Cell Surface Displaying Of Thermomyces Lanuginosus Lipase On Pichia Pastoris

Thermomyces lanuginosus lipase?TLL?,a heat-resistant,location-specific and more selective lipase,has good prospect of application in many fields,including detergents,cosmetics,bio-chemical industries and so on.At present,the commercialization of TLL is immobilized mostly,and it still needs to be immobilized after getting the free enzyme protein by isolation and purification,so the price is a bit high,which limits the wide usage.Yeast cell-surface display system is one of the most popular techniques in recent years,it merges target protein and cell wall protein together by means of genetic engineering methods so as to fix target protein on the surface of yeast cells in standard process of fermentation,which resembles the immobilization of enzyme.The Pichia pastoris cell surface display system is a kind of rapidly growing and extensive applied eukaryotic cell expression system.It not only has the advantage of simple molecular genetic manipulation,but also can achieve high density of expression under a strongly inducible AOX1 promoter,therefore,getting TLL displayed on the cell surface of Pichia pastoris provides a good idea for seeking the catalyst of Thermomyces lanuginosus lipase in high performance.Nowadays the Thermomyces lanuginosus lipase is studied less than other lipases,and the two genes of TLL1?GenBank:ABV69591.1?and TLL2?NCBI Accession: O59952?are only a few reported.In this subject we firstly selected TLL1 as the target protein.The corresponding and optimized gene,respectively with the signal peptide sequence and without the signal peptide sequence,were merged with Pichia pastoris endogenous wall protein Gcw51 p and Gcw61 p gene.Thus we constructed four display the expression plasmid,namely pPIC9k-sTLL1-GCW61 ? pPIC9k-TLL1??sig?-GCW61 ? pPIC9k-sTLL1-GCW51 ?pPIC9k-TLL1??sig?-GCW51.The four plasmids were then transferred into Pichia pastoris GS115,and the four kinds of recombinant surface display strains with high activity of high hydrolytic enzymes were obtained.Then the four strains were fermented,and the expression of lipase was studied.The results show that the Thermomyces lanuginosus lipase was successfully displayed on surface of Pichia yeast,and it show some hydrolytic activity:GS115/pPIC9k-s TLL1-GCW61,205.24U/g;GS115/pPIC9k-TLL1??sig?-GCW61,118.69U/g;GS115/pPIC9k-sTLL1-GCW51,97.33U/g;GS115/pPIC9k-TLL1??sig?-GCW51,26.50U/g.It is concluded that the display system is effective,and the display effect ofanchoring protein Gcw61 p is better than that of the anchor protein Gcw51 p,and the enzyme activity of target gene with signal peptide is higher than that of target gene without the signal peptide.Secondly we the chose TLL2 to construct GS115/pPIC9k-TLL2-GCW61 and GS115/pPIC9k-TLL2-GCW51 expression strain,and the enzyme activity of them were172.34U/g and 84.19U/g.The activity did not increase,so we chose PHKA-AOX high expression plasmid which was constructed by our laboratory to structure GS115/PHKA-AOX-TLL2-GCW61 expression strain which were screened by Tributyrin plate and shake flask.GS115/PHKA-AOX-TLL2-GCW61 was then labeled by Immunofluorescence to confirm that the lipase was displayed on the cell surface.After shaking flask fermentation of 144 h,the activity of the enzyme was up to 1964.76U/g.Fermentation broth is studied on enzymatic properties after vacuum freeze drying.The optimum substrate is p-nitrophenyl hexanoate;the optimum temperature is about 55?;the optimum pH is 9.0 and after saving 90 min in 70?,TLL2 activity still preserves 70% above,so it has the characteristics of high heat resistance.Ca²⁺,Mn²⁺,Zn²⁺ has a strong activation effect on TLL2,while Cu²⁺,Fe²⁺,Fe³⁺,K⁺,Li⁺,Na⁺,Co²⁺ has a strong inhibitory effect on it,and Ba²⁺,Mg²⁺,Ni²⁺ has little effect on the enzyme.The TLL2 displaying on cell of Pichia pastoris with high hydrolytic activity,alkali and thermal stability lays a foundation for extensive application in industry and provides the solution for the industrial application of other lipases.