Asp577 mutations enhance the catalytic efficiency of cyclodextrin glycosyltransferase from Bacillus circulans

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• 作者：Zhaofeng Li^a ; ^b ; ^c ; ¹ ; Min Huang^a ; ^b ; ¹ ; Zhengbiao Gu^a ; ^b ; ^c ; ^{zhengbiaogu@jiangnan.edu.cn" class="auth_mail" title="E-mail the corresponding author} ; Tod P. Holler^d ; Li Cheng^a ; ^b ; Yan Hong^a ; ^b ; ^c ; Caiming Li^a ; ^b ; ^{caimingli@jiangnan.edu.cn" class="auth_mail" title="E-mail the corresponding author} ; ^{licaiming2009@126.com" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Cyclodextrin glycosyltransferase ; Cyclodextrin ; Calcium-binding site
• 刊名：International Journal of Biological Macromolecules
• 出版年：2016
• 出版时间：February 2016
• 年：2016
• 卷：83
• 期：Complete
• 页码：111-116
• 全文大小：1218 K

文摘

The amino acid residue Asp 577 is located in calcium-binding site III (CaIII) of the cyclodextrin glycosyltransferase (EC 2.4.1.19, CGTase) from Bacillus circulans STB01. In the present study, the effects of replacing Asp577 with glycine, alanine, valine, leucine, and isoleucine on the catalytic efficiency of this CGTase were investigated. Two of these replacements, D577G and D577A, increased the β-cyclization activity of CGTase. Kinetic studies showed that the K_m values of D577G and D577A were 36.1% and 18.0% lower and the k_cat/K_m values were 43.9% and 23.0% higher than those of the wild-type enzyme, respectively. These mutations increased both the affinity of CGTase for maltodextrin and the catalytic efficiency of the cyclization reaction. Furthermore, although D577G and D577A only slightly enhanced β-cyclodextrin production, compared with the wild-type enzyme, their higher β-cyclization activities resulted in a significant reduction in the amount of mutant protein required during the cyclodextrin production process. Thus, the two mutants are more suitable for the industrial production of β-cyclodextrin than the wild-type enzyme. The enhancement of catalytic efficiency may be due to the smaller size of the glycine and alanine side chains, which may weaken the impact of this residue on CaIII.