Properties and gene structure of a bifunctional cellulolytic enzyme (CelA) from the extreme thermophile ‘Anaerocellum thermophilum’ with separate glycosyl hydrolase family 9 and 48 catalytic domains

Abstract

A large cellulolytic enzyme (CelA) with the ability to hydrolyse microcrystalline cellulose was isolated from the extremely thermophilic, cellulolytic bacterium 'Anaerocellum thermophilum I . Full-length CelA and a truncated enzyme species designated CelA were purified to homogeneity from culture supernatants. CelA has an apparent molecular mass of 230 kDa. The enzyme exhibited significant activity towards Avicel and was most active towards soluble substrates such as CM-cellulose (CMC) and p-glucan. Maximal activity was observed between pH values of 5 and 6 and temperatures of 95 "C (CM-cellulase) and 85 "C (Avicelase). Cellobiose, glucose and minor amounts of cellotriose were observed as end-products of Avicel degradation. The CelAencoding gene was isolated from genomic DNA of 'A. thermophilum by PCR and the nucleotide sequence was determined. The celA gene encodes a protein of 1711 amino acids (190 kDa) starting with the sequence found at the Nterminus of CelA purified from 'A. thermophilum I . Sequence analysis revealed a multidomain structure consisting of two distinct catalytic domains homologous to glycosyl hydrolase families 9 and 48 and three domains homologous to family 111 cellulose-binding domain linked by Pro-Thr-Ser-rich regions. The enzyme is most closely related to CelA of Caldicellulosiruptor sacchamlyticus (sequence identities of 96 and 97% were found for the N-and C-terminal catalytic domains, respectively). Endoglucanase CelZ of Clostridium stercorarium shows 70.4% sequence identity to the N-terminal family 9 domain and exoglucanase CelY from the same organism has 69.2% amino acid identity with the C-terminal family 48 domain. Consistent with this similarity on the primary structure level, the 90 kDa truncated derivative CelA' containing the N-terminal half of CelA exhibited endoglucanase activity and bound t o microcrystalline cellulose. Due to the significantly enhanced Avicelase activity of full-length CelA, exoglucanase activity may be ascribed to the C-terminal family 48 catalytic domain.

Lic16A of Clostridium thermocellum, a non-cellulosomal, highly complex endo-β-1,3-glucanase bound to the outer cell surface

“…The high complexity of the Lic16A structure is comparable to the most complex cellulolytic proteins from the non-cellulosomal cellulase system of Caldicellulosiruptor sp. or its relative Anaerocellum thermophilum (Gibbs et al, 2000;Zverlov et al, 1998). A comparison between the two strains and partial resequencing of sequence differences between the genes made the correctness of the unusual structure of licA reliable beyond reasonable doubt.…”

Toward an aggregated understanding of enzymatic hydrolysis of cellulose: Noncomplexed cellulase systems

“…Quantitative representation of the extent of synergism is usually expressed in terms of a ''degree of synergism'' (DS)-equal to the ratio of the activity exhibited by mixtures of components divided by the sum of the activities of separate components. Types of synergism proposed in the cellulose hydrolysis literature include: 1) endoglucanase and exoglucanase; 2) exoglucanase and exoglucanase (Fagerstam and Pettersson, 1980;Tomme et al, 1988Tomme et al, , 1990Wood and McCrae, 1986;Wood and Garcia-Campayo, 1990); 3) endoglucanase and endoglucanase Tuka et al, 1992;Walker et al, 1992); 4) exoglucanase or endoglucanase and h-glucosidase, which reduces inhibition by cellobiose (Lamed et al, 1991;Woodward, 1991); 5) intramolecular synergy between catalytic domain and CBM (Din et al, 1994) or two catalytic domains Te'o et al, 1995;Warren et al, 1987;Zverlov et al, 1998); 6) cellulose-enzyme-microbe (CEM) synergism (Lynd et al, 2002); and 7) a proximity synergism due to formation of cellulase complexes (Fierobe et al, 2001(Fierobe et al, , 2002Mandels, 1985;Schwarz, 2001). Not all synergies are necessarily operative in any given situation.…”

A diverse set of family 48 bacterial glycoside hydrolase cellulases created by structure‐guided recombination

“…In the chimeras, both these measured properties extend beyond the range of the parents. Many of the chimeras are very stable: indeed, this experiment has added 35 new Cel48 enzymes with a T opt of > 60°C to the six natural thermostable cellulases that have been characterized to date: C. thermocellum ATCC 27405 CelS [24], C. thermocellum F7 CelS [25], C. thermocellum ATCC 27405 CelY [26], Thermobifida fusca YX CelF [27], C. stercorarium CelY [28], and Anaerocellum thermophilum DSM 6725 CelA [29].…”