کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
18009 42710 2007 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Inducible and constitutive expression of glutaryl-7-aminocephalosporanic acid acylase by fusion to maltose-binding protein
موضوعات مرتبط
مهندسی و علوم پایه مهندسی شیمی بیو مهندسی (مهندسی زیستی)
پیش نمایش صفحه اول مقاله
Inducible and constitutive expression of glutaryl-7-aminocephalosporanic acid acylase by fusion to maltose-binding protein
چکیده انگلیسی
Glutaryl-7-aminocephalosporanic acid acylase (GL-7-ACA acylase, GLA) from Pseudomonas was expressed by E.coli BL21(DE3)/pET-Acy grown in a 5 L fermentor. The activity accumulation of GLA stopped at 11 h due to the formation of the excessive insoluble and inactive precursor. Therefore, maltose-binding protein (MBP) was introduced to improve the solubility of the target enzyme. Recombinant E. coli TB1/pMAL-Acy and TB1/pMKL-Acy, in which the MBP fusion GLA (MBP-GLA) were inducible expression, as well as recombinant E. coli TB1/pMKC-Acy and TB1/pMKC-AS, in which MBP-GLA were constitutive expression by deleting the gene lacIq, were constructed to assess the function of MBP, respectively. Results showed that the ratio of soluble GLA to total GLA increased from approximately 15-60% to 75-100%, when E. coli BL21 (DE3)/pET-Acy was substituted by TB1/pMKL-Acy grown at 37-28 °C. Meanwhile, MBP-GLA of almost completely soluble form could be harvested in both constitutive expression strains E. coli TB1/pMKC-Acy and TB1/pMKC-AS grown at 28 °C. It can be inferred that MBP-GLAs are more soluble than single GLA in both inducible and constitutive expression. Further researches on constitutive expression of MBP-GLA were performed and the optimal activity of acylase in recombinant E. coli TB1/pMKC-AS reached 4682 U/L at 28 °C in a 5 L fermentor after 31 h culture, in the absence of any inducer. The productivity of the functional active fusion GLA reached as high as 151.0 U/(L h), which is very promising in the industrial enzymatic production of 7-aminocephalosporanic acid.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Enzyme and Microbial Technology - Volume 40, Issue 4, 5 March 2007, Pages 555-562
نویسندگان
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