Screening And Enzymatic Properties Of A Thermostable Cellulase Producing Aspergillus Fumigatus,and Its Application Evaluation

Biological conversion of cellulose,an eminent representative of naturally available nanostructure renewable matter,to fuels and chemicals offers the high yields to products vital toecnomic success and the potential for very low costs.Worlswide research is focuss–ed to ultilize the inexhaustible natural cellulosic biomass efficiently as a raw material for the production of various environmentally friendly biofuels,especially ethanol.The objectives in this study were mainly to obtain a thermophilic fungiwith high cellulase activity and a high titer ethanol production.The detailed contents are as follows:?1?This study obtain a fungi strain–Aspergillus fumigatus LY1,which has identified by 18S rDNA,isolated from sugar cane bagasse,according to its ability to grow on CMC-Na as the sole carbon source at the temperature of 45?.Nano LC-ESI-MS/MS Analysis was used to analyze and identify the secretory proteins in the fermentation broth of LY1.The results showed that there are 120 kinds of proteins exist in the fermentation broth under the detection range of this strain,and there are 23 kinds of cellulolytic enzymes.Glycoside hydrolase?GH?family proteins accounting for 29.2%of the total protein in the crude enzyme solution.It is noteworthy that the 23 cellulose degrading enzyme proteins produced by the LY1 strain include glucanase?0.2%of total protein?,?-glucosidase?0.7%of total protein?,cellobiohydrolase?0.4%of total protein?,xylanases,glucoamylases,glycosyl hydrolases,and other cellulolytic enzymes.The 3D structure analysis of cellulase which produced by Aspergillus fumigatus LY1 also showed that it have the ability to completely degrade lignocellulose?2?Cellulase has 50? optimum temperature,optimum pH5.0,9.52 mM Km,1.11?M/mL/min Vmax,and the activity of cellulase measured by the method of DNS.The thermaland pH stability analysis showed that it remained 50%activity after 1h incubation at 60oC and pH6.0 respectively.The experimental of metal ions on the enzyme showed that the monovalent metal ions Na⁺,K⁺,and some of the divalent heavy metal ions Co²⁺,Zn²⁺,Cu²⁺had little effect on cellulase activity;1mM Mg²⁺,Ca²⁺,Mn²⁺,Cd²⁺,can promote cellulase activity.Maximum cellulolytic enzymeproduction can achieve 1.40 IU/mL which the activity increased 6%than the raw media,observed after 96h at the medium composition was as follows:straw15 g/L,glucose 2.5 g/L,soybean meal 4.5 g/L,NH4NO30.5 g/L,KH2PO4 0.5 g/L,MgSO4·7H2O 0.9 g/L,which was optimize by rotatable orthogonal central composite design.?3?A preliminary study was conducted on the pretreatment process of lignocellulose and part of the combined biofermentation process for ethanol production.The results of single factor test showed that when usingthe 4%NaOH treat the straw for 24h at the room temperature and the ratio of solid to liquid at 1:10 canget the best educt which was indicated by the final concentration of reducing sugar.S.cerevisiaewas inoculated for producing ethanol.Using HPLC to determine the sugar composition and content in thefermentation broth,the results showed that with a detection limit of 0.1%,Glucose and xylose components were detected at contents of 0.96%and 0.2%.After orthogonal experiment analysis,when pH 4.5,aerobic fermentation time 24h,anaerobic fermentation time 36h,the ethanol concentrationproduced in the fermentation liquid reached 26.791 g/L.