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

AIM: To observe effects of shear stress and TNF-? on caveolin-1 expression. METHODS: Cultured human aortic endothelial cells (HAECs) of passage 3-5 were used in the experiment. Cells were exposed to a laminar flow (shear stress 1.0 Pa) by using a parallel rectangular flow chamber for different time. Caveolin-1 mRNA and protein expression were measured by RT-PCR and Western blot, respectively. Caveolin-1 expression of the cells stimulated by TNF-? were also studied to elucidate the influence of this inflammatory factor. RESULTS: After 24 h of exposure to 1.0 Pa shear stress, both of caveolin-1 protein and mRNA expression decreased in HAECs, especially caveolin-1 mRNA expression (P