Expression Of The Inulinase Gene From Marine Derived Pichia Guilliermondii And Its Application On Ethanol Production

Inulin is present as a reserve carbohydrate in the roots and tubers of plants such as Jerusalem artichoke, chicory, dahlia, and yacon. The yields of the roots and tubers are very high. The dried materials of the tubers contain over 70% inulin. Inulin consists of linear chains ofβ-2,1-linked D-fructofuranose molecules terminated by a glucose residue through a sucrose-type linkage at the reducing end. Such inulin sources have recently received attention as a renewable raw material for fructose syrup production, ethanol fermentation, and inulooligosaccharide (IOS) production. The inulinases are classified among the hydrolases and target on theβ-2,1 linkage of inulin and hydrolyze it into fructose and glucose.Pichia guilliermondii strain 1 (the collection number:2E00048 at Marine Microorganisms Culture Collection of China), isolated from the surface of a marine alga, was found to secrete a large amount of inulinase into the medium. The inulinase gene cloned from the marine-derived yeast P. guilliermondii strain 1 was expressed in Pichia pastoris X-33 and the conditions for over expression of the inulinase were optimized. After the optimization of the conditions for production of the recombinant inulinase,79.8±3.5 U/mL of the recombinanat inulinase in the supernatant of the culture of the 150.0 mL flask at 72 h shaking culture, and 286.8±5.4 U/mL and 8873±55.3 U/mg of the recombinanat inulinase in the supernatant of the culture of 2-L fermentor were attained at 120 h of the fermentation. The recombinant inulinase was purified and characterized. The molecular weight of the purified recombinant inulinase was 57.6 kDa, which was higher than that of the native iunlinase. The optimal pH and temperature of the purified recombinant inulinase were 6.0 and 60℃, respectively. Other biochemical characteristics of the purified recombinant inulinase were the same as those of the native inulinase produced by the marine derived P. guilliermondii strain 1. The purified recombinant inulinase also had high exoinulinase activity. Therefore, the recombinant inulinase may have highly potential applications in food and pharmaceutical industries.It has been confirmed that Saccharomyces sp. WO can produce high concentration of ethanol. However, inulin could not be utilized directly by the strain WO. Therefore, the recombinant inulinase produced by P. pastoris X-33 carrying the inulinase gene was used to saccharify inulin into reducing sugars for ethanol fermentation by Saccharomyces sp. WO. During the small scale of the fermentation and 1-L fermentation,14.9±0.4%(v/v) and 14.8±0.2%(v/v) of ethanol were produced from inulin, respectively when the 11.5±0.5 U of inulinase activity per ml of the fermentation medium was added for the inulin saccharification. Only a trace amount of residual total sugar and reducing sugar was left in the fermented media, indicating that most of inulin in the fermentation media was utilized for ethanol production and cell growth.In order to make the strain WO to produce both inulinase and ethanol in one step fermentation, the INU1 gene cloned from the marine-derived Pichia guilliermondii was transformed into uracil mutant of Saccharomyces sp. WO. The transformant Inu-66 obtained could produce 33.7±0.4 U/mL of extracellular inulinase within 72 h of cultivation. It was found that 22.8±0.1 U of inulinase per one gram of inulin was suitable for inulin hydrolysis and ethanol production by the transformant Inu-66. During the small scale of fermentation,13.7±0.5%(v/v) ethanol was produced and 99.1% of the added inulin was utilized by the transformant. During the 2.0-L fermentation,14.9±0.6%(v/v) of ethanol was produced from inulin and 99.5% of the added inulin was converted into ethanol, CO2 and cell mass.50.0% Jerusalem artichoke powder and 1.0% ammonium sulfate were mixed in the 2.0-L fermentor, then, the culture of the transformant Inu-66 was added to the mixture. During the fermentation,12.1±0.3%(v/v)ethanol was produced from Jerusalem artichoke powder in one step fermentation and 90.4±0.5%(w/v)total sugar was utilized.