| Cellulose is an extremely important carbohydrate. By now, with the dwindling non-renewable resources such as oil and coal, the degradation and utilization of renewable cellulose is becoming more and more emphatic. Endoglucanase is one of complex enzyme, include endo-β-1,4-glucanase(EC 3.2.1.4), cellobiohydrolase(EC3.2.1.91) and β-glucosidase(EC 3.2.1.21). Among them, endo-β-1,4-glucanase catalyzes the first step by randomly breaking down the internal β-1,4-glycoside linkages,then through synergetic action with other two cellulolytic enzymes, it converts the cellulosic materials to glucose, which is further turned into bioethanol. Cellulase widely exists in the organism of the nature, such as bacteria, fungi, and animals. It can be used in food industry, textile industry, feed, pulp and paper industry, has potential applications of brewing and oil exploitation, especially in the biological energy industry.However, cellulase produeed by natural microorganism has low activity, as well as production cost very high, then by means of genetic engineering to improve the activity of cellulase become an effeetive way to reduce the cost of cellulase production.Pichia pastoris is a methylotrophic yeast, it can grow on medium with methanol as sole carbon and energy source. Pichia pastoris expression system is the most commonly methylated yeast expression system. Its advantages include it can post-translational modification after heterologous protein, easy to increase gene copy,less self-secreted background protein, low degree of glycosylation, low nutritional requirements, the medium can be cheap, and high cell density fermentation can be carried out. These advantages is the foundation for the industrial production of the endoglucanase, and as an extremely important tool model in modern molecular biology,more and more researchers pay attention to Pichia pastoris.In recent years,with the development of micro-organisms in food bio-engineering technology, Aspergillus oryzae as an important species in fermentation industry and food processing industry, it become a hot topic. In 2005, several scientific research institutions and companies from Japan jointly completed Aspergillus oryzae RIB40(ATCC42149) genome sequencing work. It is a mode strains to study Aspergillus oryzae physiological regulation. Sequencing results showed that it has 8 chromosomes.Aspergillus oryzae can secrete a variety of enzymes, such as cellulose enzyme, protease,pectinase, phytase, etc. According to the amino acid sequence of AoEGLA gene and yeast bias codon, the desired gene of endo- β-1,4-glucanase(AoEGLAI) was synthesized chemically using PTDS method.The purpose gene cloned in Escherichia coli and sequencing, the new β-1,4-glucanase gene was ligated into expression vector pPIC9 K, and integrated into the genome of Pichia pastoris GS115 by electroporation. The expressed enzyme was recovered from the culture supernatant and purified by fractional precipitation with ammonium sulfate and Ni affinity chromatography. As determined by SDS-PAGE, the molecular mass of the purified β-1,4-glucanase was 27 kDa. The result of enzyme activity assay and SDS-PAGE demonstrated that the recombinant β-1,4-glucanase was induced and heterologous expressed in P. pastoris. Main biochemical properties of this recombinase, such as optimal pH and pH stability, thermodependence and thermostability, and the effect of metal ions and protease, were characterized.The results show that the Km and Vmax values of the recombinant β-1,4-glucanase were 22.47 g/L and 1.22 g min-1 L-1 respectively at pH 5.0 and 37 ?C.The optimum pH and optimal temperature for AoEGLAI was 3.0 and 50°C. After incubated at 37?C for 1 h or 4h, the rest of the activity was about 90% at pH 4-8. Theβ-1,4-glucanase had the maximum activity at 30-45?C. The activity of recombinase would reduce increasingly if the temperature was over 50?C. Though it almost entirely inactivation after incubation at 70?C for 10 min, the thermal stability of the enzyme could be improved to some extent with CMC existing. Most metal ions had no impact in recombinant enzyme activity at low concentration(1m M). When the concentration increased to 10 m M, the study show that Cu2+、Ca2+、Zn+、Gr3+、Fe2+ and Fe3+ could active the endo-β-1,4-glucanase activity, and Mn2+ inhibitors to it. This enzyme have a certainresistance to trypsin, pepsin resistance was relatively poor.In recent years, there are many studies about the cellulase gene cloning and expression. However, the research about β-1,4-glucanase from Aspergillus oryzae is less. The study show that the modified gene was heterologous expressed in P. Pastoris,and the recombinant β-1,4-glucanase exhibited high stability at acidic pH. These results are conducive to further explore the application in food industry of acid cellulose. |
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