Purification, characterization, and molecular analysis of thermostable cellulases CelA and CelB from Thermotoga neapolitana

Abstract

Two thermostable endocellulases, CelA and CelB, were purified from Thermotoga neapolitana. CelA (molecular mass, 29 kDa; pI 4.6) is optimally active at pH 6.0 at 95 degreesC, while CelB (molecular mass, 30 kDa; pI 4.1) has a broader optimal pH range (pH 6.0 to 6.6) at 106 degreesC. Both enzymes are characterized by a high level of activity (high Vmax value and low apparent Km value) with carboxymethyl cellulose; the specific activities of CelA and CelB are 1,219 and 1,536 U/mg, respectively. With p-nitrophenyl cellobioside the Vmax values of CelA and CelB are 69.2 and 18.4 U/mg, respectively, while the Km values are 0.97 and 0.3 mM, respectively. The major end products of cellulose hydrolysis, glucose and cellobiose, competitively inhibit CelA, and CelB. The Ki values for CelA are 0.44 M for glucose and 2.5 mM for cellobiose; the Ki values for CelB are 0.2 M for glucose and 1.16 mM for cellobiose. CelB preferentially cleaves larger cellooligomers, producing cellobiose as the end product; it also exhibits significant transglycosylation activity. This enzyme is highly thermostable and has half-lives of 130 min at 106 degreesC and 26 min at 110 degreesC. A single clone encoding the celA and celB genes was identified by screening a T. neapolitana genomic library in Escherichia coli. The celA gene encodes a 257-amino-acid protein, while celB encodes a 274-amino-acid protein. Both proteins belong to family 12 of the glycosyl hydrolases, and the two proteins are 60% similar to each other. Northern blots of T. neapolitana mRNA revealed that celA and celB are monocistronic messages, and both genes are inducible by cellobiose and are repressed by glucose.

FIG. 1

Maps of clone p17D1 and the subclones used for sequencing. The horizontal arrows indicate the direction of transcription of celA and celB, and the bars above the clone p17K1 map show the regions used as probes for Northern blots.

FIG. 2

SDS-PAGE analysis of purified CelA and CelB from T. neapolitana. Lane M, molecular weight markers; lane 1, crude extract (10 μg); lane 2, CelA (1.0 μg); lane 3, CelB (2.0 μg).

FIG. 3

Nucleotide sequence and deduced amino acid sequence of the celA and celB genes. A putative ribosome binding site is italicized; the start and stop codons are indicated by boldface type; experimentally derived N-terminal sequences are underlined; inverted repeats are indicated by arrows; the putative signal peptide cleavage site is double underlined; and the conserved aspartic acids and glutamic acids are indicated by triangles.

FIG. 4

Northern blot analysis of RNA. (A) Regulation of celA and celB genes (with a probe specific for both celA and celB). Lanes 1 and 2, total RNA from T. neapolitana grown on cellobiose (5 and 10 μg, respectively); lanes 3 through 6, total RNA from T. neapolitana grown on glucose (5, 10, 20, and 40 μg, respectively). (B) Northern blot with celA gene-specific probe. Lane 1 contained 10 μg of RNA from T. neapolitana grown on cellobiose.