| ?-galactosidase,also known as melibiose enzyme,catalyzing ?-galactoside bond hydrolysis enzymes,is a kind of exoglycosidases widely existing in nature.Its role is to catalyze ?-galactoside bond hydrolysis,I.e.,hydrolyzing galactosidase compound with ? bond of non-reducing end,So it can hydrolyze oligosaccharides such as melibiose,raffinose,stachyose and mullein sugar,and also hydrolyze heteropolysaccharide containing ?-galactosidase keys.Today,a study of ?-galactosidase showes that the enzyme has broad application prospects in the feed,sugar,food,pharmaceutical industry and other fields.Soybean meal is the most widely used vegetable protein feed,But soybean meal contains a class of ?-galactosidase oligosaccharides anti-nutritional factors,affecting the absorption and utilization of nutrients,such as raffinose,stachyose.on the one hand,application on the ?-galactosidase can make this type of oligosaccharides used,on the other hand,it can improve the utilization of soybean meal,eliminate flatulence by reducing the anti-nutritional factors.Experimental results show that adding ?-galactosidase can increase 8% effective metabolic energy and 7% digestible protein in the Soybean Meal.At present,China's annual consumption of legumes feed is about 10 million tons,after extensive use of enzymes,representing an increase of the use of legumes 80 tons / year.Therefore,the development of ?-galactosidase as a feed additive has important social and economic value.In this experiment,Aspergillus niger yielding glucoamylase is the recipient bacteria,building ?-galactosidase expression vector for Aspergillus niger,by Agrobacterium-mediated transformation,Aspergillus niger was transformed,homologous recombination transformants were screened,the activity of the recombinant protein were detected and SDS-PAGE were analyzed,with a result of high effective expression of target gene.The purpose of this experiment is to use the food-grade Aspergillus niger expresion vector built in lab to express and secret ?-galactosidase,exploring new avenues for ?-galactosidase industrial production.The main findings are as follows:(1),Cloning of ?-galactosidase gene and construction of expression vector According Aspergillus niger ?-galactosidase gene in Genebank,primers were designed,?-galactosidase ?glA was amplified in Aspergillus niger CICC2462.For highly expressed glucoamylase gene(glaA)locus in Aspergillus niger,using gene replacement techniques to build niger expression vector pSZHG-?glA.(2)Transformation of Aspergillus niger Agrobacterium and screening of ?-galactosidase homologous recombination transformantsUtilizing freeze-thaw method,the Agrobacterium transformation of Aspergillus niger expression vector pSZHG-?glA transformed into Agrobacterium AGL1,using primers P1,P2,identifying transformants by colony PCR and obtained 3 positive transformants.Identified the three positive transformants used homologous recombination primers were identified by PCR,the result was three positive transformants were amplified 1701 bp target band,proved to be homologous recombinants,pSZHG-?glA homologous vector conversion recombination rate was 100%.(3)Expression analysis of ?-galactosidase in recombinant strainsUnder the conditions of fermentation,?-galactosidase enzyme activity in the fermentation supernatant can reach 10.2 U · mL-1,is 10.4 times of the original strain.The culture supernatant which activity reached the highest value of recombinant ?-galactosidase was detected by SDS-PAGE.and the results show that three recombinant strains were successfully secreted and expressed,significantly higher than the original strain.The results show that the study realized ?-galactosidase homologous expression in Aspergillus niger,and laid the foundation for large-scale industrial production of food-grade ?-galactosidase enzyme. |
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