FOOD SCIENCE ›› 2020, Vol. 41 ›› Issue (18): 113-119.doi: 10.7506/spkx1002-6630-20190805-058

• Bioengineering • Previous Articles     Next Articles

Heterologous Expression of Codon Optimized Thermophilic β-Mannanase Gene (manBK) and Its Application in Degradation of Konjac Mannan

LU Haiqiang, GU Xinxi, YUAN Qiaomin, TAN Suhui, TIAN Hongtao   

  1. (College of Food Science and Technology, Hebei Agricultural University, Baoding 071000, China)
  • Online:2020-09-25 Published:2020-09-18

Abstract: In this study, we optimized the codon usage of the thermophilic β-mannanase gene (manBK) and the fermentation conditions to increase its expression in Pichia pastoris. The enzymatic properties of ManBK were investigated and the effect of metal ions on the enzymatic stability was explored as well as its application potential in enzymatic hydrolysis of konjac mannan. Compared with the wild type manBK gene, 242 base pairs were modified after the optimization, and the GC content decreased to 40%–53%, on average 47%. The codon adaptation index (CAI) was 0.87. The optimized synthetic sequence showed a 76% homology to the wild-type gene, which was successfully expressed in P. pastoris GS115 using the constitutive expression vector pPIC9K. The ManBK activity attained 22 U/mL after a 72-hour incubation at 25 ℃. The ManBK had a molecular mass of approximately 45 kDa. The optimum temperature and pH value for the enzyme activity were 80 ℃ and pH 5.0, respectively. The kinetic parameters Km and Vmax of ManBK were found to be 1.40 mg/mL and 149.25 μmol/(min·mg), respectively. The enzyme was strongly activated by Fe3+ (32%), Ca2+ (52%), Cu2+ (23%) and Zn2+ (459%) but inhibited by K+, Li+, Mn2+and Cu2+. In addition, Zn2+ enhanced the thermal stability of the enzyme, which prolonged the half-life time by 116 min at 70 ℃. The optimal conditions for enzymatic hydrolysis of konjac flour were determined as follows: reaction time 1.5 h, reaction temperature 70 ℃, pH 5.0, and E/S ratio 1:1. The antioxidant activity of the konjac degradation products was 6.6 times as high as that of konjac flour. In conclusion, The ManBK has a great potential application in the preparation of konjac oligosaccharide.

Key words: mannanase; konjac degradation products; codon optimization; thermal stability; antioxidant activity

CLC Number: