Purification, characterization, and molecular analysis of thermostable cellulases CelA and CelB from Thermotoga neapolitana.
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.
Bok
J D
JD
Department of Biochemistry and Microbiology, Cook College, Rutgers University, New Brunswick, New Jersey 08901, USA.
Yernool
D A
DA
Eveleigh
D E
DE
D000595:Amino Acid Sequence; D001483:Base Sequence; D002480:Cellulase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004926:Escherichia coli; D015698:Genomic Library; D016965:Gram-Negative Anaerobic Straight, Curved, and Helical Rods / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity
D011994:Recombinant Proteins; C074775:CelA endoglucanase; D002480:Cellulase