Cloning and expression of an endocellulase gene from a novel streptomycete isolated from an East African soda lake.
Alkaline cellulase-producing actinomycete strains were isolated from mud samples collected from East African soda lakes. The strains were identified as novel Streptomyces spp. by 16S rDNA sequence analysis. A cellulase gene (cel12A) from Streptomyces sp. strain 11AG8 was cloned by expression screening of a genomic DNA library in Escherichia coli. From the nucleotide sequence of a 1.5-kb DNA fragment, an open reading frame of 1,113 nucleotides was identified encoding a protein of 371 amino acids. From computer analysis of the sequence, it was deduced that the Cel12A mature enzyme is a protein of 340 amino acids. The protein contained a catalytic domain, a glycine-rich linker region, and a cellulose-binding domain of 221, 12, and 107 amino acids, respectively. FASTA analysis of the catalytic domain of Cel12A classified the enzyme as a family 12 endoglucanase and the cellulose-binding domain as a family IIa CBD. Streptomyces rochei EglS was determined as nearest neighbor with a similarity of 75.2% and 61.0% to the catalytic domain and the cellulose-binding domain, respectively. The cell2A gene was subcloned in a Bacillus high-expression vector carrying the Bacillus amyloliquefaciens amylase regulatory sequences, and the construct was transformed to a Bacillus subtilis host strain. Crude enzyme preparations were obtained by ultrafiltration of cultures of the Bacillus subtilis recombinant strain containing the 11AG8 cell2A gene. The enzyme showed carboxymethylcellulase (CMCase) activities over a broad pH range (5-10) with an optimum activity at pH 8 and 50 degrees C. The enzyme retained more than 95% of its activity after incubation for 30 min under these conditions.
van Solingen
P
P
Genencor International B.V., Archimedesweg 30,2333 CN Leiden, The Netherlands.
Meijer
D
D
van der Kleij
W A
WA
Barnett
C
C
Bolle
R
R
Power
S D
SD
Jones
B E
BE
D000351:Africa, Eastern; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics; D001483:Base Sequence; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010641:Phenotype; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D014871:Water Microbiology
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D011994:Recombinant Proteins; D002480:Cellulase