title: [Heterologous expression, purification and characterization of exo-inulinase from Kluyveromyces marxianus YX01].
abstract: 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.
authors.forename: Yimin
authors.initials: Y
authors.lastname: Li
authors.forename: Jiaoqi
authors.initials: J
authors.lastname: Gao
authors.forename: Wenjie
authors.initials: W
authors.lastname: Yuan
authors.forename: Ruijuan
authors.initials: R
authors.lastname: Xiang
authors.forename: Shengbo
authors.initials: S
authors.lastname: Hou
mesh_terms: D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics; D007218:Industrial Microbiology; D007444:Inulin; D007716:Kluyveromyces / Q000201:enzymology* / Q000235:genetics; D010843:Pichia; D013395:Sucrose; D013696:Temperature
chemical_list: D013395:Sucrose; D007444:Inulin; D006026:Glycoside Hydrolases; C023086:inulinase