[Cloning, sequence analysis and heterologous expression of a beta-mannanase gene from Bacillus subtilis Z-2].
Mannanase, an extracellular enzyme catalyzes the hydrolysis of hemicelluloses to produce oligosaccharides, has a potential to be applied in food industries. In this study, a mannanase gene from B. subtilis Z-2 was isolated through PCR screening of a genomic DNA library. The nucleotide sequence of the mannanase gene, man, contained an open reading frame of 1.080 bp, which codes for a deduced 26 amino-acid signal peptide and a mature protein with the deduced molecular mass of 38 KDa. The man gene can both be expressed heterologously into the periplasm in plasmid pET22b(+) containing intact signal peptide (pET-NdeI18) and the pelB signal peptide of the pET22b(+)vector (pET-NcoI3). The Escherichia coli BL21 (DE3) containing pET-NcoI3 secreted about twice as much mannanase as that harboring pET-NdeI18. In E. coli DH5alpha, expression of man was under the control of the lac promoter in the pRK415 vector and was much more effective when the Shine-Dalgamo (SD) sequence was changed from GGGGAG to AAGGAG and the start codon was changed from TTG to ATG, respectively. These results suggest that genetic modification of the SD sequence and start codon is practical for high-level expression of mannanase in different bacterial strains.
Zhang
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Yan
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Zhang
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Tang
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D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D018387:Codon, Initiator / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D021382:Protein Sorting Signals / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D044902:beta-Mannosidase / Q000096:biosynthesis / Q000235:genetics*
D001426:Bacterial Proteins; D018387:Codon, Initiator; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D044902:beta-Mannosidase