Research On The Property Of Novel Cellobiose Phosphorvlase (CBP) And Clostridium Thermocellum CBP Displayed On The Surface Of Saccharomyces Cerevisiae

How to effectively use cellulose resources to produce raw material products with high efficiency is a hot issue in scientific research.At present,scientists have established a new way to produce starch from cellulose,which is of great significance for reducing cellulose waste and alleviating food crisis.In the cellulose-transformed starch pathway,cellobiose phosphorylase?CBP?plays an important role in the conversion of ?-glycosidic bonds to ?-glucosidic linkages,as the core enzyme in this system.Based on an optimized cellulose-transformed starch core on the perspective of the system,this study performed the discovery and functional identification of novel enzymes with excellent properties,and performed by surface display of Saccharomyces cerevisiae on CBP to reduce industrial production costs.The results are as follows:?1?The protein THA₁941,annotated as CBP in Thermosipho africanus TCF52 B,was searched in the KEGG database.Although this protein has very low amino acid homology compared with GH94 enzymes,its secondary structure is conserved by CBP.In the active region,there is a highly conserved??-??6 barrel domain,so THA₁941 is selected as a candidate CBP.?2?THA₁941 was cloned and expressed in E.coli BL21?DE3?,and the functional activity was identified,as well as the activity of Ta CBP was determined.The optimal temperature was 75°C and the optimum p H was 7.5.In addition,the enzyme can use cellobiose and long-chain oligosaccharides with a degree of polymerization greater than 2 as glucose receptors to release phosphates from G-1-P,indicating that Ta CBP has both cello-oligosaccharide phosphorylases.?CDP?functional activity.?3?The catalytic efficiency?Kcat/Km?of Ta CBP was measured.The results showed that cellotetraose and fiber pentamer were the best substrates for the direction and synthesis direction of phosphorylation,respectively.The catalytic rate in the synthesis direction was much greater.Catalyzed by the rate of phosphorylation,this enzyme may be a potential catalyst for the synthesis of various oligosaccharides.?4?Comparing with the ability of Ta CBP and Ct CBP in transforming starch,it has relatively strong ability to synthesize starch.Therefore,this study set as Ct CBP for display on the surface of Saccharomyces cerevisiae?5?Construction of recombinant plasmid of Ct CBP and yeast display plasmid p YD1,transformation of S.cerevisiae cell EBY100,induction expression of Ct CBP on the surface of Saccharomyces cerevisiae,determination of enzyme activity of Ct CBP display strain recombinase,60 h for the best induction time,and the optimum p H of the recombinase is 7.0,the optimum temperature is 65°C.It has good thermal stability and still possess 91% of the original activity after 6 hours of incubation at65°C.The functional identification of the novel Ta CBP was found in this study.It wasfound that it also has the functions of CBP and CDP,and has unique ability for the synthesis reaction of oligosaccharides as substrates;the surface display of Ct CBP shows the potential value of industrial applications.Cellulose-transformed starch systems provide the basis for practical applications.