Extraction of cellulose-synthesizing activity of Gluconacetobacter xylinus by alkylmaltoside

Abstract

This study reinvestigated the synthesis of cellulose in vitro with a well-known cellulose-producing bacterium, Gluconacetobacter xylinus. Alkylmaltoside detergents, which are more frequently used in recent structural biological researches, are uniquely used in this study to solubilize cellulose-synthesizing activity from the cell membrane of G. xylinus. Activity comparable to that previously reported is obtained, while the synthesized cellulose is crystallized into a non-native polymorph of cellulose (cellulose II) as well as the previous studies. In spite of this failure to recover the native activity to synthesize cellulose I microfibril in vitro, the product is a polymer with a degree of polymerization greater than 45 as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). It was thus concluded that the established protocol can solubilize cellulose-synthesizing activity of G. xylinus with polymerizing activity.

Effects of reaction conditions on cellulose structures synthesized in vitro by bacterial cellulose synthases

“…The cellulosic aggregates were surrounded by a second phase, most likely corresponding to residual proteins and lipids from the crude enzyme preparation. Similar cellulosic structures have been observed previously as products of in vitro synthesis with bacterial cellulose synthases (Hashimoto et al, 2011;Lin, Brown, Cooper, & Delmer, 1985). When the samples were washed several times with water (6×H 2 O-washed samples), the proportion of the non-cellulosic phase was reduced ( Fig.…”

“…These results clearly show that the in vitro synthesized cellulose crystallized into the energetically most favourable crystal structure even without the application of any chemical for its purification. Synthesis products with the cellulose II crystal structure have been reported also earlier as the result of in vitro synthesis by bacterial systems (Bureau & Brown, 1987;Hashimoto et al, 2011).…”

“…The cellulose-synthesizing activity can be extracted from bacteria by using detergents (Aloni, Cohen, Benziman, & Delmer, 1983;Hashimoto et al, 2011), which solubilize at least the CesA, CesB, and CesD proteins but not CesC (Hashimoto et al, 2011). In this way, synthesis experiments can be carried out under circumstances that would not be tolerated by living bacterial cells.…”

Synthase-dependent exopolysaccharide secretion in Gram-negative bacteria

“…Synthase-dependent exopolysaccharide secretion can occur in the presence or absence of a lipid acceptor molecule, depending on the polysaccharide [23][24][25]. In these systems, it appears that a membrane-embedded glycosyl transferase is able to facilitate simultaneous polymer formation and translocation across the inner membrane [25].…”

Degradation and Synthesis of β-Glucans by a Magnaporthe oryzae Endotransglucosylase, a Member of the Glycoside Hydrolase 7 Family

“…Electron Microscopy and Electron Diffraction-Electron microscopic observations and electron diffraction of insoluble material (synthesized cellulose) were performed as described previously (29). The insoluble product was applied to a carboncoated copper grid that was glow-discharged before use.…”