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Structural insights into the affinity of Cel7A carbohydrate-binding module for lignin.

Strobel KL, Pfeiffer KA, Blanch HW, Clark DS - J. Biol. Chem. (2015)

Bottom Line: Cellulose and lignin affinity of the 31 mutants were highly correlated, although several mutants displayed selective reductions in lignin or cellulose affinity.Four mutants with increased cellulose selectivity (Q2A, H4A, V18A, and P30A) did not exhibit improved hydrolysis of cellulose in the presence of lignin.Further reduction in lignin affinity while maintaining a high level of cellulose affinity is thus necessary to generate an enzyme with improved hydrolysis capability.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Chemical and Biomolecular Engineering, University of California Berkeley, Berkeley, California 94720.

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Enzyme partition coefficients (α) for Avicel and lignin calculated from binding experiments at room temperature and an initial enzyme concentration of 125 nm. Values presented are means of duplicate samples and errors are S.D.
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Figure 2: Enzyme partition coefficients (α) for Avicel and lignin calculated from binding experiments at room temperature and an initial enzyme concentration of 125 nm. Values presented are means of duplicate samples and errors are S.D.

Mentions: Unpurified, buffer-exchanged mutants were normalized to the same concentration of active Cel7A. Binding to Avicel and lignin was measured in 50 mm sodium acetate buffer, pH 5, using 15 mg/ml of Avicel or lignin and 125 nm enzyme. Experiments were conducted in a total volume of 70 μl in a rotating shaker (300 rpm) at room temperature. An initial time course with the wild type enzyme was measured to determine the time required for binding to reach equilibrium. Equilibrium was reached after ∼30 min. Subsequent assays were performed for 1 h. After equilibration, solids were separated by centrifugation and the supernatant was analyzed for free, active enzyme using 4-methylumbelliferyl β-d-cellobioside. The amount of bound enzyme was calculated from the difference between added enzyme and enzyme remaining in solution. The values plotted in Fig. 2 are bound enzyme divided by enzyme remaining in solution. At the low enzyme concentrations employed, these values approximate the partition coefficient, α, as defined in Equation 1. The values reported are averages of duplicate experiments and the errors are calculated as standard deviations.


Structural insights into the affinity of Cel7A carbohydrate-binding module for lignin.

Strobel KL, Pfeiffer KA, Blanch HW, Clark DS - J. Biol. Chem. (2015)

Enzyme partition coefficients (α) for Avicel and lignin calculated from binding experiments at room temperature and an initial enzyme concentration of 125 nm. Values presented are means of duplicate samples and errors are S.D.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4566252&req=5

Figure 2: Enzyme partition coefficients (α) for Avicel and lignin calculated from binding experiments at room temperature and an initial enzyme concentration of 125 nm. Values presented are means of duplicate samples and errors are S.D.
Mentions: Unpurified, buffer-exchanged mutants were normalized to the same concentration of active Cel7A. Binding to Avicel and lignin was measured in 50 mm sodium acetate buffer, pH 5, using 15 mg/ml of Avicel or lignin and 125 nm enzyme. Experiments were conducted in a total volume of 70 μl in a rotating shaker (300 rpm) at room temperature. An initial time course with the wild type enzyme was measured to determine the time required for binding to reach equilibrium. Equilibrium was reached after ∼30 min. Subsequent assays were performed for 1 h. After equilibration, solids were separated by centrifugation and the supernatant was analyzed for free, active enzyme using 4-methylumbelliferyl β-d-cellobioside. The amount of bound enzyme was calculated from the difference between added enzyme and enzyme remaining in solution. The values plotted in Fig. 2 are bound enzyme divided by enzyme remaining in solution. At the low enzyme concentrations employed, these values approximate the partition coefficient, α, as defined in Equation 1. The values reported are averages of duplicate experiments and the errors are calculated as standard deviations.

Bottom Line: Cellulose and lignin affinity of the 31 mutants were highly correlated, although several mutants displayed selective reductions in lignin or cellulose affinity.Four mutants with increased cellulose selectivity (Q2A, H4A, V18A, and P30A) did not exhibit improved hydrolysis of cellulose in the presence of lignin.Further reduction in lignin affinity while maintaining a high level of cellulose affinity is thus necessary to generate an enzyme with improved hydrolysis capability.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Chemical and Biomolecular Engineering, University of California Berkeley, Berkeley, California 94720.

Show MeSH