To improve the inulinase application in biotechnology, the characteristic of inulinase from Kluyveromyces marxianus YX01 was investigated. The inu gene of K. marxianus YX01 was transformed into Pichiapastoris GS115 host cells with molecular biology techniques. Then we achieved the heterologous expression of exo-inulinase whose molecular mass was about 86.0 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Furthermore, six His-tag was added to the inulinase and a two-step method was applied in the purification of inulinase, including concentration via dialysis by polyethylene glycol 20 000 and metal Ni-NTA Agarose affinity adsorption. The purification factor of purified protein was 3.6 and the recovery rate of enzyme activity was 33.1%. We characterized the purified inulinase. The optimum temperature was 60 degrees C and pH was 4.62. When inulin and sucrose were used as substrates, the K(m) and V(max) values were 80.53 g/L vs 4.49 g/(L x min) and 183.10 g/L vs 20.20 g/(L x min), respectively. In addition, metal ions including Mn2+, Ca2+, Cu2+, Zn2+ and Fe2+ exhibited different degrees of inhibition on the enzyme activity, and Cu2+, Zn2+ and Fe2+ exhibited the most significant inhibition. Our findings might lay a good foundation for industrial application of inulinase.
Glycoside Hydrolases ; chemistry ; genetics ; Industrial Microbiology ; Inulin ; Kluyveromyces ; enzymology ; genetics ; Pichia ; Sucrose ; Temperature

Kluyveromyces marxianus, as unconventional yeast, attracts more and more attention in the biofuel fermentation. Although this sort of yeasts can ferment pentose sugars, the fermentation capacity differs largely. Xylose and arabinose fermentation by three K. marxianus strains (K. m 9009, K. m 1911 and K. m 1727) were compared at different temperatures. The results showed that the fermentation performance of the three strains had significant difference under different fermentation temperatures. Especially, the sugar consumption rate and alcohol yield of K. m 9009 and K. m 1727 at 40 ℃ were better than 30 ℃. This results fully reflect the fermentation advantages of K. marxianus yeast under high-temperature. On this basis, five genes (XR, XDH, XK, AR and LAD) coding key metabolic enzymes in three different yeasts were amplified by PCR, and the sequence were compared by Clustalx 2.1. The results showed that the amino acid sequences coding key enzymes have similarity of over 98% with the reference sequences reported in the literature. Furthermore, the difference of amino acid was not at the key site of its enzyme, so the differences between three stains were not caused by the gene level, but by transcribed or translation regulation level. By real-time PCR experiment, we determined the gene expression levels of four key enzymes (XR, XDH, XK and ADH) in the xylose metabolism pathway of K. m 1727 and K. m 1911 at different fermentation time points. The results showed that, for thermotolerant yeast K. m 1727, the low expression level of XDH and XK genes was the main factors leading to accumulation of xylitol. In addition, according to the pathway of Zygosaccharomyces bailii, which have been reported in NCBI and KEGG, the xylose and arabinose metabolic pathways of K. marxianus were identified, which laid foundation for further improving the pentose fermentation ability by metabolic engineering.