Web Release Date: December 3,
Quantification of Cell and Cellulase Mass Concentrations during Anaerobic Cellulose Fermentation: Development of an Enzyme-Linked Immunosorbent Assay-Based Method with Application to Clostridium thermocellum Batch Cultures
and
Thayer School of Engineering and Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755
Received for review April 26, 2002. Accepted October 14, 2002.
Abstract:
A methodology was developed to determine the mass
concentrations of cellulase and cells applicable to studies
of microbial cellulose utilization in systems for which a
substantial fraction of cellulase is cell-associated. Antibodies raised against a 14-amino acid synthetic peptide
with sequence taken from the cohesin domain of the
scaffoldin protein of Clostridium thermocellum ATCC
27405 were used to develop an indirect ELISA protocol.
Six cellulase calibration standards were prepared using
affinity digestion (Morag, E.; Bayer, E. A.; Lamed, R.
Enzyme Microb. Technol. 1992, 14, 289-292.). These
included supernatant and pellet samples from an Avicel-grown culture with fractional cellulose conversion (
) =
0.98, as well as supernatant, pellet, cell-associated, and
cellulose-associated samples from an Avicel-grown culture
with = 0.8. All six standards displayed a very similar
absorbance versus concentration relationship when subjected to ELISA, essentially identical SDS-PAGE banding
patterns, and similar cellulase specific activity in relation
to both other purified cellulase preparations and crude
samples. Coefficients of variation for cellulase concentration measurements were 5.2% for supernatant samples
and 5.9% for pellet samples. The ELISA method was
applied to batch cultures of C. thermocellum grown on
Avicel. Cell concentration was calculated from the pellet
protein concentration and the cell protein fraction of a
cellobiose-grown control. Two alternative methods appeared to overpredict the cell concentration and were not
capable of quantifying cells as distinct from cellulase.
Cellulase protein production by Avicel-grown batch cultures represented ~20% of cell mass exclusive of cellulase. It is concluded that the reported protocols establish
a reasonable methodological basis for quantitative determination of the mass concentration of cellulase protein
produced by C. thermocellum and for calculation of cell
mass concentration as distinct from cellulase concentration.
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