α-半乳糖苷酶基因工程菌的开发

北京理工大学珠海学院16届本科生毕业论文Development of a-galactosidase producing strainAbstracta -Galactosidase (EC 3.2.1.22),is one kind of enzyme that hydrolyze non-reducingterminal a-D-galactosidic bonds in a-D-galactoside.The enzyme is currently widely used infood and feed industry.However,due to the low production capacity and high cost ofa-galactosidase,it cannot meet the needs of industrial applications.In order to reduce theproducing cost,this research aims to construct engineered Pichia pastoris strain with highlevel secretory expression of a-galactosidase through molecular biology.In this research,five a-galactosidases (AB,AC,AA,AZ and AF)from different specieswere investigated and their expression vectors were constructed,named pPICz a A-AB,pPICz a A-AC,pPICz a A-AA,pPICz a A-AZ and pPICz a A-AF,respectively.Among thesegenes,AA strain showed the highest activity and the activity of the fermentation broth reachedto 912U/mL.Subsequently,two methods were applied to improve the expression ofa-galactosidases including increasing gene copy number and co-expressing of chaperoneHACI.The result showed that co-expression of HACI had obvious improvement ona-galactosidases expression while increasing gene copy number had no influence.The activityof AA/HACI strain was up to 4663U/mL in 161h,which was 5.1 times compared to AA strainand was the highest productivity of a-galactosidases up to date.In this work,gene screening and protein high level expression strategy were applied toconstruct engineered Ppastoris strain with high level secretory expression of a-galactosidase.The activity of fermentation broth was up to 4663U/mL,the highest productivity up to date.This work lays the foundation of the industrial production and application for a-galactosidase.Keywords:a-galactosidase;Pichia pastoris expression system;gene screening;high proteinexpression目录1d言1