title: Engineering of a Bacillus amyloliquefaciens Strain with High Neutral Protease Producing Capacity and Optimization of Its Fermentation Conditions.
abstract: The neutral protease has high potential for industrial applications, and attempts to improve enzyme expression level have important application values. In the present study, a neutral protease-encoding gene, Banpr, was cloned from Bacillus amyloliquefaciens strain K11, and a genetic manipulation method specific for this difficult-to-transform strain was developed for the high-level expression of neutral protease. The recombinant plasmid pUB110-Banpr was constructed in Bacillus subtilis strain WB600 and then transformed into strain K11 under optimized conditions. A positive transformant 110N-6 with the highest protease secreting capacity on skim milk plates and great genetic stability for more than 100 generations was selected for further study. Optimization of the fermentation conditions increased the enzyme activity of strain 110N-6 to 8995 ± 250 U/ml in flask culture and 28084 ± 1282 U/ml in 15-l fermentor, which are significantly higher than that of the native strain K11 and industrial strain B. subtilis AS.1398, respectively. The high expression level and extreme genetic stability make B. amyloliquefaciens strain 110N-6 more favorable for mass production of neutral protease for industrial uses.
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, P. R. China.
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, P. R. China.
authors.forename: Lian
authors.initials: L
authors.lastname: Yang
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, P. R. China.
authors.forename: Yanhai
authors.initials: Y
authors.lastname: Ping
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, P. R. China.
authors.forename: Yingguo
authors.initials: Y
authors.lastname: Bai
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, P. R. China.
authors.forename: Huiying
authors.initials: H
authors.lastname: Luo
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, P. R. China.
authors.forename: Huoqing
authors.initials: H
authors.lastname: Huang
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, P. R. China.
authors.forename: Bin
authors.initials: B
authors.lastname: Yao
mesh_terms: D001407:Bacillus / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation / Q000235:genetics / Q000502:physiology*; D010447:Peptide Hydrolases / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids
chemical_list: D001426:Bacterial Proteins; D010447:Peptide Hydrolases