Production of High Concentrations of Fuel Ethanol from Inulin

两种菊粉酶，P-IA（MW 70，000）和P-IB从菊粉酶高产突变株尼日尔817的培养滤液中，经DEAE-Cellulofine A-500和Q-SepharoseHP层析，纯化出P-IA和P-IB两种酶，其比活力分别为350 U/mg和340 U/mg，并且缺乏对蔗糖、棉子糖或莱万的活性。菊粉水解的主要产物为菊粉三糖和β-四糖。因此，这两种菊粉酶对菊粉都表现出内型作用模式。在40 ℃、pH5.0条件下，菊粉的表观K_m值分别为0.48mM和0.50mM。将尼日尔817在30 ℃下在浸没培养物中生长5天。菊粉消化液培养物（150 ml）中添加45 g菊粉、0.45 g（NH_4）_2SO_4和0.15 g KH_2PO_4。用耐乙醇菌株酿酒酵母1200接种培养基（pH 5.0），并在30 ℃下进行发酵。发酵15小时后，向培养物中加入另外30 g菊粉。酿酒酵母1200在发酵期间利用了65 g菊粉的99%，并且在发酵的3天内分别从菊苣和大丽花菊粉产生20.4%和21.0%（v/v）的乙醇。乙醇的最大体积生产率分别为6.2和6.0克/升/小时菊苣和大丽花菊糖，分别。菊糖转化为乙醇的转化率为理论乙醇产率的83 - 84%。这种高浓度的乙醇也是从含菊粉的农作物耶路撒冷蓟中生产的。

Two inulinases, P-IA (MW 70,000) and P-IB (MW 68,000), were purified from the culture filtrate of an inulinase-hyperproducing mutant, Aspergillus niger 817, grown on sucrose by DEAE-Cellulofine A-500 and Q-Sepharose HP chromatographies.The specific activities were 350 U/mg for P-IA and 340 U/mg for P-IB.The enzymes were active only toward inulin, and lacked activity toward sucrose, raffinose or levan. The main products of inulin hydrolysis were inulotriose and -tetraose. Thus, both inulinases exhibited the endotype mode of action on inulin. The apparent K_m values for inulin were 0.48 mM for P-IA and 0.50 mM for P-IB at 40ﾟC and pH 5.0.Pure nonhydrolyzed inulin was directly converted to ethanol in a simultaneous saccharification and fermentation process. A.niger 817 was grown in a submerged culture at 30ﾟC for 5 days. The inulin-digestive liquid culture (150 ml) was supplemented with 45 g of inulin, 0.45 g of (NH_4)_2SO_4, and 0.15 g of KH_2PO_4. The medium (pH 5.0) was inoculated with an ethanol-tolerant strain, Saccharomyces cerevisiae 1200, and fermentation was conducted at 30ﾟC.An additional 30 g of inulin was added to the culture after 15 h of fermentation. S.cerevisiae 1200 utilized 99% of the 65 g of inulin during the fermentation, and produced 20.4 and 21.0% (v/v) ethanol from chicory and dahlia inulins, respectively, within 3 days of fermentation. The maximum volumetric productivities of ethanol were 6.2 and 6.0 g/liter/h for chicory and dahlia inulins, respectively. The conversion efficiency of inulin to ethanol was 83 to 84% of the theoretical ethanol yield. Such high concentrations of ethanol were also produced from a inulin-containing agricultural crop, Jerusalem artichoke.