title: The alkaline pectate lyase PEL168 of Bacillus subtilis heterologously expressed in Pichia pastoris is more stable and efficient for degumming ramie fiber.
abstract: BACKGROUND
  The conventional degumming process of ramie with alkaline treatment at high temperature causes severe environmental pollution. Pectate lyases can be used to remove pectin from ramie in a degumming process with reduced environmental pollution and energy consumption. Pectate lyase PEL168 from Bacillus subtilis has been previously characterized and the protein structure was resolved. However, Bacillus is not a suitable host for pectate lyases during the degumming process since most Bacillus produce cellulases endogenously with a detrimental effect to the fiber. Pichia pastoris, which does not express endogenous cellulases and has high secretion capability, will be an ideal host for the expression. No previous work was reported concerning the heterologous expression of pectate lyase PEL168 in P. pastoris with an aim for industrial application in ramie bio-degumming.
  
  
  RESULTS
  The gene pel168 was expressed in P. pastoris in this study. The recombinant protein PEL168 in P. pastoris (PEL168P) showed two bands of 48.6 kDa and 51.4 kDa on SDS-PAGE whereas the enzyme expressed in E. coli (PEL168E) was the same as predicted with a band of 46 kDa. Deglycosylation digestion suggested that PEL168P was glycosylated. The optimum reaction temperature of the two PEL168s was 50°C, and the optimum pH 9.5. After preincubation at 60°C for 20 min, PEL168E completely lost its activity, whereas PEL168P kept 26% of the residual activity. PEL168P had a specific activity of 1320 U/mg with a Km of 0.09 mg/ml and a Vmax of 18.13 μmol/min. K⁺, Li⁺, Ni²⁺ and Sr²⁺ showed little or no inhibitory effect on PEL168P activity, and Ca²⁺ enhanced enzyme activity by 38%. PEL168P can remove the pectin from ramie effectively in a degumming process. A 1.5 fold increase of PEL168 enzyme expression in P. pastoris was achieved by further codon optimization.
  
  
  CONCLUSIONS
  Pectate lyase PEL168 with an available protein structure can be heterologously expressed in P. pastoris. The characterized recombinant PEL168P can be used to remove pectin from ramie efficiently and the expression level of PEL168 in P. pastoris was increased markedly by codon optimization. Therefore, PEL168 is an ideal candidate for further optimization and engineering for bio-degumming.
authors.affiliation: College of Life Sciences, Hubei University, Wuhan 430062, China.
authors.forename: Chengjie
authors.initials: C
authors.lastname: Zhang
authors.forename: Jia
authors.initials: J
authors.lastname: Yao
authors.forename: Cheng
authors.initials: C
authors.lastname: Zhou
authors.forename: Liangwei
authors.initials: L
authors.lastname: Mao
authors.forename: Guimin
authors.initials: G
authors.lastname: Zhang
authors.forename: Yanhe
authors.initials: Y
authors.lastname: Ma
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D032402:Boehmeria / Q000737:chemistry / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D003001:Cloning, Molecular; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D010368:Pectins / Q000378:metabolism*; D010843:Pichia / Q000378:metabolism*; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D007477:Ions; D008670:Metals; D011994:Recombinant Proteins; D010368:Pectins; D011133:Polysaccharide-Lyases; C023911:pectate lyase; D002118:Calcium