| As an important class of starch derivatives,cyclodextrins(CD)are produced by cyclodextrin glycosyltransferase(CGTase)from starch,and are widely used in food,medicine,materials,cosmetics and other fields.CGTase cannot act on the branch points in starch,which limits the utilization of starch substrate during the production of CD.Therefore,it is considered to add pullulanase in the reaction to solve this problem.However,CD is an inhibitor of pullulanase,which makes pullulanase unable to effectively perform debranching during the process of synergistic conversion with CGTase to produce CD.Based on the above problems,the thesis wants to modify pullulanase through site-directed mutagenesis to weaken the inhibitory effect of CD on it.Screening pullulanase with CD ligand in the PDB database,pullulanase from Bacillus subtilis str.168(PulA)was found and recombinantly expressed.The binding mechanism and structural basis of PulA and CD were explored in this thesis.Mutants with weakened inhibitory effect of CD were obtained by site-directed mutagenesis.The enzyme activity of the mutants,the changes in the inhibitory effect of CD on the mutants,the effect of the mutations on the stability of the enzyme structure and ligand binding,and other enzymatic properties were investigated.Finally,the effective mutants were used in simultaneous transformation process with CGTase to produce CD to investigate its application properties.The main research contents are as follows:(1)Recombinant expression and enzymatic properties characterization of PulA.The secretory expression vector p ET-20b-pul A with encoding gene of pul A from B.subtilis str.168was constructed and heterologous expressed in E.coli BL21(DE3).The fermentation broth of the recombinant bacteria was purified by two steps of ammonium sulfate precipitation and nickel affinity chromatography to obtain the PulA in electrophoretic pure with a molecular weight of about 83 k Da.The results from enzymatic properties determination showed that PulA had hydrolysis activity and reverse hydrolysis activity.The optimum temperature and p H were40℃and 6.0,respectively.The specific activity of PulA was 136.1 U/mg at optimal conditions.When PulA andβ-CGTase acted synergistically on potato starch,tapioca starch,and corn starch,the improvement of substrate utilization and product conversion rate was very limited.(2)Study on the interaction between PulA and CD.α-CD,β-CD,andγ-CD all had inhibitory effects on PulA,and the inhibitory effect and inhibitory rate were both shown asβ-CD>α-CD>γ-CD.For the three CD,the inhibitory effect increased with the increase of CD concentration,and decreased with the increase of substrate concentration.Inhibition kinetics results showed that the inhibition mode ofα-CD,β-CD,andγ-CD on PulA were all in competitive inhibition,and the inhibition constants(K_i)were 2.25,1.04,and 5.73 m M,respectively.The results from isothermal titration calorimetry(ITC)assay showed that the main driving force for the binding between PulA and cyclodextrin was hydrophobic interaction,and the dissociation constants(K_D)of the three CD and PulA were 4.59,3.84,and 15.30μM,respectively.The results from crystal structure analysis and molecular docking showed that the common amino acid residues bound to three CD in PulA were W437,D465,R468,F476,H477,N526,and E579,respectively.(3)The study on breaking the binding structure of the aromatic amino acid residues at acceptor subsites in PulA to CD.W437 and F476 at acceptor subsites in PulA were absolutely conserved in pullulanases from different sources,and they interacted with CD to form a stable"sandwich"structure by stacking force and hydrophobic interaction,respectively.To breaking the stable binding of aromatic amino acid residues of W437 and F476 at the acceptor subsites in PulA to CD inhibitors,W437G,W437P,W437R,W437F,F476Y,F476V,F476H,F476C,F476D,and F476A mutants were constructed and heterologously expressed.The optimal temperature and optimal p H of mutants remained to be 40℃ and 6.0,respectively.The results from enzyme activity determination showed that the mutation of W437 almost lost the hydrolysis activity.Results from circular dichroism spectroscopy,ITC,and molecular docking assay showed that the secondary structures of W437 