Isolation And Identification Of Anaerobic Ethanol-producing Cellulolytic Bacterial Consortia And Cloning And Expression Of The β-galactosidase

As the non-renewable resources such as oil and natural gas, are gradually exhausted, the renewable cellulosic ethanol has become one of the hot topics in the world. The bottleneck of the cellulosic ethanol is lacking efficient strains which can produce ethanol by decomposing cellulose and the high cost. Cellulose-decomposing by traditional chemical technology need harsh reaction conditions, and usually cause serious environmental pollution. Therefore, the key of cellulosic ethanol is to breeding strains with high cellulose-decomposing and ethanol-producing ability.By enriching with cellulosic substrates as the sole carbon source, a thermopHilic anaerobic bacterial consortia YTY-70was isolated from a Hot Spring in YongTai. The optimum growth temperature of the bacterial consortia is70℃, optimum pH is7.0. With filter paper as the sole carbon source, the growth of the bacteria density reaches its maximum after72hours. When the CMC, filter paper, bagasse, mushroom barrel were used as the carbon source respectively, the ethanol content in fermentation supernatant were detected by GC after a week cultivation, the results show ethanol yields were0.124mL/L,0.127mL/L,0.122mL/L,0.124mL/L respectively. Extraction of total bacterial consortia DNA, amplify the conserved sequence by16S primers, Connect the fragment to the PMD-18T vector, transformed into E. coli, then the positive clones were identified. The results show that the vast majority of bacteria are Caldicellulosiruptor. These cellulose-decomposing bacteria can not only produce ethanol but also produce hydrogen, acetic acid thermopHilic cellulase,galactosidase, and so on, which have a good value of research and application.There are unique metabolic pathways of high temperature and thermostable enzymes in thermopHilic bacteria, some thermostable enzymes also have been isolated. However, the culture conditions of thermopHilic bacteria are harsh, and the growth is slower, the thermopHilic enzymes of them are complex, low production and poor tolerance of acetate, which hinder its large-scale production and application. In this study, we cloned a thermostable β-galactosidase gene from thermopHilic anaerobic bacterial consortia YTY-70, the β-galactosidase gene was cloned into the pGEX4T-2vector and transformed into E. coli, the recombinant β-galactosidase was heterologously overexpressed. Purified by Glutathione SepHarose4B, we got about2.19mg of GST-P-galactosidase fusion protein, and the purified recombinant β-galactosidase was characterized. The results showed that the optimal condition of the enzymatic reaction was75℃, pH7.0. The reaction temperature range is very wide, it had good enzyme activity at temperature range from50℃to90℃. Metal ions Ba2+、K+、Na+、Mg2+、Zn2+and EDTA can slightly inhibit the activity of the recombinant enzyme (1.4%±4.4%), and the enzyme activity can be promoted by Fe2+and Ca2+. Under the optimum reaction conditions, the enzyme specific activity up to260U/mg. By analyzing the encoded amino acid sequence, the β-galactosidase belongs to glycosyl hydrolase family1.Comparing with other P-galactosidase from different sp, the identity is low.