title: Cloning, expression and characterization of a cold-adapted endo-1, 4-β-glucanase fromCitrobacter farmeriA1, a symbiotic bacterium ofReticulitermes labralis.
abstract: BACKGROUND
  Many biotechnological and industrial applications can benefit from cold-adapted EglCs through increased efficiency of catalytic processes at low temperature. In our previous study,Citrobacter farmeriA1 which was isolated from a wood-inhabiting termiteReticulitermes labraliscould secrete a cold-adapted EglC. However, its EglC was difficult to purify for enzymatic properties detection because of its low activity (0.8 U/ml). The objective of the present study was to clone and express theC. farmeri EglCgene inEscherichia colito improve production level and determine the enzymatic properties of the recombinant enzyme.
  
  
  METHODS
  TheEglCgene was cloned fromC. farmeriA1 by thermal asymmetric interlaced PCR.EglCwas transformed into vector pET22b and functionally expressed inE. coli. The recombination protein EglC22b was purified for properties detection.
  
  
  RESULTS
  SDS-PAGE revealed that the molecular mass of the recombinant endoglucanase was approximately 42 kDa. The activity of theE. colipET22b-EglC crude extract was 9.5 U/ml. Additionally, it was active at pH 6.5-8.0 with an optimum pH of 7.0. The recombinant enzyme had an optimal temperature of 30-40 °C and exhibited >50% relative activity even at 5 °C, whereas it lost approximately 90% of its activity after incubation at 60 °C for 30 min. Its activity was enhanced by Co2+and Fe3+, but inhibited by Cd2+, Zn2+, Li+, Triton X-100, DMSO, acetonitrile, Tween 80, SDS, and EDTA.
  
  
  CONCLUSIONS
  These biochemical properties indicate that the recombinant enzyme is a cold-adapted endoglucanase that can be used for various industrial applications.
authors.affiliation: Institute of Ensiling and Processing of Grass, Nanjing Agricultural University , Nanjing , China.
authors.forename: Xi
authors.initials: X
authors.lastname: Bai
authors.affiliation: Institute of Ensiling and Processing of Grass, Nanjing Agricultural University , Nanjing , China.
authors.forename: Xianjun
authors.initials: X
authors.lastname: Yuan
authors.affiliation: College of Animal Science, University of Science and Technology of Anhui , Fengyang , China.
authors.forename: Aiyou
authors.initials: A
authors.lastname: Wen
authors.affiliation: Institute of Ensiling and Processing of Grass, Nanjing Agricultural University , Nanjing , China.
authors.forename: Junfeng
authors.initials: J
authors.lastname: Li
authors.affiliation: Institute of Agricultural Resource and Environment, Jiangsu Academy of Agricultural Sciences , Nanjing , China.
authors.forename: Yunfeng
authors.initials: Y
authors.lastname: Bai
authors.affiliation: Institute of Ensiling and Processing of Grass, Nanjing Agricultural University , Nanjing , China.
authors.forename: Tao
authors.initials: T
authors.lastname: Shao
keywords: Citrobacter farmeri; Cold-adapted; Endoglucanase; Escherichia coli; Expression; Properties