Studies On Mutagenesis Of Cyclodextirn Glucanotransferase At The Calcium Binding Sites For Enhancement Of Specificity

Cyclodextrins have characteristics of capacity to encapsulate molecules because of theirthree-dimension structure, which expand the application of cyclodextrins in the industriesrelated to food, pharmaceuticals, agriculture, etc. Cyclodextrin glucanotransferase (CGTase,EC2.4.1.19) is an extracellular enzyme capable of producing cyclodextrins through anintramolecular transglycosylation reaction, which is wildly used in production ofcyclodextrins. One disadvantage of CGTase is that wild-type CGTase produce a mixture of α-,β-and γ-cyclodextrins, which is not favorable to separation and purification. Thedisadvantage increased production cost of cyclodextrins, which limited its industrialutilization. Thus, it is highly desirable to improve specificity of CGTase to adapt to thedevelopment of industrialization. In the present research, the product of specificity of CGTasefrom Bacillus circulans STB01was improved through site-directed mutagenesis.Firstly, Sequence comparisons were performed using the sequence alignment program.Based on the results and crystal structure analysis, calcium binding sites widely exit inα-amylase family. The studies indicated that calcium binding site CaII exist commonly inCGTases and α-amylases, which most amino acids at this calcium binding site is highlyconserved. It was found that calcium binding site CaII located in the active center and hadvery important roles for CGTases. Mutations at calcium binding site CaII resulted decrease incyclization activity. Calcium binding sites CaI and CaIII have discrimination betweendifferent types of CGTases, which may be the reason that CGTases have differentcharacteristics. Calcium binding site CaI appears as U-shap where calcium binds in themiddle of the pocket. The residue31at the top of U-shap have clear discrimination amongdifferent types of CGTases: The residue31in α/β、β/α-CGTase is Ser, while in β-、β/γ-CGTaseis Ala. Calcium binding sites CaIII only exists in a few CGTases. According to study, Calciumbinding sites might be related to cyclization activity, thermostability, and product specificityof CGTases, which provided the directions for enhancement of β-cyclodextrin specificity.Secondly, the residue31locates at top of U-shap, which could improve cyclizationactivity and product specificity of CGTase. Thus, the cgt gene from B. circulans STB01withappropriate modification at residue31was cloned and transformed into the host B.subtilis WB600. The results indicated that the replacement of Ala31by Arg, Pro and Threnhanced β-cyclodextrin specificity of CGTase. The mutants A31R, A31P and A31Tdisplayed25.8%,16.1%and9.7%increase in production of β-cyclodextrin. Otherwise, themutation A31S enhanced α-cyclodextrin specificity of CGTase while the mutation A31Eincreased γ-cyclodextrin specificity of CGTase. The increased β-cyclodextrin specificity ofmutants might because appropriate mutations stabilized calcium binding sites and specificconformation of CGTase, which resulted β-CGTase produced much β-cyclodextrin. The enhancement of α-or γ-cyclodextrin specificity of these mutants might be a result of clashingwith the calcium ions and residues around calcium binding sites, which were not suitable forproduction of β-cyclodextrin. The product inhibition of cyclodextrin weakened whilepromoted α-or γ-cyclodextrins production. Moreover, in order to verify stability of calciumbinding sites affected specificity of CGTase, the mutants N29K, A31G from β-CGTase andmutants T31A, T31S from α-CGTase were constructed and analyzed, which furtherconformed that much stability of calcium binding sites in β-CGTase were more suitable forβ-cyclodextrin production.Finally, Calcium binding site CaIII only exists in few CGTases. The replacement of Ala315by hydrophilic residues increased β-cyclodextrin specificity. The mutants A315R andA315D showed12%and10%shift in β-cyclodextrin activities compared to wild-typeCGTase. Furthermore, for the mutants A315R and A315D, the production of β-cyclodextrinincreased15.1%and10.8%, respectively. The mutation of Ala315into Thr, Val and Ileshowed no significant effect. The enhancement of β-cyclodextrin specificity for the mutantsmight be due to hydrophilic residues tended to distribute surface of CGTase and benefited forstability of calcium binding sites, Compared to wild-type CGTase, the mutants with higherβ-cyclodextrin specificity of CGTase were more suitable for industrial production ofβ-cyclodextrin. Besides, some mutations of Asp577could affect specificity of CGTase. Theresults suggested that calcium binding site CaIII was important for specificity of CGTase. Inaddition, appropriate mutations of calcium binding sites CaI and CaIII could change theresponse of calcium ions, it indicated that calcium binding sites were crucial to CGTase.