Synthesis and secretion of a heat-stable carboxymethylcellulose from Clostridium thermocellum in Bacillus subtilis and Bacillus stearothermophilus

Abstract

The cellulase gene celA of Clostridium thermocellum coding for the thermostable endoglucanase A was transferred from Escherichia coli to Bacillus subtilis 168 and B. stearothermophilus CU21 using plasmids derived from the Bacillus vector pUB110. When the structural part of the gene was joined to a pUB110 promoter the recombinant plasmids (pSE102, pSE105) were stably maintained and expressed carboxymethylcellulose (CMCase) activity. In B. stearothermophilus CU21 (pSE105) the clostridial CMCase was produced over a wide temperature range up to the maximal growth temperature (68 degrees C). In contrast to E. coli, all of the CMCase synthesized in bacilli was released into the culture medium. About 50% of the extracellular protein secreted by B. subtilis 168 (pSE102) carrying the celA gene consisted of endoglucanase A. These findings demonstrate the feasibility of producing cellulolytic enzymes from thermophilic anaerobes in bacilli.