title: [High expression and characterization of N-acyl-homoserine lactonase-xylanase recombinant protein].
abstract: OBJECTIVE
  The fusion protein SL2B with both xylanase and N-acyl-homoserine lactonase activities was expressed in Pichia pastori. Characterization of the purified xylanase and N-acyl-homoserine lactonase fusion protein SL2B was investigated.
  
  
  METHODS
  The fusion gene sl2b was amplified from the N-acyl-homoserine lactonase gene aiiA-B546 and the xylanase gene xynAS27cd via overlap PCR technique. After the recombinant vector pPIC9/sl2b was transformed into P. pastoris, transformants with both xylanase and N-acyl-homoserine lactonase activity were screened. The purified SL2B was obtained with ammonium sulfate precipitation and molecular sieve. Both N-acyl-homoserine lactonase and xylanase activities of SL2B were characterized.
  
  
  RESULTS
  The purified SL2B showed that the xylanase had optimal pH and temperature at pH 6.5 and 60 degrees C, respectively. The enzyme was stable between pH 6.0 and 8.0, retained over 80% enzyme activity between 50 and 65 degrees C. It resisted various neutral proteases and chemical reagents. With oat spelt xylan as substrate, the Km value of SL2B was 2.9 mg/L. The N-acyl-homoserine lactonase had optimal pH and temperature at pH 8.0 and 30 degrees C, respectively. The enzyme was stable between pH 4.0 and 10.0, retained over 80% enzyme activity between 0 and 50 degrees C. It resisted various neutral proteases and chemical reagents. The fusion protein can hydrolyze many N-acyl homoserine lactones substrates. With N-(3-oxo-octanoyl)-L-homoserine lactone as substrate, the Km value of SL2B was 0.050 mmol/L.
  
  
  CONCLUSIONS
  High level expression is achieved by fusing N-acyl-homoserine lactonase to the xylanase.
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China. mei_chao_zhang@163.com
authors.forename: Meichao
authors.initials: M
authors.lastname: Zhang
authors.forename: Yanan
authors.initials: Y
authors.lastname: Cao
authors.forename: Bin
authors.initials: B
authors.lastname: Yao
authors.forename: Zhigang
authors.initials: Z
authors.lastname: Zhou
mesh_terms: D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000378:metabolism; D043364:Endo-1,4-beta Xylanases / Q000235:genetics* / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*
chemical_list: D011993:Recombinant Fusion Proteins; D002265:Carboxylic Ester Hydrolases; C432003:N-acyl homoserine lactonase; D043364:Endo-1,4-beta Xylanases