Cloning And Expression Of Trichoderma Viride Endoglucanase Genes In Lacticacid Bacteria Andanalysis Of Enzyme Activity

Cellulose,as a substance widely found in nature,has a huge potential for application.Jilin Province,as one of the provinces with relatively abundant soybean resources,wastes too much resources each year during the processing of edible oil and soy products.The resulting by-products contain a lot of cellulose and some other nutrients such as protein,fat,minerals,vitamins,essential amino acids,etc.In addition,the presence of cellulose also affect the body's absorption of these nutrients.Due to technical means and cost issues,these by-products can not be efficiently used,resulting in a waste of resources.In addition,the process of dealing with by-products,not only consumes a lot of manpower and material resources,more serious is the impact on the natural environment.Recognized as a safe bacteria and with probiotic function,Lactobacillus is now accepted by the vast number of consumers,the combination of the by-product of the recycling of cellulose and lactic acid bacteria not only solve the problem of waste of resources,while playing Prebiotic effects of lactic acid bacteria.In this study,three endoglucanase genes EG?,EG?,EGV from Trichoderma viride AS3.3711 were used to clone the Usp45 signal from the perspective of biotechnology and food science Peptide gene and fusion with three endoglucanases in the food-grade Lactococcus lactis expression vector pNZ8149 / NZ3900,and three recombinant strains were obtained: pNZ8149-S-EGI / NZ3900,pNZ8149-S-EGIII / NZ3900,pNZ8149-S-EGV / NZ3900.For the late development of cellulose-related products to provide a breeding base.In this study,endoglucanase was cloned and expressed in Lactococcus lactis for the first time.Total RNA was extracted from Trichoderma viride AS3.3711 strain,and three internal steels were obtained by reverse transcription cloning.The length of the EG?,EG a? nd EGV fragments was 1380 bp,1257bp and 744 bp respectively.At the same time,the length of the Usp45 signal peptide gene was 81 bp,cloned and ligated into the pMD18-T vector,and then ligated with endoglucan S-EG?,S-EG?,S-EGV were constructed and then ligated with pNZ8149 lactobacillus expression vector to construct pNZ8149-S-EG?,pNZ8149-S-EG?,pNZ8149-S And the expression vector was: 2kV,25?F,200?,time 4ms.The constructed vector was transferred into Lactococcus lactis NZ3900.SDS-PAGE analysis was carried out by SDS-PAGE analysis.,And the target bands of about 48 kDa,45kDa and 25 kDa were obtained respectively.The enzyme activity of the enzyme was analyzed.The results were as follows: Congo red staining and 3,5-dinitro-nitric acid Acid(DNS)method,were used to obtain its optimum temperature,pH,and enzyme activity.By measuring the enzyme activity of three recombinant strain,the optimum temperature is 50 ?;The optimum pH of EG?and EG? is 5,the optimum pH of EG is 5.5,the definition ?of enzyme activity at optimum temperature and the optimum pH of the amount of enzyme which catalyze the hydrolysis of cellulose to glucose 30 min 1ug an enzyme activity unit(U),enzyme activity EG?,EG?,EG ?were 20.1U / mL,10.8 U / mL,18.7 U / mL;the results showed that,EG?> EG?> EG?,EG about twice the EG?.