Site-directed Mutagenesis Of Chitinase From Bacillus Thuringiensis WB7

Chitinases(EC 3.2.1.14)are glycosyl hydrolases that catalyze the hydrolysis ofβ-(1,4)-glycosidic bonds in chitin,the major structural polysaccharide presenting in the cuticle and gut peritrophic matrix of insects.In this study,six active site residues. including phenylalanine 201(F201),glycin 203(G203),aspartic acid 205(D205), aspartic acid 207(D207),tryptophan 208(W208)and glutamine 209(E209),in catalytic domain of chitinase from Bacillus thuringiensis WB7,were substituted with other amino acids by site-directed mutagenesis.Twelve mutant proteins,pET-F201L, pET-F201Y,pET-G203A,pET-G203D,pET-D205E,pET-D205N,pET-D207E,pET-D207N, pET-W208C,pET-W208R,pET-E209D and pET-E209Q as well as the wild-Wpe enzyme,were produced in E.coli BL21(DE3)using pET-29a as expression vector.The detection of enzymatic activity showed that the mutants pET-F201L,pET-G203D, pET-D205N,pET-D207E,pET-D207N,pET-W208C,pET-E209D were devoid of activity,and that the loss of the enzymatic activity for pET-F201Y, pET-G203A,pET-D205E,pET-W208R and pET-E209Q was 72%,70%,48%,31% and 29%,respectively,compared with the wild-type enzyme.The pH-activity profile indicated that the optimal pH for pET-F201 Y,pET-G203A,pET-D205E,pET-W208R, pET-E209Q and the wild-type enzyme was pH 8.0 whereas the valid pH range was reduced bv about 1 unit for pET-D205E,and by about 2 units for pET-G203A and pET-F201Y.The mutant pET-G203A also resulted in a reduction of thermostability, for the optimal temperature for other mutants as well as wild-type enzyme was 60℃. but 50℃for pET-G203A,whose enzymatic activity,would greatly be destroyed as the temperature reached 60℃.It was suggested that G203 may be crucial for maintaining the three dimensional structure of the protein.D207 was supposed to be ionized at reaction pHs,and the negative charge it carried formed an ion-pair to help stabilize the positively charged oxazoline ion intermediate formed as carbonyl oxygen of the N-acetyl group reacted with the C-1 of N-acetylglucosamine at the-1 position of the substrate in the reaction.D207 then continued to promote the reaction to further stabilize the transition state.Another possible function of D207 was to enhance the acidity,of E209 protonated.D205 also played an important role in catalysis because it was supposed that there was hydrogen bonding between D205 and D207,which may influence the ionization state of D207 and then influence the ionization state of the active center. W208 may be involved in interactions with sugars by stacking interactions and hydrogen bonding,which probably participate in the recognition and binding between the chitinase and substract.It was supposed that E209 may serve as a general acid to provide proton in the catalysis of the chitinase,which perhaps take part in the first and most important step in the catalysis,namely the protonation of the susceptible glycosidic bond,and may participate in the following step as a proton acceptor from water. The role of F201 in the catalysis was not clear now.