Synthesis of Fibrils in Vitro by a Solubilized Cellulose Synthase from Acetobacter xylinum

Abstract

A digitonin-solubilized cellulose synthase was prepared from Acetobacter xylinum. When this enzyme was incubated under conditions known to lead to active synthesis of 1,4-β-D-glucan polymer (cellulose), electron microscopy revealed that clusters of fibrils were assembled within minutes. Individual fibrils are 17 ± 2 angstroms in diameter. Evidence that the fibrils were freshly synthesized and cellulosic in nature was their incorporation of the tritium from UDP-[³H]glucose (UDP, uridine 5'-diphosphate), their binding of gold-labeled cellobiohydrolase, and an electron diffraction pattern with 004, 200, and 012 reflections (characteristic of cellulose synthesized in vivo) but missing 110 and 110 reflections. The small size of the fibrils is atypical of native A. xylinum cellulose microfibrils. The fibrils synthesized in vitro resemble, in morphology and size, the fibrillar cellulose produced when A. xylinum is cultured in the presence of agents that interfere with the normal process of crystallization of the microfibrils. The solubilized enzyme unit may therefore be producing a basic fibrillar structure that, in vivo, interacts laterally with other fibrils to produce native cellulose microfibrils.