Construction And Characterization Of CBP/PGP Substrate Shuttle For Cellulose Transstarch Critical Enzyme

In recent years,a new field of cellulose degradation and Utilization-cellulose into edible starch,considering the current world food security situation,cellulose to starch has far-reaching practical significance.However,the existing enzymatic cellulosic starch-trans-starch system is restricted by cellobiose phosphorylase(CBP)and glucan phosphorylase(PGP)in the starch synthesis step,which have a single source and limited catalytic efficiency,which seriously restrict the practical production and application of starch-trans-starch system.Considering that CBP and PGP are excellent enzymes obtained by a large number of screening,and the relationship between the two enzymes is sequential reaction,this study expects to construct CBP-PGP artificial multi-enzyme complex and fusion enzyme to achieve high synergistic effect of enzyme combination-substrate shuttle effect,and improve the conversion efficiency of the system.The main findings of this study are:(1)In the study of substrate shuttle construction by artificial multi-enzyme complex strategy,PDZ(D1),GBD(D2),SH3(D3)were firstly connected to the C-terminal of CBP by flexible ligand peptide(F),and CBP-D1,CBP-D2,CBP-D3 were constructed.The peptide ligands(L1,L2,L3)corresponding to the protein domain were linked to the C-terminal of PGP by flexible ligand ligands,and PGP-L1,PGP-L2 and PGP-L3 were constructed.After calculating the optimum p H,optimum temperature,semi-inactivation time and specific activity,it was found that the optimum p H of CBP-D1,CBP-D2 and CBP-D3 were all 7.5,the optimum temperature was 45,the semi-inactivation time was 2 minutes,2 minutes and 4 minutes,the relative activity was 0.0003,0.0003,0.0010 and 45 minutes respectively.Time and specific activity of 0.0287 micromol/min/mg,the fusion protein domain seriously affected the activity of CBP,CBP-D3 was the relative optimal fusion enzyme;PGP-L1,PGP-L2,PGP-L3 were the optimal p H 5.5;the optimal temperature was 30;half inactivation time was 12 minutes,9 minutes,16 minutes;relative activity was 0.0003 micromol/min,0.0006 micromol,respectively.With reference to the half-inactivation time of PGP for 18 minutes and the specific activity of 0.0199 micromol/min/mg,PGP-L3 was determined to be the optimal fusion enzyme.However,CBP-D3 and PGPFL3 did not produce starch in vitro.(2)Because of the serious influence of protein domain on CBP,peptide ligands with small molecular weight were fused into CBP by flexible ligand ligands,and CBP-L1,CBP-L2 and CBP-L3 were constructed.The peptide ligands which had no effect on CBP were expected to be selected.After calculating the optimum p H,optimum temperature,semi-inactivation time and specific activity,it was found that the optimum p H of CBP-L1,CBP-L2 and CBP-L3 were 7.5,the optimum temperature was 45,the semi-inactivation time was 3 minutes,2 minutes and 2 minutes,the relative activity was 0.0013,0.0006 and 0.0002 micromol/min,respectively,and the semi-inactivation time was 45 minutes.The time and specific activity of 0.0287 micromol/min/mg still seriously affected the activity of CBP after the fusion peptide ligand.CBP-L1 was the relatively optimal fusion enzyme,but no starch formation was observed in the in vitro starch transfer experiments of CBP-L1 and PGP.CBP-R1-L1,CBP-R2-L1 and CBP-Rf-L1 were constructed by using rigid ligand peptide R1,R2 and R.flavefaciens ligand peptide Rf.The optimum p H,optimum temperature,semi-inactivation time and specific activity of the fusion enzyme were calculated by changing the ligand peptide.The results showed that the optimum p H of CBP-R1-L1,CBP-R2-L2 and CBP-Rf-L3 were 7.5.The optimum temperature was 45,the half-inactivation time was 5 min,4 min and 5 min,the relative activities were 0.0017,0.0009,0.0012 and 0.0287,respectively,and the half-inactivation time of 45 min and the specific activity of 0.0287,respectively.1L1 is more suitable for CBP than other connections.However,no starch production was observed in CBP-L1 and PGP in vitro.(3)The fusion enzymes of CBP at N-terminal,PGP at C-terminal and PGP at N-terminal and CBP at C-terminal were constructed by flexible ligation peptide.The optimum p H values of CBP-PGP and PGP-CBP were 7.5 and 6.5,respectively.The optimum temperatures were 45 and 60,and the half-inactivation time was 5 minutes and 30 minutes respectively.PGP enzyme activity: the optimum p H of CBP-PGP and PGP-CBP were 7.5 and 5.5,respectively.The optimum temperature was 30,35,and the half-inactivation time was 2 min and 14 min,respectively.The specific activity of PGP-CBP was 0.0258 micromol/min/mg,10% lower than that of CBP,0.0097 micromol/min/mg lower than that of PGP,and 51% lower than that of PGP.Under the same molar concentration of transstarch reaction system,the transstarch rate of PGP-CBP was 12.79%,the transstarch rate of CBP and PGP free enzyme was 16.84%.The substrate shuttle effect of PGP-CBP was determined by detecting G-1-P and introducing PGM competitive intermediates.The total catalytic activity of PGP-CBP fusion enzyme system decreased only by 1.58% after adding 200 U of PGM for 16 h.In this paper,two key enzymes,CBP and PGP,which restrict the cellulose starch conversion system,were constructed to improve the conversion efficiency of the system and solve the increasingly tense food security situation by constructing artificial multi-enzyme complex and fusion enzyme.The effects of ligand type and ligand type on CBP were investigated,and the advantages of rigid ligand type on the construction of fusion enzyme were proved.Although the first strategy could not achieve substrate shuttle,the fusion enzyme PGP-CBP showed higher CBP activity and PGP activity,and the starch conversion rate was not significantly different from that of free enzyme.PGP-CBP realizes substrate shuttle,which provides the possibility for the practical production and application of cellulose to starch,and is beneficial to the target.