Emily Kwan

Cellobiohydrolase A (CbhA) from the cellulolytic bacterium Cellulomonas fimi is a β‐1,4‐exoceilobiohydrolase analogous to Trichoderma reesei CBH II

The gene cbhA from the cellulolytic bacterium Cellulomonas fimi encodes a protein of 872 amino acids designated cellobiohydrolase A (CbhA). Mature CbhA contains 832 amino acid residues and has a predicted molecular mass of 85 349 Da. It is composed of five domains: an N‐terminal catalytic domain, three repeated sequences of 95 amino acids, and a C‐terminal cellulose‐binding domain typical of other C. fimi glycanases. The structure and enzymatic activities of the CbhA cataiytic domain are closely related to those of CBH ll, an exocelloblohydrolase in the glycosyl hydrolase family B from the fungus Trichoderma reesel. CbhA is the first such enzyme to be characterized in bacteria. The data support the proposal that extended loops around the active site distinguish exohydrolases from endohydrolases in this enzyme family.

Crystallization and preliminary X-ray diffraction analysis of the catalytic domain of Cex, an exo-β-1,4-glucanase and β-1,4-xylanase from the bacterium Cellulomonas fimi☆

Single crystals of the catalytic domain of Cex, an exo-beta-1,4-glucanase and beta-1,4-xylanase from the cellulolytic bacterium Cellulomonas fimi, have been grown in the presence of polyethylene glycol 4000 using the vapour diffusion technique. The crystals, which diffract to better than 2.0 A resolution, belong to space group P4(1)2(1)2 or P4(3)2(1)2 and have cell constants: a = b = 88.21 A, c = 81.10 A; alpha = beta = gamma = 90 degrees.

Attack of carboxymethylcellulose at opposite ends by two cellobiohydrolases from Cellulomonas fimi

The patterns of reducing sugar release following sequential addition of two Cellulomonas fimi cellobiohydrolases to carboxymethylcellulose corroborate previous evidence for preferential attack of β-1,4-glucans at opposite ends. In other experiments, sequential additions involving a single cellobiohydrolase and an Agrobacterium β-glucosidase indicate that CbhA attacks preferentially from the non-reducing end, CbhB from the reducing end. We suggest that all aerobic bacteria and fungi involved in cellulose hydrolysis produce a similar pair of cellobiohydrolases: a family 6 cellobiohydrolase that attacks from the non-reducing end and a family 48 (bacterial) or family 7 (fungal) cellobiohydrolase that attacks from the reducing end.

Glycosynthase-mediated assembly of xylanase substrates and inhibitors.

An exo‐β‐xylosidase mutant with glycosynthase activity was created to aid in the synthesis of xylanase substrates and inhibitors. Simple monosaccharides were easily elaborated into di‐, tri‐ and tetrasaccharides by using this enzyme. Some products proved to be surprisingly potent inhibitors of xylanases from glycoside hydrolase families 10 and 11.

Production of a self-activating CBM-factor X fusion protein in a stable transformed Sf9 insect cell line using high cell density perfusion culture

Factor Xa is a serine protease, whose high selectivity can be used to cleave protein tags from recombinant proteins. A fusion protein comprised of a self-activating form of factor X linked to a cellulose-binding module, saCBMFX, was produced in a stable transformed Sf9 insect cell line. The activity of the insect cell produced saCBMFX was higher than the equivalent mammalian cell produced material. A 1.5 l batch fermentation reached a maximum cell concentration of 1.6 × 107 cells ml−1 and a final saCBMFX concentration of 4 mg l−1. The production of saCBMFX by this cell line was also analyzed in a 1.5 l perfusion system using an ultrasonic filter as a cell-retention device for flow rates up to 3.5 l day−1. The cell-retention efficiency of an air backflush mode of acoustic filter operation was greater than 95% and eliminated the need to pump the relatively shear sensitive insect cells. In the perfusion system over 4 × 107 Sf9 cells ml−1 were obtained with a viability greater than 80%. With a doubling of viable cell concentration from 1.5 to 3 × 107 cells ml−1 the saCBMFX production rate was doubled to 6 mg l−1 day−1. The saCBMFX volumetric productivity of the perfusion system was higher than the batch fermentations (0.6 mg l−1 day−1) by an order of magnitude.