The Chickpeas Spore Kluyveromyces Inulinase Secretory Expression And Applied Research

Strains of Kluyveromyces lactis which secrete high level of inulinase were constructed by transforming of expression vector pUKD-S-αF-PinuT. The disruption of Klhapl increased heterologous expression of inulinase gene Kcinu derived from Kluyveromyces cicerisporus CBS4857. Inulinase activity in the supernatant of Klhap1Δstrain was 391 U/ml after cultured for 120 h, which was 2.2-fold that of the wildtype host. Comparing with the wildtype host, the relative KcINUl mRNA level of Klhap1Δstrain was increased significantly in the indicated culture time. After 50 generations of continuous cultured in non-selective medium, the percentage of expression plasmid-carrying cells were 99% and 89% for Klhap1Δand wildtype strains respectively. Based on these results, the disruption of Klhapl gene facilitated the high and stable expression of inulinase in K. lactis by certain mechanism.Saccharomyces cerevisiae strain Y16hap1Δwas obtained by homologous recombination of disruption cassette into strain Y16. Transcription level of different promoter in the Y16 and Y16 was determined by lacZ report gene.β-Galactosidase activities directed by PinuS and Ptef promoter were higher than other promoter tested. Transcription level of PinuL promoter in Y16hap1Δwas lower than that in the Y16 which is opposite to the result in K. lactis. This suggested that the function of Klhapl is different to that of Schapl in S. cerevisiae.The research gives us a better understanding about the property of heterologous inulinase expression in K. lactis and S. cerevisia and facilliates the development of yeast expression system. It indicated heterolgous target proteins could be more efficiently produced by using Klhap1ΔKluyveromyces lactic as host.Four recombinant S. cerevisiae of inulinase expression were constructed by introduction of episomal plasmid. Inulinase activities secreted by Y16/pHC-PinuLT were highest among these recombinant yeasts which reach 57U/ml after cultured for 96h. The inulinase activities were 2-fold that of Y16hap1Δ/pHC-PinuLT. The result show the disruption of ScHAPl gene impaired heterologous expression of inulinase. Inulinase activities secreted by Y16/pHC-PinuLT could be elevated by addition of urea to the raw Jerusalem artichoke medium. Recombinant diploid S. cerevisiae XN5-aF-inu 41 of inulinase secretion were constructed by integration of Kcinu into the rDNA locus in chromosome. Inulinase activity was 29 U/ml for XN5-aF-inu 41 after cultured in YEPI medium[1%(w/v) Yeast Extract,2%(w/v) Polypeptone, 10%(w/v) Inulin] for 72h. The inulinase produced by XN5-αF-inu 41 was sufficient to hydrolyze 95% of the initial inulin to reducing sugar in 48h. The results contribute to the development of ethanol production by inulinase secretion S. cerevisiae strains using Jerusalem artichoke as raw material. The genetic engineering stain of S. cerevisiae that is unable to consume fructose was then constructed by double disruption of the hexokinase gene hxkl and hxk2 in Y16/pHC-PInuLT strain constructed in the first part of the study. Inulinase activity in the culture supernatant of recombinant Schxk1Δhxk2Δstrain was 30.9 U ml-1 after cultured 96 h which was 2.4-fold that of Y16/pHC-PInuLT. After 48 h of fermentation in the medium initially containing 10%(w/v) inulin as sole carbon source by the recombinant Schxk1Δhxk2Δstrain, the fructose was accumulated to 9.0%(w/v) in the fermentation supernatant and the glucose content in the culture medium was less than 0.005%(w/v). When using wild Jerusalem artichoke as sole fermentation medium component without any supplementary, the fructose concentration of the fermentation supernatant reached 9.2%(w/v) and the glucose content was 0.10%(w/v) after 24 h incubation. The result suggested that fructose production of high purity can be achieved by one step fermentation of recombinant Schxk1Δhxk2Δyeast strain in the culture medium of Jerusalem artichoke as the sole component.Ethanol production from raw Jerusalem artichoke tubers by simultaneous saccharification and fermentation using yeast Y179 was studied. Kluyveromyces cicerisporus Strain Y179 could express high level of inulinase. The optimization of the fermentation process was achieved by research the impact of different fermentation parameter on ethanol production. Optimized fermentation conditions were as follow:Y179 was cultivated in YEPD medium for 36h.25%(w/v) wild artichoke medium was inoculated with 6%(v/v) inoculum of the 36h-precultured Y179 culture. After 8h of fermentation in rotary shaker, Y179 was then transferred to stationary cultivation at 30℃. Ethanol reached 9-11%(v/v) after 40h of fermentation. The highest conversion ration of carbohydrate to alcohol is about 75%. Jerusalem artichoke medium of 22%(w/v) total sugar content in a 5-L fermenter was inoculated with 10%(v/v) inoculum of 36 h-precultured Y179 cells. The batch fermentation was conducted at 30℃and with a stirring speed of 300 rpm under anaerobic condition. After 144 h of batch fermentation, ethanol concentration obtained from the fermentation broth was 12.34%(v/v) and the conversion efficiency of carbohydrate to ethanol was 86.9% of the theoretical ethanol yield. The cells utilized 93.6% of the total sugar during the fermentation.Ethanol production by inulinase-secretion S. cerevisiae Y16/pHC-PInuLT using crude Jerusalem artichoke medium of 22%(w/v) total sugar content was conducted with magnetic stirrer in anaerobic condition at 30℃. inoculated with 10%(v/v) inoculum of 24 h-precultured Y179 cells. After 48h of batch fermentation, ethanol concentration was 10.5%(v/v) and the conversion efficiency of carbohydrate to ethanol was 85% of the theoretical ethanol yield.The results of ethanol fermentation showed that K. cicerisporus Y179 and recombinant inulinase-secretion S. cerevisiae Y16/pHC-PInuLT are promising candidates for industrial application of fuel ethanol production in simultaneous saccharification and fermentation using crude Jerusalem artichoke.Ethanol and fructose production by S. cerevisiae and K. cicerisporus with inuliase activities is promising method which is simple and low cost for industrial application.