title: High-level expression of nattokinase in Bacillus licheniformis by manipulating signal peptide and signal peptidase.
abstract: OBJECTIVE
  Nattokinase is an enzyme produced by Bacillus licheniformis and has potential to be used as a drug for treating cardiovascular disease due to its beneficial effects of preventing fibrin clots etc. However, the low activity and titre of this protein produced by B. licheniformis often hinders its application of commercial production. The aim of this work is to improve the nattokinase production by manipulating signal peptides and signal peptidases in B. licheniformis.
  
  
  RESULTS
  The P43 promoter, amyL terminator and AprN target gene were used to form the nattokinase expression vector, pHY-SP-NK, which was transformed into B. licheniformis and nattokinase was expressed successfully. A library containing 81 predicted signal peptides was constructed for nattokinase expression in B. licheniformis, with the maximum activity being obtained under the signal peptide of AprE. Among four type I signal peptidases genes (sipS, sipT, sipV, sipW) in B. licheniformis, the deletion of sipV resulted in a highest decrease in nattokinase activity. Overexpression of sipV in B. licheniformis led to a nattokinase activity of 35·60 FU ml(-1) , a 4·68-fold improvement over activity produced by the initial strain.
  
  
  CONCLUSIONS
  This work demonstrates the potential of B. licheniformis for industrial production of nattokinase through manipulation of signal peptides and signal peptidases expression.
  
  
  CONCLUSIONS
  This study has screened the signal peptides of extracellular proteins of B. licheniformis for nattokinase production. Four kinds of Type I signal peptidases genes have been detected respectively in B. licheniformis to identify which one played the vital role for nattokinase production. This study provided a promising strain for industry production of nattokinase.
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, College of Life Sciences, Hubei University, Wuhan, China.|State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China.
authors.forename: D
authors.initials: D
authors.lastname: Cai
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China.|College of Food Science and Technology, Huazhong Agricultural University, Wuhan, China.
authors.forename: X
authors.initials: X
authors.lastname: Wei
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, College of Life Sciences, Hubei University, Wuhan, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Qiu
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China.
authors.forename: J
authors.initials: J
authors.lastname: Chen
authors.affiliation: College of Food Science and Technology, Huazhong Agricultural University, Wuhan, China.|Department of Food Science and Human Nutrition, Iowa State University, Ames, IA, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Wen
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, College of Life Sciences, Hubei University, Wuhan, China.|State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China.
authors.forename: S
authors.initials: S
authors.lastname: Chen
mesh_terms: D000069976:Bacillus licheniformis / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics; D005343:Fibrinolytic Agents / Q000378:metabolism; D005822:Genetic Vectors; D007218:Industrial Microbiology; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases; D013381:Subtilisins / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005343:Fibrinolytic Agents; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D013381:Subtilisins; C026515:type I signal peptidase; C053771:nattokinase
keywords: Bacillus licheniformis; nattokinase; secretion; signal peptidase; signal peptide