Site-Directed Mutations in Alanine 223 and Glycine 255 in the Acceptor Site of γ-Cyclodextrin Glucanotransferase from Alkalophilic Bacillus clarkii 7364 Affect Cyclodextrin Production

Abstract

A cyclodextrin glucanotransferase (CGTase) from Bacillus clarkii 7364 converts starch into gamma-cyclodextrin (gamma-CD) with high specificity. Comparison of the deduced amino acid sequence of this CGTase with those of other typical CGTases revealed that several amino acids are deleted or substituted with others at several subsites. Of these amino acids, Ala223 at subsite +2 and Gly255 at subsite +3 in the acceptor site of the enzyme were replaced by several amino acids through site-directed mutagenesis. The replacement of Ala223 by lysine, arginine and histidine strongly enhanced the gamma-CD-forming activity in the neutral pH range. On the other hand, all mutants obtained on replacing Gly255 with the above amino acids showed significant decreases in the gamma-CD-forming activity. Taking into account both the kinetic parameters and pKa values of the side chains of the three basic amino acids, the protonation state of the amino groups in their side chains at subsite +2 seems to enhance the hydrogen bonding interaction between these basic amino acids and the glucose residues of linear oligosaccharides. The enhancement of the interaction may play an important role by helping the substrate reach subsite +1, hence increasing the gamma-CD-forming activity and kcat value.

Engineering of cyclodextrin glucanotransferases and the impact for biotechnological applications

“…Engineering CD specificity by mutating conserved acceptor subsites residues of CGTases is not advisable as this typically results in highly hydrolytic CGTases (Table 2) that form small quantities of CDs (Shim et al 2004; Kelly et al 2007). Mutating the nonconserved residue 232 (Lys or Ala) at acceptor subsite +2 slightly altered CD ratios but also lowered CD yields (Nakagawa et al 2006; Kelly et al 2008a). Figure 3 shows the CGTase residues/regions important for reaction specificity.…”

Stepwise error‐prone PCR and DNA shuffling changed the pH activity range and product specificity of the cyclodextrin glucanotransferase from an alkaliphilicBacillus sp

“…The CD 8 -synthesizing activity of the wild-type enzyme was 6.1 ± 0.45 nmol/ min and its CD 7 :CD 8 [37,61]. A site saturation mutagenesis at this position has been performed with the c-CGTase from Bacillus clarkii 7364 [28]. The changes did not include substitutions with apolar amino acids like Val.…”

“…Protein engineering of CGTases has been performed previously to improve their substrate and product specificity, as well as their thermostability [23]. By site-directed mutagenesis, the product specificity of a- [24][25][26], b- [27,28] and c-CGTases [29][30][31] has been enhanced successfully. Replacement of amino-acids at the subsite À3 [25,27,31], À6 [24], and À7 [26] Abbreviations: CD, cyclodextrin; CGTase, cyclodextrin glucanotransferase has been obtained by introduction of an additional salt bridge [32].…”

Purification and Properties of a New Thermostable Cyclodextrin Glucanotransferase from Bacillus pseudalcaliphilus 8SB

“…The studied CGTase could be compared to γ-CGTase from B. clarkii 7364 converting 13.7% of pre-gelatinized potato starch [10] and γ-CGTase 825-6 producing primarily γ-CD at pH 10.0, namely 7.2 mg ml −1 γ-CD and 3.5 mg ml −1 β-CD from 10% soluble starch with conversion of 10.7% [11]. The advantage of the new CGTase was its effective action on raw insoluble starch (50.7% conversion) at high temperatures of 60-65°C and a relatively high content of the formed γ-CD (3.7 mg ml −1 ), unlike the most other studies where the high production of γ-CD has been achieved by using of special reaction conditions, such as a heat or pullulanase pre-treatment of the starch; addition of some additives such as glucose [19], glycyrrhzic acid [20], toluene, cyclododecanone [19], and ethanol [21,22]; or by genetic manipulations [23,24].…”