Sonstruction Of Novel Efficicent Cell-surface Display Systems On Pichia Pastoris Cell Surface And Directed Evolution Of Candida Antarctica Lipase B

With the application of recombinant DNA technology, microbial cell-surface display systems have been used in various microorganisms, such as in bacteria, in phage and especially in yeast. Microbial cell-surface display system has been widely used in biotechnologieal research and industrial applications, including:identifying Protein-Protein inieraetions, displaying polypeptide libraries as selection devices, mapping functional protein epitopes, immobilizing proteins and enzymes as whole-cell biocataysts, producing antibodies and live vaccines, making bioadsorbents for the removal of harmful chemicals and heavy metals, therefore, which has becoming a research hotspot in the field of biotechnology in recent years.The lipase B from Candida antarctica is one of a very important lipase, which have some good propertirs of biocatalysis and postion selection. In this paper, we cloned the gene calb and expressed the functional protein in Pichia pastoris systems including the P. pastoris cell-surface display system. The methylotrophic yeast is a sigal-cell eukaryote microorganism and has been widely used to express various recombinant proteins for both basic laboratory research and industrial manufacture. It became a host for cell-surface display system. For the rich yeast display system, using P. pastoris cell-surface to display Candida antarctica lipase B. While this papper by constructing different yeast cell-surface diplay system by various anchor proteins, then, using these systems to screen of some higher activity whole-cell catalysts. On the other hand, using directed evolution technology to improve the thermal stability and activity, furthermore, using the whole-cell biocatalysts to synthsize the short chain fatty acid esters in non-aqueous phase. And then to lower the production cost of enzyme preparation for its large-scale lay the foundation for industrial applications. Specific study as follows:PartⅠConstructing and comparing the yeast display systems by usingα-agglutinin and flocculation functional domain(flolp)This part constructed two new type yeast display systems, including theα-agglutinin system and flolp system in Phichia pastoris cell surface. Combined with Our laboratory had constructed the FS system. Then using the lipase B from Candida antarctica as a model protein to check the 3 dispalying vectors in yeast. Lipase hydrolysis activity and FLAG tags and other means to detect the protein in the yeast cell wall anchoring conditions. FLAG tag sequence and the N-terminal protein CALB fusion, when the recombinant yeast cells can be detected when the green fluorescent CALB has been successfully verified the anchoring on the surface. Will be verified by the positive plate and the fluorescent recombinant shake flask fermentation by three kinds of whole-cell catalyst, was carried out further analysis of enzymatic properties.Part II Constructing and analysizing the Pir-type and GPI-type displaying CALB on Pichia cell surfaceThis part has cloned the gene Pir1, Pir4 and Sedl from Saccharomyces cerevisiae and ligated into Pichia pastoris expression vectors pPIC9K to construct 3 different yeast cell surface display systems, namely, pKPirl, pKPir4 and pKSed1 which all using-factor secretion signal peptide. Then tagged with a FLAG Candida antarctica lipase B protein with three kinds of C-terminal N-anchored fusion protein and used immunofluorescence microscopy, that the newly constructed yeast display common carrier is successful. Protein electrophoresis and Western blot demonstrated an initial Sedl-CALB fusion protein can be mild-alkali extraction from the cell wall, further evidence of the successful construction of a yeast display system.Part III Improving the thermostability and activity of lipase CALB by use of directed evolution technologyUsing the method of error-prone PCR to random mutation CALB,then, using 96 plate high-throughput screening method of activity of about 2,000 recombinant yeasts. The outcome of screening is obtained a strain which has higher hydrolytic activity than the wild type lipase.The sequence of the mutant was found 4 amino acids mutant (Thr57Ala,Ala87Thr Arg168Lys and Gly226Arg), using bioinformatics analysis shows that the spatial structure of point mutations around the active site, which may be beneficial to its combination with the relevant substrate, thereby improving its hydrolytic activity.Then, using target sites of regional saturation mutagenesis in the yeast cell-surface system, the lipase CALB was redesigned by using directed evolution technology. At 60℃,using tributyrin emulsion plate reorganization of about 5,000 yeast mutants were screened and received two higher thermal stability of lipase producing strains. Sequencing indicated that KFS-mCALB7 mutation of 7 amino acids (Pro218Asn,Leu219Lys,Phe220Thr, Val221Ser,Leu278Gly,Ala279Met and Ala281Ile),KFS-mCALB168 also has three amino acid (Thr57Ala, Ala87Thr and Arg168Lys) mutations arise. Further use of its simulation of the spatial structure analysis revealed that two strains were 3 and 4 hydrogen bonds increases, which may be mutants of thermal stability is an important reason.Part IV Comparing the yeast whole-cell catalysts in non-aqueous synthetic activity This section, we analyzed the different types of anchoring proteins CALB-displayed whole-cell catalysts in non-aqueous phase, which can catalyze the synthesis of short chain fatty acid ester. The data shows the KNS-CALB strain that has advantage in the synthesis and initial access to ethyl acetate. Some synthetic activty of KNS-CALB is superior to the commercialization of the same type of lipase. In addtion, in the aqueous phase of the excellent heat-resistant mutants in the organic phase of thermal stability of the mutants were found that the thermostability has no significant increase. The outcome indicated that directional transformation of thermal lipase in the aqueous phase does not reflect the organic phase with a certain correlation. Therefore, it is necessary to establish a high-throughput screening in the organic phase, thermal mutants of the methodology for lipase in organic synthesis basis.