Studies On Site-directed Mutagenesis Of Cyclodextrin Glycosyltransferase To Decrease Product Inhibition

With a unique hydrophilic surface and hydrophobic cavity, cyclodextrin can encapsulate lots of guest molecules. Therefore, it could be widely used in food, pharmaceuticals and cosmetic industries. Meanwhile, Cyclodextrin glycosyltransferase(C GTase, EC 2.4.19), enzyme for industrial cyclodextrin production, has become a hot area of research currently. In the process of enzymatic cyclodextrin production, the cyclization activity of CGTase would be inhibited by cyclodextrin when its concentration reached a threshold value. With the combination of cyclodextrin and CGTase, cyclization reaction would be inhibited, leading to the low yield of starch conversion and high cost of cyclodextrin production. Hence, it is crucial that solving the problem- inhibition of cyclodextrins to CGTases. In this paper, product inhibition patterns of two CGTases from different resources(α-CGTase from P. macerans JFB05-01 and β-CGTase from B. circulans STB01) were studied. β-cyclodextrin is the most widely used product in industrial applications. By analyzing inhibition patterns of C GTases from different resources, mutants were constructed based on β-CGTase to decrease the inhibition of β-cyclodextrin. Finally, simulation of industrial cyclodextrin production was conducted by using mutant CGTases. The main results are listed below:(1) Cyclization kinetic properties of C GTases can be well described by Michaelis-Menten equation with maltodextrin(DE 5) as substrate. The Lineweaver-Burk plots show that CGTases from different resources had different inhibition patterns, when adding cyclodextrins in the substrate as inhibitors. α-, β- and γ-cyclodextrins were competitive inhibitors for α-CGTase from P. macerans JFB05-01. However, β-CGTase from B. circulans STB01 represents a mixed inhibition pattern(competitive and noncompetitive inhibition) by adding cyclodextrins. It demonstrated that product inhibition pattern was decided by the resource of CGTase. Thus, our research found cyclodextrin had same inhibition type for each CGTase when determining different cyclization activities. Moreover, our study also discovered each kind of CD had the biggest impact to its corresponding cyclization reaction, like β-cyclodextrin has the strongest inhibition towards β-cyclization activity.(2) 5 mutant β-CGTases, A599 N, A599 V, A599N/Y633 A, A599V/Y633 A and MBS2(structure of β-CGTase in maltose binding site 2 was replaced by peptide chain of α-CGTase from the same region) were obtained by mutating β-CGTase at the maltose binding site 2. The 5 mutant β-CGTases’ enzymatic properties and product inhibition patterns with β-cyclodextrin as inhibitor were studied. O ur results showed that the mutation at maltose binding site 2 remarkably weakened noncompetitive inhibition and slightly decreased competitive inhibition, which the Ki values increased 2-4 fold. The crystal structure modeling of mutant β-CGTases elaborated mutation at maltose binding site 2 weakened the hydrogen bonds and Van der Vaal force between cyclodextrins and enzyme leading to a relieved product inhibition. The cyclization activities and catalytic efficiency were also influenced by mutation. Compared with wild type β-CGTase, total cyclization activity of A599 N, A599 V and A599V/Y633 A declined 15.6%, 76.8% and 70.1% respectively, while total cyclization activity of A599N/Y633 A and MBS2 increased 22.4% and 4.3%. Catalytic efficiency(Kcat/Km) only reserved 23.3% for A599 V, 9.8% for A599V/Y633 A, 64.7% for A599 N a nd 31.6% for A599N/Y633 A. MBS2 is the best, which 98% catalytic efficiency was kept. It assumed that single/double mutation might reduce the bind force between enzyme and substrate. Besides, mutations at maltose binding site 2 had mild influence on enzyme thermo stability and product specificity.(3) Lower inhibited mutants, A599 N, A599 V, A599N/Y633 A, A599V/Y633 A and MBS2 were used for β-cyclodextrin production. It was found that A599 N, A599N/Y633 A and MBS2 could obviously increase cyclodextrin yield when using soluble starch(5%, w/v) as substrate, without any organic solvent. Compared with wild-type β-CGTase, the soluble starch conversion increased 13.1%, 15.8% and 19.7% when using A599 N, A599N/Y633 A and MBS2 for enzyme reaction. MBS2 also could increase the starch conversion when using normal corn starch(20%, w/v) as substrate with cyclohexane in reaction system. But only MBS2 could increase the corn starch co nversion 15.6% when adding cyclohexane. A599 N, A599N/Y633 A decreased starch conversion. MBS2 is a very promising CGTase for cyclodextrin industrial production.