112. Bureau, T. E. and R. M. Brown, Jr.
1987. In vitro synthesis of cellulose II from a cytoplasmic
membrane fraction of Acetobacter xylinum, Proc. Nat. Acad.
Sci. U.S.A. 84:6985-6989.
The cytoplasmic and outer membranes of Acetobacter
xylinum (ATCC 53582) were isolated by discontinuous sucrose
density ultracentrifugation. Both lysozyme (EC 184.108.40.206) and
trypsin (EC 220.127.116.11) were required for efficient crude membrane
separation. Primary dehydrogenases and NADH oxidase were used
as cytoplasmic membrane markers, and 2-keto-3-deoxyoctulosonic
acid was used to identify the outer membranes. Cellulose synthetase
(UDP-glucose: l ,4--D-glucan 4--D-glucosyltransferase ; EC 18.104.22.168)
activity was assayed as the conversion of radioactivity from UDP-[14C]glucose
into an alkali-insoluble -1,4-D-[14C]glucan. This
activity was predominantly found in the cytoplasmic membrane.
The cellulose nature of the product was demonstrated by (i)
enzymatic hydrolysis followed by TLC, (ii) methylation
analysis followed by TLC, and (iii) GC/MS. Further, the
weight-average and number-average degree of polymerization of
the in vitro product, determined by high-performance
gel permeation chromatography, were 4820 and 5270, respectively.
In addition, x-ray diffraction analysis indicated that the in
vitro product is cellulose II, which is in contrast to the
in vivo product--namely, cellulose I.