title: Cloning, expression and characterization of a pectate lyase from Paenibacillus sp. 0602 in recombinant Escherichia coli.
abstract: BACKGROUND
  Biotechnological applications of microbial pectate lyases (Pels) in plant fiber processing are considered as environmentally friendly. As such, they become promising substitutes for conventional chemical degumming process. Since applications of Pels in various fields are widening, it is necessary to explore new pectolytic microorganisms and enzymes for efficient and effective usage. Here, we describe the cloning, expression, characterization and application of the recombinant Pel protein from a pectolytic bacterium of the genus Paenibacillus in Escherichia coli.
  
  
  RESULTS
  A Pel gene (pelN) was cloned using degenerate PCR and inverse PCR from the chromosomal DNA of Paenibacillus sp. 0602. The open reading frame of pelN encodes a 30 amino acid signal peptide and a 445 amino acid mature protein belonging to the polysaccharide lyase family 1. The maximum Pel activity produced by E. coli in shake flasks reached 2,467.4 U mL⁻¹, and the purified recombinant enzyme exhibits a specific activity of 2,060 U mg⁻¹ on polygalacturonic acid (PGA). The maximum activity was observed in a buffer with 5 mM Ca²⁺ at pH 9.8 and 65°C. PelN displays a half-life of around 9 h and 42 h at 50°C and 45°C, respectively. The biochemical treatment achieved the maximal reduction of percentage weight (30.5%) of the ramie bast fiber.
  
  
  CONCLUSIONS
  This work represents the first study that describes the extracellular expression of a Pel gene from Paenibacillus species in E. coli. The high yield of the extracellular overexpression, relevant thermostability and efficient degumming using combined treatments indicate its strong potential for large-scale industrial production.
authors.forename: Xiaoman
authors.initials: X
authors.lastname: Li
authors.forename: Huilin
authors.initials: H
authors.lastname: Wang
authors.forename: Cheng
authors.initials: C
authors.lastname: Zhou
authors.affiliation: National Engineering Laboratory for Industrial Enzymes and Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China. mayanhe@im.ac.cn.
authors.forename: Yanhe
authors.initials: Y
authors.lastname: Ma
authors.forename: Jian
authors.initials: J
authors.lastname: Li
authors.forename: Jiangning
authors.initials: J
authors.lastname: Song
mesh_terms: D000595:Amino Acid Sequence; D032402:Boehmeria; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D056507:Paenibacillus / Q000201:enzymology* / Q000235:genetics; D011133:Polysaccharide-Lyases / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D013379:Substrate Specificity; D013783:Textile Industry
chemical_list: D004269:DNA, Bacterial; D011994:Recombinant Proteins; D011133:Polysaccharide-Lyases; C023911:pectate lyase