A single mutation in cyclodextrin glycosyltransferase from Paenibacillus barengoltzii changes cyclodextrin and maltooligosaccharides production

Abstract

Cyclodextrin glycosyltransferases (CGTases) are bacterial enzymes that catalyze starch conversion into cyclodextrins, which have several biotechnological applications including solubilization of hydrophobic compounds, masking of unpleasant odors and flavors in pharmaceutical preparations, and removal of cholesterol from food. Additionally, CGTases produce maltooligosaccharides, which are linear molecules with potential benefits for human health. Current research efforts are concentrated in the development of engineered enzymes with improved yield and/or particular product specificity. In this work, we analyzed the role of four residues of the CGTase from Paenibacillus barengoltzii as determinants of product specificity. Single mutations were introduced in the CGTase-encoding gene to obtain mutants A137V, A144V, L280A and M329I and the activity of recombinant proteins was evaluated. The residue at position 137 proved to be relevant for CGTase activity. Molecular dynamics studies demonstrated additionally that mutation A137V produces a perturbation in the catalytic site of the CGTase, which correlates with a 10-fold reduction in its catalytic efficiency. Moreover, this mutant showed increased production of maltooligosaccharides with a high degree of polymerization, mostly maltopentaose to maltoheptaose. Our results highlight the role of residue 137 as a determinant of product specificity in this CGTase and may be applied to the rational design of saccharide-producing enzymes.

Improving Galactooligosaccharide Synthesis Efficiency of β-Galactosidase Bgal1-3 by Reshaping the Active Site with an Intelligent Hydrophobic Amino Acid Scanning

“…Analysis of the microenvironments showed that the hydrophobicity around these two positions was significantly increased after the mutations (Figure 7B−D), which may reduce the diffusion of water in the active site and thus boost transglycosylation reaction. 8,31,34,38,39 Interestingly, C510 and H512 are located at the end of a cleft leading to the active site and look like a pair of gatekeepers (Figure 7B). Previous studies have shown that water channels can be formed based on such architectures, in which water molecules could be transferred from the protein surface to the active site.…”

“…This indicated that the mutations caused very little perturbation to the overall structure and affected the enzymatic property through subtle molecular adjustments. Analysis of the microenvironments showed that the hydrophobicity around these two positions was significantly increased after the mutations (Figure B–D), which may reduce the diffusion of water in the active site and thus boost transglycosylation reaction. ,,,, …”

CGTase, a novel antimicrobial protein from Bacillus cereus YUPP‐10, suppresses Verticillium dahliae and mediates plant defence responses

“…Paenibacillus spp. (Castillo et al, 2018). CGTase can catalyse four reactions: cyclization, coupling, disproportionation, and hydrolysis (Li et al, 2014b), though the hydrolysis activity is relatively weak (Costa et al, 2009).…”

Increasing γ-CD conversion rates by improving thermostability of Bacillus sp. FJAT-44876 γ-CGTase

“…In summary, γ-CGTase with improved thermostability successfully increased yields of γ-CD and total CDs. In others work, protein engineering for improving yield of CDs payed more attention on changing product specificity or improving cyclization activity (Castillo et al, 2018;Lee et al, 2002;Wang et al, 2020). There also had limited studies to increase the yield of CDs by improving the thermostability.…”