title: [Expression and secretion regulation of Bacillus naganoensis pullulanase in recombinant Escherichia coli].
abstract: OBJECTIVE
  To increase extracellular productivityof pullulanase, pullulanase gene from Bacillus naganoensis JNB-1 was expressed in recombinant Escherichia coli, followed by optimizing induction conditions and applying chemical additives.
  
  
  METHODS
  We amplified pullulanase gene pul from B. naganoensis genome by PCR and constructed recombinant E. coli BL21/pET-20b-pul. Optimal induction conditions and additive parameters of glycine and Na+ were determined by measuring the extracellular pullulanase activity.
  
  
  RESULTS
  Pullulanase was expressed in E. coli with the molecular weight of 119 kDa. Under optimal induction conditions, i. e. induction was initiated with 0.4 mmol/L isopropyl beta-D-1-Thiogalactopyranoside (IPTG) at 20 degrees C when OD600 of bacteria culture reached 1.2, total pullulanase activity including intracellular and extracellular enzyme reached 10.8 U/mL. Addition of glycine and Na+ enhanced the secretion of pullulanase. With the supplementation of 0.08 mol/L glycine and 0.2 mol/L Na+, extracellular pullulanase activity was increased up to 8.1 U/mL, 10.3 times of that without additives.
  
  
  CONCLUSIONS
  A promising resource of pullulanase was achieved by construction of recombinant E. coli for industrial production of pullulanase, and additionally the efficient regulation method with chemical additives was developed for pullulanase secretion, which would also be useful for high-level extracellular production of recombinant enzymes.
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China. yanweihades@163.com
authors.forename: Wei
authors.initials: W
authors.lastname: Yan
authors.forename: Yao
authors.initials: Y
authors.lastname: Nie
authors.forename: Yan
authors.initials: Y
authors.lastname: Xu
mesh_terms: D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D058947:Bacterial Secretion Systems; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D021381:Protein Transport
chemical_list: D001426:Bacterial Proteins; D058947:Bacterial Secretion Systems; D006026:Glycoside Hydrolases; C100181:pullulanase