黑曲霉内切菊粉酶基因在毕赤酵母中的表达及应用研究

利用PCR方法从黑曲霉（Aspergillus niger）基因组DNA中扩增内切菊粉酶（endo I）全长基因（1551 bp）,经序列分析后将其连接到表达载体p PIC9K上,得到重组表达载体p PIC9K-endo I。重组质粒经Sac I线性化并电转化到毕赤酵母GS115,阳性转化子进行发酵产酶后考察其酶学性质。酶学性质研究表明,重组酶的最适p H和最适温度分别为5和60℃,重组酶在55℃下保温9 h后,重组菊粉内切酶仍残余83.2%的活性,表现出极高的热稳定性,Cu²⁺、Ag⁺对重组酶有明显的抑制作用。对内切菊粉酶水解菊粉的过程研究表明,重组内切菊粉酶能在8 h内将4%（w/v）菊粉水解为3⁶个聚合度的低聚果糖,水解11 h后,低聚果糖得率达到63.5%,本研究为进一步开发以菊粉为原料生产低聚果糖的应用奠定了基础。 

Overexpression of the endo-inulinase gene from Aspergillus niger in Pichia pastoris and its application research

The endo- inulinase gene( 1551 bp) was amplified from the total DNA of A. niger by PCR and subsequently cloned into p PIC9 K. The recombinant plasmid was linearized by Sac I and transformed into P.pastoris GS115 by electroporation,yielding the recombinant strain P. pastoris GS115/p PIC9K-endo I. Then right transformants were isolated and the recombinant enzyme derived from fermentation was characterized.Its optimal p H and optimum temperature of the recombinant enzyme was 5 and 60 ℃,respectively,moreover83.2% of the original enzymatic activity remained after the incubation of recombinant enzyme at 55 ℃ for 9hours. It was shown that Cu²⁺,Ag⁺ strongly inhibited the recombinant enzyme activity. The hydrolytic process analyses showed that the recombinant endo- inulinase could hydrolyze 4 %( w / v) inulin into DP3- 6 of fructooligosaccharides in 8 h. The conversion rate reached 63.5% in 11 h. The research laid a foundation for the produce of fructooligosaccharides.