Cloning and expression of Asperguillus endo-chitosanase gene inEscherichia coli
10.3969/j.issn.2095-4344.2014.34.014
- VernacularTitle:曲霉菌内切型壳聚糖酶基因克隆及在大肠杆菌中的表达
- Author:
Huading LU
;
Liyi LIAN
;
Mingwei CHEN
;
Yuhu DAI
- Publication Type:Journal Article
- Keywords:
biocompatible materials;
chitosan;
genes
- From:
Chinese Journal of Tissue Engineering Research
2014;(34):5490-5496
- CountryChina
- Language:Chinese
-
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.
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.
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
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.
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
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
temperature of 30-50℃.
