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Title: THE FIBRONECTIN TYPE 3-LIKE REPEAT FROM THE CLOSTRIDIUM THERMOCELLUM CELLOBIOHYDROLASE CBHA PROMOTES HYDROLYSIS OF CELLULOSE BY MODIFYING ITS SURFACE

Author
item KATAEVA, IRINA - UNIV OF GEORGIA
item SEIDEL III, RONALD - UNIV OF GEORGIA
item SHAH, ASHIT - UNIV OF GEORGIA
item WEST, LARRY - UNIV OF GEORGIA
item Li, Xin Liang
item LJUNGDAHL, LARS - UNIV OF GEORGIA

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/28/2002
Publication Date: 9/1/2002
Citation: KATAEVA, I.A., SEIDEL III, R.D., SHAH, A., WEST, L.T., LI, X., LJUNGDAHL, L.G. THE FIBRONECTIN TYPE 3-LIKE REPEAT FROM THE CLOSTRIDIUM THERMOCELLUM CELLOBIOHYDROLASE CBHA PROMOTES HYDROLYSIS OF CELLULOSE BY MODIFYING ITS SURFACE. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. 2002. V. 68. P. 4292-4300.

Interpretive Summary: Efficient conversion of lignocellulosic biomass such as straw and corn stalks to fermentable sugars has been recognized as the major bottleneck for the economical production of biofuels and feedstock chemicals from these almost infinite renewable resources. There are three major constitutes, cellulose, hemicelluloses, and lignin, commonly found in lignocellulosic biomass. Efficient degradation of the three constitutes requires a variety of biological functions because some of these constitutes form crystalline and network structures. Microorganisms have developed very different strategies for the degradation of plant cell walls. Various biologically functional molecules related to lignocellulose degradation have been identified. Some molecules have only one function while others may have multiple functions. Well-recognized functions include catalysis, substrate-binding, docking, and scaffolding as well as thermostabilization. This paper reports for the first time a new module, fibronectin type 3-like domain, that modifies the surface of cellulose and enhances the substrate hydrolysis by catalytic domains. This domain was commonly found in enzymes involved in polysaccharide hydrolysis but its function was not known. The discovery may allow us to design improved cellulase products which convert lignocellulosic biomass to fermentable sugars at a more efficient rate.

Technical Abstract: Fibronectin type 3 homology domains (Fn3), as found in the cellobiohydrolase CbhA of Clostridium thermocellum, are common among bacterial extracellular glycohydrolases. The function of these domains is not clear. CbhA is modular and composed of an N-terminal family IV carbohydrate-binding domain (CBDIV), an immunoglobulin-like domain, a family 9 glycosyl hydrolase catalytic domain (Gh9), two Fn3-like domains (Fn31,2), a family III carbohydrate-binding domain (CBDIII), and a dockerin domain. Efficiency of cellulose hydrolysis by truncated forms of CbhA increased in the following order: Gh9 (lowest efficiency), Gh9-Fn31,2 (more efficient), and Gh9-Fn31,2-CBDIII (greatest efficiency). Thermostability of the above constructs decreased in the following order: Gh9 (most stable), Gh9-Fn31,2, and then Gh9-Fn31,2-CBDIII (least stable). Mixing of Orpinomyces endoglucanase CelE with Fn31,2 or Fb31,2-CBDIII increased efficiency of hydrolysis of acid-swollen cellulose (ASC) and filter paper. Scanning electron microscopic studies of filter paper treated with Fn31,2, Fn31,2-CBDIII, or CBDIII showed that the surface of the cellulose fibers had been loosened up and crenellated by Fn31,2 and Fn31,2-CBDIII and to a lesser extent by CBDIII. X-ray diffraction analysis did not reveal changes in the crystallinity of the filter paper. CBDIII bound to ASC and filter paper with capacities of 2.45 and 0.73 umoles g**-1 and relative affinities (Kr) of 1.12 and 2.13 liters g**-1, respectively. Fn31,2 bound weakly to both celluloses. Fn31,2-CBD bound to ASC and filter paper with capacities of 3.22 and 0.81 umoles g**-1 and Krs of 1.14 and 1.98 liters g**-1, respectively. Fn31,2 and CBDIII contained 2 and 1 mol of calcium per mol, respectively. The results suggest that Fn31,2 aids the hydrolysis of cellulose by modifying its surface. This effect is enhanced by the presence of CBDIII, which increases the concentration of Fn31,2 on the cellulose surface.