Expression of Synthetic Gene Encoding Acidic, Thermostable Endo-1,4-β-Xylanase for Application in Feed Industry

Abstract

      Xylanase is widely used as feed additive to reduce viscosity and to improve digestibility. Thermostability and activity at low pH of feed grade xylanase are important characteristics that allow it to withstand pelleting process at high temperature and to remain active in acidic environment of animal stomach. This study was aimed at producing recombinant xylanase for application in feed industry. The GenBank sequence FJ212324 of 636 nucleotides encoding endo-1,4-β-xylanase Xyl11B from Bispora sp. MEY-1 was optimized for codon usage in Pichia pastoris and synthesized by GenScript. The synthetic sequence was inserted to pPICZαA and transformed into P. pastoris X33 for heterologous extracellular enzyme production. Recombinant P. pastoris X33 strain showed xylanase activity of 96 IU.ml^-1 after 10 days of cultivation using flask culture in mineral medium with methanol as sole carbon source. The purified enzyme had a molecular weight of 30 kDa. Endo-1,4-β-xylanase was most active at 65^oC and pH 3.0. It was thermostable and retained 90% of activity after 20 min of incubation at 70^oC. Recombinant enzyme retained more than 90% activity at a wide range of pH from 1 to 8.5. The enzyme was resistant to the proteolytic activity of pepsin. The enzyme activity was inhibited by the presence of SDS. Metal ions, such as Mn²⁺ , Cu²⁺ negatively affected the enzyme activity. Endo-1,4-β-xylanase obtained met the important requirements for feed grade enzyme. With high level of extracellular enzyme production. The recombinant P. pastoris strain could be a potential candidate for industrial application.