Purification, Characterization, and Molecular Analysis of Thermostable Cellulases CelA and CelB fromThermotoga neapolitana

Abstract

ABSTRACTTwo thermostable endocellulases, CelA and CelB, were purified fromThermotoga neapolitana. CelA (molecular mass, 29 kDa; pI 4.6) is optimally active at pH 6.0 at 95°C, while CelB (molecular mass, 30 kDa; pI 4.1) has a broader optimal pH range (pH 6.0 to 6.6) at 106°C. Both enzymes are characterized by a high level of activity (highVmaxvalue and low apparentKmvalue) with carboxymethyl cellulose; the specific activities of CelA and CelB are 1,219 and 1,536 U/mg, respectively. Withp-nitrophenyl cellobioside theVmaxvalues of CelA and CelB are 69.2 and 18.4 U/mg, respectively, while theKmvalues are 0.97 and 0.3 mM, respectively. The major end products of cellulose hydrolysis, glucose and cellobiose, competitively inhibit CelA, and CelB. TheKivalues for CelA are 0.44 M for glucose and 2.5 mM for cellobiose; theKivalues 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°C and 26 min at 110°C. A single clone encoding thecelAandcelBgenes was identified by screening aT. neapolitanagenomic library inEscherichia coli. ThecelAgene encodes a 257-amino-acid protein, whilecelBencodes 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 ofT. neapolitanamRNA revealed thatcelAandcelBare monocistronic messages, and both genes are inducible by cellobiose and are repressed by glucose.