Biomedical subjects

Lee R Lynd

Publications and source records attributed to Lee R Lynd.

22 records · Page 2Linked to original sources

PubMed2003

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.

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 Avicelgrown culture with fractional cellulose conversion (X) = 0.98, as well as supernatant, pellet, cell-associated, and cellulose-associated samples from an Avicel-grown culture with X = 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 approximately 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.

Anaerobiosis↗

PubMed2002

Quantitative determination of cellulase concentration as distinct from cell concentration in studies of microbial cellulose utilization: analytical framework and methodological approach.

In analyzing microbial cellulose utilization, it would be useful to independently measure the mass concentration of cells and cellulase enzymes. Such measurements would allow investigation of the allocation of cellular resources between synthesis of cells and cellulase, in vivo cell- and cellulase-specific cellulose hydrolysis rates, and bioenergetics. Methodological protocols are not established for independent determination of cell and cellulase concentrations for the common case in which a substantial fraction of cellulase is attached to the cell surface. Alternative analytical approaches by which to develop such protocols are examined from the perspective of error minimization. For cell concentration measurement, acceptable accuracy is expected when the concentrations of a cell-specific component (e.g., DNA) is determined or when total protein is determined in conjunction with a measurement specific to cellulase. For cellulase concentration measurement, acceptable accuracy is expected when a measurement specific to cellulase such as ELISA is used. Several analytical approaches are rejected based on large expected errors.

Bacteria↗

PubMed2002

Microbial cellulose utilization: fundamentals and biotechnology.

Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for "consolidated bioprocessing" (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts.

Bacteria↗

PubMedDate not supplied

Determination of the number-average degree of polymerization of cellodextrins and cellulose with application to enzymatic hydrolysis.

A rapid and accurate method for determining the number-average degree of polymerization (DP(n)) was established for insoluble cellulose and soluble cellodextrins as the ratio of glucosyl monomer concentration determined by the phenol-sulfuric acid method divided by the reducing-end concentration determined by a modified 2,2'-bicinchoninate (BCA) method. The modified BCA method, featuring incubation at 75 degrees C for 30 min, did not result in beta-glucosidic bond cleavage, whereas substantial cleavage was observed at higher temperature. Solubilization of insoluble cellulose in cold phosphoric acid prior to measurement of the reducing-end concentration by the BCA method was found not to be necessary for several model celluloses such as microcrystalline cellulose, but such solubilization was required for large fibers of cellulose such as Whatman No. 1 filter paper. The phenol-sulfuric acid method can be used for measuring the glucosyl monomer concentration of soluble cellodextrins, and also for insoluble cellulose if preceded by a liquefaction step. Standard deviations of < or =2% were obtained for both reducing and glucosyl monomer determination and of < or =3% for overall determination of DP. By use of the reported method, hydrolysis of phosphoric acid-swollen cellulose (PASC) by the Trichoderma reesei cellulase system was shown to result in a rapid decrease in DP as hydrolysis proceeded. By contrast, the DP of Avicel remained nearly constant during hydrolysis. The specific enzymatic cellulose hydrolysis rate is 100-fold higher for PASC as compared to Avicel.

Cellulose↗