Studies On Alkaliphilic Mechnism Of Alkaliphilic β-Mannanase From Alkaliphilic Bacillus SP.16-5

The mechanism of high alkali-tolerant proteins' stability is a hot topic currently, but owing to complex nature of the mechanism, there are few reports about it The mechanism of high alkali-tolerant β-mannanase from alkaliphilic Bacillus sp N16-5 was studied in this research work, and the mechanism was learned partly from our work.The gene of alkali-tolerant β-mannanase was subcloned from a large plasmid containing the gene, and expressed the gene in E.coli, which is the basic work for selecting mutants by error-prone PCR.Error-prone PCR was performed by using standard PCR buffer with CT-rich dNTP mixtures and high concentration Mg²⁺. The high-through selections were performed to select a large number of mutants. A mutant with optimum pH change from 9.5 to 8.5 was selected, and its gene was sequenced and compared with the original one, the change was due to two amino acid substitutions, from A to V and from T to A. In order to determine which substitution is the reason for the change, chimera of the single substitution was constructed, the result was that the substitution of A to V determined the change of optimum pH; Two helixes in the surface of the enzyme was disappeared in the mutant by predicting and comparing the three-dimension structures of the original and mutant β -mannanase, and they were important for stability of the enzyme under alkali conditions.The sequence of the gene was also compared with other genes of β-mannanase, and the unique characteristic giving the reason of the alkali-tolerant mechanism of the enzyme was not found.