Cloning and expression of Asperguillus endo-chitosanase gene inEscherichia coli
Lu Hua-ding
Lian Li-yi
Chen Ming-wei
Dai Yu-hu
Abstract：BACKGROUND:Chitosanase is an enzyme for efficient and special hydrolysis of chitoan, and hence its effective and stable expression with enzymatic activity wil contribute to improving gene therapeutic effect. <br> OBJECTIVE: To construct a chitosanase gene for the efficient and specifical hydrolysis of chitosan, and to investigate its expression inEscherichia coli and the main influencing factors of enzymatic activity. <br> METHODS:According to the sequences of endo-chitosanase ofAspergilus sp. CJ22-326 provided in Genbank (EU302818), primers were designed and synthesized. The Asperguilus endo-chitosanase gene was amplified by successive extension PCR. And then the recombinant pET28a-His6-CSN was constructed and expressed in <br> Escherichia coli BL21. Finaly the recombinant His6-CSN fusion protein was analyzed by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE), the western blot and dinitrosalicylic acid assay for detecting the enzyme activity of eluted His6-CSN fusion protein. The influence of different pH value and temperature on the enzyme activity of the recombinant chitosanase was investigated. <br> RESULTS AND CONCLUSION: SDS-PAGE showed that 29 kDa proteins were expressed and the western blot assay showed that His6-CSN expressed successfuly in the host. Dinitrosalicylic acid assay determined the <br> enzymatic activity of His6-CSN was significantly higher than that of lysozyme, but lower than that of chitosanase from Streptomyces griseus (P < 0.05). The recombinant chitosanase displayed the maximal activity at temperature of 50℃ and pH value of 6.0. There were a higher enzymatic activity remaining at pH value of 4.0-7.0 and <br> temperature of 30-50℃.
Keywords：biocompatible materialschitosangenes
Publication Date：2014-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：7( 5490-5496 )