mutants changed significantly and the structural stability was severely destroyed,thus losing the ligand binding capacity and causing the loss of enzyme activity.The enzyme activities of F476 mutants including F476Y,F476V,F476H,F476C,F476D,and F476A,were all reduced,which were 35.3%,1.6%,32.8%,31.0%,2.7%,and 7.8%of PulA,respectively.Results from inhibition kinetics showed the inhibitory constants(K_i)of the F476 mutants were 3.9,1.3,4.2,3.5,1.4,and 1.6 m M,respectively,which were all higher than the K_i value of PulA,suggesting that the inhibitory effect of CD on the mutants was significantly lower than that of PulA.Among of mutants,F476C and F476H have better apparent activities with 29.9 and 35.7 U/mg,respectively,which both higher than that of PulA.The results of ITC showed that the dissociation constants(K_D)of F476C and F476H withβ-CD were 32.1 and 22.7μM,respectively,which were both higher than that of PulA,further indicating that the inhibitory effect of CD on mutants was weakened compared to that of PulA.The best mutant F476H was used to produceβ-CD combined withβ-CGTase.Compared with the conversion rate from reaction without debranching enzymes,the conversion rates from F476H andβ-CGTase on potato starch,tapioca starch,and corn starch were increased by 20.1%,30.3%,and 29.0%,respectively.(4)Research on the contribution of non-conserved amino acid residues on the inhibitory effect by CD on PulA.E579 at acceptor subsites in PulA interacted with CD by hydrogen bond and it was not conserved in pullulanases from different sources.Results from molecular docking between D465 saturation mutants andβ-CD showed that the binding free energy of E579A,E579G,E579Q,E579D,E579P,E579C,E579F,and E579S toβ-CD was higher than that of PulA.Therefore,the above mutants were constructed and expressed heterologously.Enzyme activities of these mutants were determined to be 18.7,29.5,83.3,61.6,3.9,3.2,24.5,and 18.2U/mg,respectively,which were all lower than that of PulA at 136.1 U/mg.The inhibition rates of 10 m Mβ-CD on mutants PulA,E579P,E579Q,E579S,E579D,E579F,E579C,E579A,and E579G were 85%,6%,36%,78%,72%,26%,12%,89%,and 85%,respectively.The inhibitory effects ofβ-CD on mutants were reduced except for the mutants E579A and E579G.It is a pity that the apparent activities of the mutants were lower than that of PulA.The results from circular dichroism and fluorescence spectroscopy showed that the mutation of E579 destroyed the secondary and tertiary structure of the enzyme,which reduced the enzyme activity,indicating that this site is essential for maintaining the catalytic structure stability of the enzyme.(5)Research on the contribution of conserved amino acid residues on the inhibitory effect of CD on PulA.D465 at acceptor subsite in PulA interacted with CD by hydrogen bond and it was absolutely conserved in all pullulanases from different sources.To break the hydrogen bond binding between D465 and CD,mutants D465E,D465N,D465A,D465G,D465K,D465R,and D465I were constructed and heterologous expressed.The electrophoretic pure mutants with a molecular weight of about 83 k Da were obtained using the same two-step purification method as PulA.The enzyme activities of mutants were lower than those of PulA,and showed as 89.4,18.3,3.0,2.4,1.5,1.2,and 1.6 U/mg,respectively.The optimum temperature of mutants changed slightly,and the optimum p H remained to be 6.0.The inhibition rate of 10 m Mβ-CD on mutants D465E,D465N,D465A,D465G,D465R,D465K,and D465I were 68%,66%,49%,40%,3%,21%,and 1%,respectively,which all lower than that of PulA.and D465I,respectively,suggesting that the inhibitory effect of CD on D465 mutants was decreased.Among of them,mutant D465E had the highest apparent activity.Inhibition kinetic results showed that the inhibitory constant K_i ofβ-CD on D465E was 1.28 m M,which was lower than that of PulA,further indicating that the inhibitory effect of CD on mutant D465E was reduced.The best mutant D465E was used to produceβ-CD combined withβ-CGTase.Compared with the conversion rate from reaction without debranching enzymes,the conversion rates from D465E andβ-CGTase on potato starch,tapioca starch,and corn starch were increased by 13.6%,22.5%,and 23.1%,respectively. |
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