Abstract:[Objective] We overexpressed β-xylosidase with heterologous expression by constructing a recombinant Pichia pastoris. [Methods] According to the codon usage frequency of highly expressed genes in P. pastoris, the Thermomyces lanuginosus β-xylosidase (Xyl43) gene was optimized and expressed in P. pastoris GS115, followed by characterization of Xyl43. Then, single factor experiments were used to optimize the fermentation conditions, in a 5-L fermenter. [Results] The optimized Xyl43 gene changed greatly with 78.17% of the sequence homology, and the GC content reduced from 52.8% to 44.6% with 222 bases substituted. The optimized gene was transplanted with an expression vector pPIC9K-OptXyl43 into P. pastoris GS115 to produce transformants. Then, a high xylosidase activity secreting recombinant P. pastoris GS115-Xyl43 was selected from the transformants on G-418 resistant plates, followed by shake flask cultivation. Basic enzyme properties of the recombinant xylosidase was analyzed as below: the protein molecular weight 51.5 kD, the optimal reaction temperature 55 °C, the optimum pH 7.0, and kinetic parameters Km=2.93 mmol/L and Vmax=157.9 μmol/(min·mg). β-xylosidase fermentation was optimized in shake flask as follows: methanol supply 1% (each 24 h), shaking speed 250 r/min, incubation time 144 h, incubation temperature 28 °C and initial pH 6.0. Under the optimal condition, the extracellular enzyme activity reached 42 U/mL with a protein content of 0.54 g/L. Further, in a 5-L fermenter, P. pastoris GS115-Xyl43 achieved 222.2 U/mL of xylosidase at 156 h (methanol induction for 96 h), with protein concentration at 2.36 g/L, which was 4.3 fold more than that in the shake-flask fermentation. [Conclusion] β-xylosidase can be expressed in P. pastoris GS115 with high level production and can be used as a candidate in various industrial applications.
