Cloning of an endo-(1-->4)-beta-glucanase gene, celA, from the rumen bacterium Clostridium sp. ('C. longisporum') and characterization of its product, CelA, in Escherichia coli.
A genomic library of Clostridium sp. ('C. longisporum') ATCC 49440 in the host Escherichia coli was screened for endo-beta-glucanases, and plasmids pCM64 and pCM4 were isolated. The nucleotide sequence of a 3620 bp fragment was found to contain a 1548 bp open reading frame (ORF), termed celA, which encodes an endo-(1-->4)-beta-glucanase, CelA, assigned to family A4. N-terminal amino acid sequence determination revealed that pCM64 encoded the full-length celA gene, including a signal sequence, while pCM4 carried a 5'-truncated celA gene expressed as an N-terminal fusion protein, CelA delta N', without a signal sequence. CelA was secreted into the periplasm in E. coli. In this organism, proteolytic cleavage of CelA at or near a putative linker region resulted in the appearance of two active polypeptides of molecular masses 57 and 47 kDa. The former was the full-length enzyme while the latter consisted of the catalytic domain from which the cellulose-binding domain (CBD) had been removed (CelA delta CBD). The intracellularly-located CelA delta N' was not subject to proteolytic degradation. The pH and temperature optima of CelA were pH 4.8 and 43 degrees C, respectively. CelA hydrolysed barley beta-glucan, lichenan, carboxymethylcellulose and xylan. It showed preferential activity against the larger cellooligosaccharides (cellohexaose and cellopentaose); cellotetraose was the smallest substrate degraded completely.
Mittendorf
V
V
Department of Microbiology, University of Cape Town, Rondebosch, South Africa.
Thomson
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D002480:Cellulase / Q000037:antagonists & inhibitors / Q000235:genetics*; D002482:Cellulose / Q000378:metabolism; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D015698:Genomic Library; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D012417:Rumen / Q000382:microbiology; D013379:Substrate Specificity; D013696:Temperature
D002413:Cations, Divalent; D004269:DNA, Bacterial; D002482:Cellulose; C074775:CelA endoglucanase; D002480:Cellulase