Seo-Hee Kang

Construction and characterization of chimeric cellulases with enhanced catalytic activity towards insoluble cellulosic substrates

The chimeric proteins viz. CBM3-Cel9A, CBM4-Cel9A and CBM30-Cel9A, are constructed by fusion of family 3, 4, and 30 cellulose binding modules (CBMs) to N-terminus of family 9 endoglucanase (Cel9A) from Alicyclobacillus acidocaldrious. The chimeric enzymes were successfully expressed in Escherichia coli and purified to homogeneity. The chimeric enzymes showed significant increase in Avicel (8-12 folds) and filter paper (7-10 folds) degradation activities compared to Cel9A endoglucanase. Computational protein modeling and simulation on the chimeric enzymes were applied to analyze the fused CBMs effect on the increased insoluble cellulosic substrates degradation activity. Thin layer chromatography analysis of the enzymatic hydrolysis products and distribution of reducing sugars between soluble and insoluble fractions indicated processive cleavage of insoluble cellulosic substrates by the chimeras. The fused CBMs played a critical accessory role for the Cel9A catalytic domain and changed its character to facilitate the processive cleavage of insoluble cellulosic substrates.

Improving the catalytic activity of cyclohexanone monooxygenase-based whole-cell biocatalysts under substrate toxic conditions

The catalytic activity of oxygenase-based whole-cell biocatalysts is heavily influenced by substrate and product toxicities due to cell membrane permeabilization and protein denaturation effects of the organic substrates and products. Therefore, stability of oxygenase-based whole-cell biocatalysts against solvent stress was investigated with recombinant Escherichia coli BL21 and Corynebacterium glutamicum ATCC13032 expressing the chnB gene of cyclohexanone monooxygenase of Acinetobacter calcoaceticus NCIMB 9871. The cyclohexanone oxygenation activity of the recombinant biocatalysts rapidly decreased as cyclohexanone concentration increased from 2.4 to 26 g/L. However, treatment of the recombinant cells with non-lethal doses of cyclohexanone or preadaptation to the toxic substrate led to the oxygenation activity being relatively maintained. For instance, the oxygenation activity of cyclohexanone-treated E. coli cells was ca. 13 U per g dry cells at the substrate concentration of 26 g/L, which was almost 5-fold higher than that of the cyclohexanone-nontreated cells. In addition, biocatalytic activity was better maintained when the genes encoding chaperones (i.e., GroEL-ES and DnaKJ-GrpE) were coexpressed with the chnB gene. The positive effects of chaperones on the catalytic activity of the recombinant E. coli-based biocatalyst appeared to be related with expression level of biotransformation enzymes rather than with solvent stress-response metabolism. Overall, molecular chaperones, of which expression can be induced by solvent treatment, were involved in catalytic stability of whole-cell biocatalysts during biotransformations involving toxic compounds as the reactants.