Mutations from a family-shuffling-library reveal amino acid residues responsible for the thermostability of endoglucanase CelA from Clostridium thermocellum

Abstract

We constructed a library of chimeras from the major endoglucanase, CelA, of Clostridium thermocellum and a less stable endoglucanase CelB from Clostridium josui with multiple point mutations using low-fidelity family-shuffling method. Mutations that inactivated the enzyme were rapidly eliminated with high-throughput screening. The activities and thermostabilities of selected variants were evaluated, and four amino acid substitutions, K249R, P258S, S329N and E355G, were identified as having significant impact on the thermostability of CelA without affecting enzymatic activity. In the crystal structure of CelA, most of them are away from the activity cleft and are responsible for the stabilization of secondary structures.

Polarity Alteration of a Calcium Site Induces a Hydrophobic Interaction Network and Enhances Cel9A Endoglucanase Thermostability

“…The thermal resistance of various enzymes depends on the various molecular interactions needed to achieve the rigid structure (5,6,8,39). Hydrophobic and electrostatic interactions are the two key factors that allow superslow protein unfolding for hyperthermal proteins to function at extreme temperatures (37).…”

“…Hence, identifying thermostable forms of cellulases or developing methods to improve the thermostability would benefit cellulase application on an industrial scale. Numerous studies on the thermostability of endoglucanase modules involved in cellulose degradation have been conducted (4)(5)(6). The endoglucanase catalytic domains of glycoside hydrolase family 9 (GH9) are arranged with specific carbohydrate-binding domains and/or immunoglobulin (Ig)-like domains to ensure stability and activity (4,7).…”

“…The N-terminal ␤-sheet, which is considered critical for the hyperthermostability of GH5 (glycoside hydrolase family 5) Pyrococcus horikoshii endoglucanase, may help stabilize the core barrel catalytic structure (5). The residues responsible for the thermostability of GH8 (glycoside hydrolase family 8) Clostridium thermocellum endoglucanase CelA provide the stability of secondary structures (6). Hence, rigid packing may be the key to converting mesostable endoglucanases into thermostable ones (5,6,8).…”

“…The residues responsible for the thermostability of GH8 (glycoside hydrolase family 8) Clostridium thermocellum endoglucanase CelA provide the stability of secondary structures (6). Hence, rigid packing may be the key to converting mesostable endoglucanases into thermostable ones (5,6,8).…”

“…The endoglucanases operate with an inversion of anomeric stereochemistry in hydrolyzing the glycosidic bonds of soluble cellulose derivatives as described in the CAZypedia database (http://www.cazypedia.org/index.php /Glycoside_Hydrolase_Family_9). The endoglucanase from Alicyclobacillus acidocaldarius is composed of a catalytic domain which folds into a typical GH9 (␣/␣) 6 -barrel comprising an open active-site cleft and an Ig-like domain at the N terminus (7,17). The enzyme is most active against substrates containing ␤-1,4-linked glucans, including carboxymethylcellulose, lichenan, and p-nitrophenyl (pNP)-cellooligosaccharides (16).…”