Improvement of expression level of keratinase Sfp2 from Streptomyces fradiae by site-directed mutagenesis of its N-terminal pro-sequence

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• 作者：Junxia Li (1)
  Dongdong Chen (1)
  Zhanqiao Yu (1)
  Longmei Zhao (1)
  Rijun Zhang (1)
  
• 关键词：Expression level ; Keratinase ; Pro ; region ; Site ; directed mutagenesis ; Streptomyces fradiae
• 刊名：Biotechnology Letters
• 出版年：2013
• 出版时间：May 2013
• 年：2013
• 卷：35
• 期：5
• 页码：743-749
• 全文大小：219KB
• 参考文献：1. Brandelli A (2008) Bacterial keratinases: useful enzymes for bioprocessing agroindustrial wastes and beyond. Food Bioproc Tech 1:105-16 CrossRef
  2. Bressollier P, Letourneau F, Urdaci M, Verneuil B (1999) Purification and characterization of a keratinolytic serine proteinase from / Streptomyces albidoflavus. Appl Environ Microbiol 65:2570-576
  3. Fang N, Zhong CQ, Liang X, Tang XF, Tang B (2010) Improvement of extracellular production of a thermophilic subtilase expressed in / Escherichia coli by random mutagenesis of its / N-terminal propeptide. Appl Microbiol Biotechnol 85:1473-481 CrossRef
  4. Fu X, Inouye M, Shinde U (2000) Folding pathway mediated by an intramolecular chaperone. J Biol Chem 275:16871-6878 CrossRef
  5. Jaouadi B, Aghajari N, Haser R, Bejar S (2010) Enhancement of the thermostability and the catalytic efficiency of / Bacillus pumilus CBS protease by site-directed mutagenesis. Biochimie 92:360-69 CrossRef
  6. Kieser T, Hopwood DA, Wright HM, Thompson CJ (1982) pIJ101, a multi-copy broad host-range / Streptomyces plasmid: functional analysis and development of DNA cloning vectors. Mol Genet Genomic 185:223-38 CrossRef
  7. Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA (2000) Practical / Streptomyces genetics. John Innes Foundation, Norwich
  8. Kitadokoro K, Tsuzuki H, Okamoto H, Sato T (1994) Crystal structure analysis of a serine proteinase from / Streptomyces fradiae at 0.16?nm resolution and molecular modeling of an acidic-amino-acid-specific proteinase. Eur J Biochem 224:735-42 CrossRef
  9. Li J, Shi PJ, Zhang WZ, Han XY, Xu LL, Zhang HT, Yao B, Fan YL (2005) Gene cloning and expression of serine protease Sfp2 from / Streptomyces fradiae var.k11. Chin J Biotechnol 21:782-88 In Chinese
  10. Li J, Shi PJ, Han XY, Meng K, Yang PL, Wang YR, Luo HY, Wu NF, Yao B, Fan YL (2007) Functional expression of the keratinolytic serine protease gene / sfp2 from / Streptomyces fradiae var. k11 in / Pichia pastoris. Protein Expr Purif 54:79-6 CrossRef
  11. Mitsuiki S, Ichikawa M, Oka T, Sakai M, Moriyama Y, Sameshima Y, Goto M, Furukawa K (2004) Molecular characterization of a keratinolytic enzyme from an alkaliphilic / Nocardiopsis sp. TOA-1. Enzyme Microbiol Tech 34:482-89 CrossRef
  12. Pulido MA, Tanaka S, Sringiew C, You DJ, Matsumura H, Koga Y, Takano K, Kanaya S (2007) Requirement of left-handed glycine residue for high stability of the Tk-subtilisin propeptide as revealed by mutational and crystallographic analyses. J Mol Biol 374:1359-373 CrossRef
  13. Sharma R, Murty NAR, Gupta R (2011) Molecular characterization of / N-terminal pro-sequence of keratinase Ker P from / Pseudomonas aeruginosa: identification of region with chaperone activity. Appl Biochem Biotech 165:892-01 CrossRef
  14. Sidhu SS, Kalmar GB, Willis LG, Borgford TJ (1995) Protease evolution in / Streptomyces griseus. J Biol Chem 270:7594-600 CrossRef
  15. Takahashi M, Hasuura Y, Nakamori S, Takagi H (2001) Improved autoprocessing efficiency of mutant subtilisins E with altered specificity by engineering of the pro-region. J Biochem 130:99-06 CrossRef
  16. Yabuta Y, Takagi H, Inouye M, Shinde U (2001) Folding pathway mediated by an intramolecular chaperone. J Biol Chem 276:44427-4434 CrossRef
  
• 作者单位：Junxia Li (1)
  Dongdong Chen (1)
  Zhanqiao Yu (1)
  Longmei Zhao (1)
  Rijun Zhang (1)
  
  1. State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, 100193, China
  
• ISSN：1573-6776

文摘

The keratinase Sfp2, produced by Streptomyces fradiae var. k11, is a serine alkaline protease first synthesized as pre-pro-mature precursor, of which the N-terminal propeptide must be autocatalytically cleaved on the C-terminal of P1 amino acid to produce mature enzyme. Single amino acid substitutions were introduced at positions ? and ? to improve the expression level of mature Sfp2. The specific activity of L(?)F mutant (48935 U/mg) was nine times that of wild-type Sfp2, whereas the mutants L(?)D, L(?)G, L(?)H, K(?)E, and K(?)L had 2-2?% of the specific activity of wild-type. The yield of mature Sfp2 of L(?)F mutant was estimated to be 800?μg/mg total protein and 112?mg/l culture supernatant, nine and twice that of wild-type, respectively. The L(?)F mutant exhibited similar enzymatic properties to wild-type.