Heterologous Expression Of Glucosidase And Characterization Of Enzyme Activity

Objective: significance: the rise and development of the new renewable energy industry is imminent in the rapid development of the times,the sharp decrease of traditional energy and the rising of the price,the large emission of greenhouse gases,and the global climate change.Among them,the most common and abundant renewable resources of lignocellulose,which are mainly agricultural and forestry wastes,can be used not only for animal husbandry,biofuel and food additives,but also in industrial production and medical and health industries.The rational and efficient utilization of lignocellulose can not only alleviate environmental pressure,but also drive economic growth.However,the characteristics of lignocellulose itself make it difficult to completely decompose and make full use of it.The main reasons are as follows: 1)the close links between cellulose,lignin and hemicellulose that make up lignocellulose lead to poor degradation;2)the yield of enzymes needed for cellulose degradation fermentation is too low to meet the large demand for industrial development;3)the degradation efficiency of single glucosidase is low.Therefore,the aim of this study is to search for a variety of highly efficient glycosidases,and to optimize and reconstruct their genes in order to produce target proteins that can be expressed in large quantities and are easy to collect and purify,and to characterize the enzymatic properties of the fusion proteases obtained by heterologous expression.In order to achieve the efficient use of lignocellulose.Material methods: in this paper,the recombinant expression vector of the target protein was constructed by using molecular biological methods.Two heterologous expression systems of Escherichia coli and Pichia pastoris were used as the heterologous expression of glycosidase to express host cells.The expression of glucosidase in different sources of different sources was expressed,and the enzyme properties of the enzyme were used by the DNS method.Detailed characterization of the product?optimum p H and temperature,p H and thermal stability as well as the influence of two valence metal ions?were carried out.Results: the target protein was successfully expressed in heterologous cells.The optimum reaction p H values of three kinds of cellulase?p JL-14,p JL-36,A10?,manna polysaccharase?C6?and pectinase?p JL-44?were 5.5,5,4,4 and 4 respectively,both were weak acidity,and were stable in the neutral and weak alkaline environment.The optimum reaction temperature and thermal stability are different: the optimum reaction temperature of p JL-14 and p JL-36 is 45?and 40?,it is more stable below 35? and 30?,and the optimum reaction temperature of C6 is 60? it is more stable below 40?.The optimum reaction temperature of A10 is 80?,and it is more stable below 60?,and the optimum reaction temperature of p JL-44 is 60?.It is stable under 25 centigrade.It can be seen that the thermostability and optimal reaction temperature of glycosidase from the same source and from different sources are different.In the common two valence metal ions,Co²⁺,Mn²⁺ and Fe²⁺ played an obvious role in promoting their enzyme activities,while Cu²⁺ and Pb²⁺ had a significant inhibitory effect.PJL-44 is slightly different from other enzymes.Ni²⁺ and Ba²⁺ have obvious effects on their enzyme activities,while Cu²⁺ and Mn²⁺ have a significant inhibitory effect.Conclusion: the experimental results show that these glucosidase is an acidic polysaccharide hydrolase with better alkali stability,and the difference in the optimum reaction temperature and thermal stability among the glucosidase may be related to the source and enzyme structure,and the effect of metal ions on isozyme is different.In view of the good stability of these enzymes,they have potential to develop commercial enzymes.