Biotechnology Bulletin ›› 2014, Vol. 0 ›› Issue (6): 181-186.

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Cloning,Expression of Thermostable β-mannanase and the Preparation of Mannooligosaccharide

Ni Yujia1,Zhou Minyu2,Ouyang Jia1,3,Zheng Zhaojuan1,Yong Qiang1   

  1. (1. College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037;2. College of Forest Resource & Environment, Nanjing Forestry University, Nanjing 210037;3. Jiangsu Key Lab of Biomass-based Green Fuels and Chemicals, Nanjing 210037)
  • Received:2013-11-20 Online:2014-06-25 Published:2014-06-25

Abstract: According to the Pichia pastoris’s codon preference, a DNA sequence encoding Aspergillus niger BK01 thermophilic β-mannanase gene was designed and synthesized. Firstly, it was inserted into pPICZαA and resulted in recombinant expression vector pPICZαA-man. Then, pPICZαA-man was linearized and transformed into different hosts by electrotransformation. An optimal recombinant stain KM71-MAN was obtained by screening activity. Using recombinant strain KM71-MAN, recombinant mannanase was overexpressed and its activity in the culture medium reached 2 318.85 IU/mL in a 3 L fermentor. Recombinant enzyme had an apparent molecular size of about 40 kD by SDS-PAGE, and optimal activity at pH 5.0 and 80℃. It was highly thermostable, retaining 43% of enzyme activity after 44 h of exposure at 70℃ and pH 5.0. Moreover, it remained over 85% activity from pH 3.0 to pH 7.0 after treating at 50℃ for 70 h. Using this crude enzyme, the main hydrolysis products yielded from konjak gum were mannobiose and mannohexaose and the yield of mannooligosaccharides was 55.6%. The recombinant enzyme exhibited good thermal and pH stability, which indicated that the recombinant yeast has potential value in preparation of konjac gum mannooligosaccharides.

Key words: β-mannanase, Codon preference, Pichia pastoris, Enzymatic properties, Mannooligosaccharide