The Surface Display And Characterization Of Mannanase In Escherichia Coli

Beta mannan is one of the non-starch polysaccharides widely existed in various plant cell walls in nature,which is a polymer connected by beta 1,4-D-pyran mannose.Beta mannanase is a kind of mult-functional growth agent,it can promote animal' s growth by increasing secretion of the insulin-like growth factor?IGF-I?,the synthesis of protein.Therefore mannanase is important in animal fodder.The mannanase gene from Bacillus Pumilus AGI16498.1 has 1104bp,encoding a protein that consisted of 368 amino acids.Microorganism surface displayed is using genetic engineering methods to realize the exogenous peptide or protein structure domain displayed on the microorganism surface in the form of integration.Recently,surface-displayed enzymes have been widely used as whole-cell biocatalysts in industry.Because the advantages such as improved enzyme stability and cost-effective downstream processing.Based on the important applications of mannanase,this study fusion sf-GFP realized a surface displayed in Escherichia Coli and secretion of the mannanase gene from Bacillus Pumilus AGI16498.1.The main contents are as follows:1.The synthesis of the Mannanase primer According to the gene of E.coli expression vector pET-23a,mannanase and sf-GFP,the forward and reverse primers were designed.6 X His tag is in the vector,which facilitated the recombinant protein of Western blot detection and purification.Synthesize the gene by using the methods of PCR.2.Construction of the recombinant vectorAfter recycle mannanase gene was cloned to pET-23a vector,carried with sf-GFP which was already digested,using the way without enzymes.3.Transformed into Escherichia Coli and screening recombinantAfter transformed Escherichia Coli competent cell,when cultured to a certain OD value a preliminary screening was done by Escherichia Coli colony PCR.Obtain the whole cell for SDS-PAGE detection and western blot identification screening recombinant Escherichia Coli.The correct recombinant Escherichia Coli was named pET23a-sfGFP-MAN.4.Surface display of MannanaseManaged the cultured bacteria,obtained the protein from supernatant,outer membrane,periplasm space,lining and whole cell.Using SDS-PAGE detection and western blot identification of mannanase surface display orientation.Because anchor protein sf-GFP has green fluorescent,it can carry fusion protein to display on the surface of outer membrane.So,the E.coli surface displayed mannanase can observe fluorescence condition by confocal fluorescence microscope.And using flow cytometry?FCM?to analysis fluorescence of secreted mannanase on E.coli.5.Characterization of the whole cell surface displayed mannanaseAfter determination,the mannanase' s optimum temperature was 55?,the optimum pH was 5.5.It was stable within the range of pH 4.5-9.The residual activity was more than 70%after incubated in 45?,50? for 3 h.The surface displayed mannanase was activated by Cu²⁺,K+,Cd²⁺.6.Characterization of the mannanaseThe secretion mannanse was collected,purified by Ni-NTA affinity chromatography and protein purification apparatus.After determination,the mannanase' s optimum temperature was 45?,the optimum pH was 6.5.It was stable within the range of pH 5-8.The residual activity was more than 80%after incubation in 45?,50? for 3 h.Mannanase was strong activated by K+and Ca²⁺?...