Calorimetric versus Growth Microbial Analysis of Cellulase Enzymes Acting on Cellulose

Assay of cellulase enzymology on cellulose was investigated by two methods: (i) plate colony counting to determine microbial growth and (ii) microbial calorimetry. These methods were chosen because they accept raw samples and have the potential to be far more specific than spectrophotometric reducing sugar assays. Microbial calorimetry requires ca. 0.5 to 1 h and 10 to 100 μM concentrations of cellulolytic lower sugars (glucose and cellobiose). Growth assay (liquid culture, plating, colony counting) requires 15 to 20 h and ca. 0.5 mM sugars. Microbial calorimetry requires simply aerobic metabolism, whereas growth assay requires completion of the cell cycle. A stripping technique is described for use in conjunction with the calorimetric method to enable separate analysis of the two sugars. Mixtures of glucose and cellobiose are equilibrated with Escherichia coli and spun out to remove glucose. The supernatant is calorimetrically combusted with Klebsiella sp. to quantitate cellobiose, and the same organism combusting the nonstripped mixture gives heat proportional to the sum of the two sugars. Calorimetry of cellulolysis products from individual exo- and endocellulases, and from their reconstituted mixture, was carried out to develop a microbial calorimetric means for demonstrating enzyme synergism.

[1]  W. Hempfling,et al.  Correlation between changes in metabolite concentrations and rate of ion transport following glucose addition to Escherichia coli B. , 1967, Biochimica et biophysica acta.

[2]  L-Sorbose metabolism in Klebsiella pneumoniae and Sor+ derivatives of Escherichia coli K-12 and chemotaxis toward sorbose , 1984, Journal of bacteriology.

[3]  R. Lovrien,et al.  Microcalorimetry of microorganism metabolism of monosaccharides and simple aromatic compounds , 1980 .

[4]  T. Bauchop,et al.  Glycosidases of the rumen anaerobic fungus Neocallimastix frontalis grown on cellulosic substrates , 1985, Applied and environmental microbiology.

[5]  M. F. Mallette.  A pH 7 Buffer Devoid of Nitrogen, Sulfur, and Phosphorus for Use in Bacteriological Systems , 1967, Journal of bacteriology.

[6]  Irving M. Klotz,et al.  Introduction to Biomolecular Energetics , 1986 .

[7]  D. Mountfort,et al.  Production and regulation of cellulase by two strains of the rumen anaerobic fungus Neocallimastix frontalis , 1985, Applied and environmental microbiology.

[8]  F. Millero,et al.  Microcalorimetric Measurements of Glucose Metabolism by Marine Bacterium Vibrio alginolyticus , 1982, Applied and environmental microbiology.

[9]  F. Neidhardt,et al.  Culture Medium for Enterobacteria , 1974, Journal of bacteriology.

[10]  R. Shrager,et al.  Simultaneous measurements of proton motive force, delta pH, membrane potential, and H+/O ratios in intact Escherichia coli. , 1983, Biophysical journal.

[11]  I. Wadsö,et al.  Heats of Hydrolysis of Acetates and Thiolacetates in Aqueous Solution. , 1962 .

[12]  J. Ashworth,et al.  The anaplerotic fixation of carbon dioxide by Escherichia coli , 1966, Proceedings of the Royal Society of London. Series B. Biological Sciences.

[13]  R. Spiro,et al.  [1] Analysis of sugars found in glycoproteins , 1966 .

[14]  T. Gusek,et al.  Cellulase and protease specific activities of commercially available cellulase preparations , 1985 .

[15]  B. D. Davis,et al.  MUTANTS OF ESCHERICHIA COLI REQUIRING METHIONINE OR VITAMIN B12 , 1950, Journal of bacteriology.

[16]  R. Thauer,et al.  Energy conservation in chemotrophic anaerobic bacteria , 1977, Bacteriological reviews.

[17]  P. Anderson,et al.  Resolution of the multienzyme cellulase complex of Trichoderma reesei QM 9414 , 1984 .

[18]  E. Lin,et al.  Replacement of a Phosphoenolpyruvate-dependent Phosphotransferase by a Nicotinamide Adenine Dinucleotide-linked Dehydrogenase for the Utilization of Mannitol , 1967, Journal of bacteriology.

[19]  R. Roberts.  Studies of biosynthesis in escherichia coli , 1955 .

[20]  R. Hammerstedt,et al.  Calorimetric techniques for metabolic studies of cells and organisms under normal conditions and stress. , 1983, The Journal of experimental zoology.