Double mutations enhance β-cyclization activity of cyclodextrin glycosyltransferase from Bacillus circulans

Publication date: Available online 14 December 2016 Source:Journal of Molecular Catalysis B: Enzymatic Author(s): Min Huang, Junyan Ren, Caiming Li, Zhengbiao Gu, Yan Hong, Li Cheng, Zhaofeng Li A major drawback to the industrial production of β-cyclodextrin is the limited β-cyclization activity of cyclodextrin glycosyltransferase (CGTase). Here, we construct mutants of the β-CGTase from Bacillus circulans strain STB01 that contain single substitutions at Tyr89 and double substitutions at Tyr89 and Asp577. The results show that the double mutants Y89G/D577R, Y89D/D577R, and Y89N/D577R display enhanced β-cyclization activity, and have higher β-cyclization activity than that of the three single Tyr89 mutants. The double mutant Y89D/D577R exhibited the highest β-cyclization activity and β-cyclodextrin production, increasing 35.1% and 12.4% compared with those of the wild-type CGTase, respectively. The β-cyclization activity of double mutant Y89D/D577R is also higher than that of the single mutant D577R, which had the highest β-cyclization activity among the mutants prepared in our previous studies. The enhanced β-cyclization activity of these mutants may be a result of intermolecular interactions that stabilize intermediates in the β-cyclization reaction. Thus, double mutant Y89D/D577R is much more suitable for industrial β-cyclodextrin production than the wild-type enzyme. Graphical abstract

Source: Journal of Molecular Catalysis B: Enzymatic - December 13, 2016 Category: Biochemistry Source Type: research