Heterelogous expression of Kluyveromyces marxianus inulinase gene and optimization of reaction parameters for oligofructose preparation from inulin

Abstract:

[Objective] Oligofructose is a promising food additive and health product. Enzymatically hydrolyzing the inulin by inulinase to produce oligofructoses was regarded as a promising, green and environment friendly technics. The aim of this study is to achieve an efficient production of Kluyveromyces marxianus inulinase and optimize the reaction parameters for oligofructose production from inulin. [Methods] The inulinase gene was cloned from K. marxianus strain CICC 1726 and expressed in Pichia pastoris cells. The reaction parameters such as pH, temperature, metal ions, substrate concentration and the amount of enzyme on the hydrolyzing of inulin to produce oligofructose were optimized. [Results] The inulinase activity of Pichia recombinants in 10 L fermenter reached 1 570 U/mL and the protein content in fermentation broth reached 2.75 g/L. In a 1 L reaction volume, when the parameters were pH 5.0, tempreture 50 °C, 0.2 mmol/L Mg2+ and 8% substrate, the inulin was properly hydrolyzed, with the content of C1 and C2 sugar was 9.25% and the oilgofructose (C3?C8) was 90.75% in the hydrolysates, among which the content of C3–C5 fraction was 72.92%. [Conclusion] This study has realized high-level heterologous expression of inulinase and facilitated the bulk preparation of oligofructose from inulin.

Key words:Inulinase, Oligofructose, High-level expression, Parameter optimization