Gene cloning, DNA sequencing, and expression of thermostable beta-mannanase from Bacillus stearothermophilus.
A DNA genomic library constructed from Bacillus stearothermophilus, a gram-positive, facultative thermophilic aerobe that secretes a thermostable beta-mannanase, was screened for mannan hydrolytic activity. Recombinant beta-mannanase activity was detected on the basis of the clearing of halos around Escherichia coli colonies grown on a dye-labelled substrate, Remazol brilliant blue-locust bean gum. The nucleotide sequence of the mannanase gene, manF, corresponded to an open reading frame of 2,085 bp that codes for a 32-amino-acid signal peptide and a mature protein with a molecular mass of 76,089 Da. From sequence analysis, ManF belongs to glycosyl hydrolase family 5 and exhibits higher similarity to eukaryotic than to bacterial mannanases. The manF coding sequence was subcloned into the pH6EX3 expression plasmid and expressed in E. coli as a recombinant fusion protein containing a hexahistidine N-terminal sequence. The fusion protein has thermostability similar to the native enzyme and was purified by Ni2+ affinity chromatography. The values for the kinetic parameters Vmax and Km were 384 U/mg and 2.4 mg/ml, respectively, for the recombinant mannanase and were comparable to those of the native enzyme.
Ethier
N
N
Département de Biochimie, Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada H3C 3J7.
Talbot
G
G
Sygusch
J
J
D001419:Bacteria / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli; D005658:Fungi / Q000201:enzymology / Q000235:genetics; D015698:Genomic Library; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics*; D006358:Hot Temperature; D008361:Mannosidases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D044902:beta-Mannosidase
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D008361:Mannosidases; D044902:beta-Mannosidase