this study. It is interesting to analyze the temporal development of SAXS feature of other in vitro systems for comparison.

In situ measurement of cellulose biosynthesis using small angle X-ray scattering

255th National Meeting (2018)

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