title: Improved extracellular expression and high-cell-density fed-batch fermentation of chitosanase from Aspergillus Fumigatus in Escherichia coli.
abstract: Chitosanase (CSN) from Aspergillus fumigatus has good thermal stability, wide pH range duration, and effective hydrolysis for chitosan. Inhere, CSN was successfully expressed in Escherichia coli followed by extracellular secretion under the guidance of an N-terminal signal peptide PelB, which effectively prompted its secretion out of E. coli cells. To facilitate its later purification, N-terminal or C-terminal 6xHis epitope tag was added to the PelB-CSN protein complex. Our results indicated that PelB-CSN without 6xHis-tag (PelB-CSN) or with N-terminal 6xHis-tag (PelB-CSN-N) can both be effectively secreted into the medium, while CSN with 6xHis-tag anchored at C-terminus was expressed as inclusion bodies. Process optimization strategies were further developed to improve the secretion efficiency of recombinant PelB-CSN-N in E. coli. Under the induction of 10 g/L lactose in shake-flask culture, the extracellular activity of CSN reached 6015 U/mL at 25 °C in TB medium containing 1 % glycine. Moreover, a fed-batch fermentation strategy for high-cell-density cultivation was applied in a 5-L fermenter, increasing the extracellular CSN activity to 14,000 U/mL in 2-day fermentation with the optimal addition of lactose and glycine.
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, The College of Life Science, Hubei University, Wuhan, 430062, People's Republic of China.
authors.forename: Liang
authors.initials: L
authors.lastname: Huang
authors.affiliation: Tianjin Institutes of Industrial Biotechnology, Chinese Academy of Science, Tianjin, 300308, People's Republic of China.
authors.forename: Qinhong
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, The College of Life Science, Hubei University, Wuhan, 430062, People's Republic of China.
authors.forename: Sijing
authors.initials: S
authors.lastname: Jiang
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, The College of Life Science, Hubei University, Wuhan, 430062, People's Republic of China. zyl609@sina.com.
authors.forename: Yuling
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, The College of Life Science, Hubei University, Wuhan, 430062, People's Republic of China. zhangguimin6@hotmail.com.
authors.forename: Guimin
authors.initials: G
authors.lastname: Zhang
authors.affiliation: Tianjin Institutes of Industrial Biotechnology, Chinese Academy of Science, Tianjin, 300308, People's Republic of China.
authors.forename: Yanhe
authors.initials: Y
authors.lastname: Ma
mesh_terms: D001232:Aspergillus fumigatus / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development*; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C019868:chitosanase
keywords: Chitosanase; Fed-batch fermentation; Secretory expression