Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Sep 13;88(17):e0102722.
doi: 10.1128/aem.01027-22. Epub 2022 Aug 11.

Engineering of Cyclodextrin Glycosyltransferase through a Size/Polarity Guided Triple-Code Strategy with Enhanced α-Glycosyl Hesperidin Synthesis Ability

Affiliations

Engineering of Cyclodextrin Glycosyltransferase through a Size/Polarity Guided Triple-Code Strategy with Enhanced α-Glycosyl Hesperidin Synthesis Ability

Hanchi Chen et al. Appl Environ Microbiol. .

Abstract

Hesperidin, a flavonoid enriched in citrus peel, can be enzymatically glycosylated using CGTase with significantly improved water solubility. However, the reaction catalyzed by wild-type CGTase is rather inefficient, reflected in the poor production rate and yield. By focusing on the aglycon attacking step, seven residues were selected for mutagenesis in order to improve the transglycosylation efficiency. Due to the lack of high-throughput screening technology regarding to the studied reaction, we developed a size/polarity guided triple-code strategy in order to reduce the library size. The selected residues were replaced by three rationally chosen amino acids with either changed size or polarity, leading to an extremely condensed library with only 32 mutants to be screened. Twenty-five percent of the constructed mutants were proved to be positive, suggesting the high quality of the constructed library. Specific transglycosylation activity of the best mutant Y217F was assayed to be 935.7 U/g, and its kcat/KmA is 6.43 times greater than that of the wild type. Homology modeling and docking computation suggest the source of notably enhanced catalytic efficiency is resulted from the combination of ligand transfer and binding effect. IMPORTANCE Size/polarity guided triple-code strategy, a novel semirational mutagenesis strategy, was developed in this study and employed to engineer the aglycon attacking site of CGTase. Screening pressure was set as improved hesperidin glucoside synthesis ability, and eight positive mutants were obtained by screening only 32 mutants. The high quality of the designed library confirms the effectiveness of the developed strategy is potentially valuable to future mutagenesis studies. Mechanisms of positive effect were explained. The best mutant exhibits 6.43 times enhanced kcat/KmA value and confirmed to be a superior whole-cell catalyst with potential application value in synthesizing hesperidin glucosides.

Keywords: cyclodextrin glycosyltransferase; hesperidin; mutagenesis strategy; transglycosylation.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Aglycon attacking site defined as the residues within 4 Å radius of the two glucose units (azure) in the +1 and +2 subsites. Other than the catalytic triad (yellow), the residues in the head pocket plus G283 (gray) were subjected to mutagenesis.
FIG 2
FIG 2
Thirty-two mutants were constructed based on the size/polarity guided triple-code strategy and cultured in shake flasks. First-round screening determines positive mutants (red arrow) based on the relative WCTAs compared with the wild type. Enzyme activities were assayed at pH 10.0, 40°C, with 50 mL shake flask cultured cells.
FIG 3
FIG 3
Eight mutants obtained by the first-round screening together with the wild type were purified and assayed with their STA. Enzyme activities were assayed at pH 10.0, 40°C, with 20 μL purified enzyme.
FIG 4
FIG 4
Whole cell catalyzed hesperidin glycosylation by Y217F (red triangle, formula image) and wild-type (blue circle, formula image). GH: hesperidin glucoside, G2H: hesperidin maltoside, G3H: hesperidin maltotrioside.
FIG 5
FIG 5
Relative locations of F205, Y217, and G283 to the catalytic pocket.
FIG 6
FIG 6
Hesperidin docked with the enzyme-glycosyl covalent complex. The distance between bond forming atoms (d = 2.8 Å) is within the van der Waals contact distance.
FIG 7
FIG 7
Enzyme-ligand interactions before and after mutation. (a) wild type; (b) F205 mutants (F205H, orange; F205V, yellow; F205L, green); (c) Y217 mutants (Y217F, green; Y217S, yellow); (d) G283 mutants (G283T, navy; G283L, yellow; G283V, green).
FIG 8
FIG 8
Changes of binding energy (ΔΔG) before and after mutagenesis.

Similar articles

Cited by

References

    1. Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B. 2014. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42:D490–D495. 10.1093/nar/gkt1178. - DOI - PMC - PubMed
    1. Janeček Š, Svensson B, MacGregor E. 2014. α-Amylase: an enzyme specificity found in various families of glycoside hydrolases. Cell Mol Life Sci 71:1149–1170. 10.1007/s00018-013-1388-z. - DOI - PMC - PubMed
    1. Uitdehaag JC, van der Veen BA, Dijkhuizen L, Dijkstra BW. 2002. Catalytic mechanism and product specificity of cyclodextrin glycosyltransferase, a prototypical transglycosylase from the α-amylase family. Enzyme Microb Technol 30:295–304. 10.1016/S0141-0229(01)00498-7. - DOI
    1. Uitdehaag JC, Van Alebeek GJW, van der Veen BA, Dijkhuizen L, Dijkstra BW. 2000. Structures of maltohexaose and maltoheptaose bound at the donor sites of cyclodextrin glycosyltransferase give insight into the mechanisms of transglycosylation activity and cyclodextrin size specificity. Biochemistry 39:7772–7780. 10.1021/bi000340x. - DOI - PubMed
    1. Szerman N, Schroh I, Rossi AL, Rosso AM, Krymkiewicz N, Ferrarotti SA. 2007. Cyclodextrin production by cyclodextrin glycosyltransferase from Bacillus circulans DF 9R. Bioresour Technol 98:2886–2891. 10.1016/j.biortech.2006.09.056. - DOI - PubMed

Publication types

LinkOut - more resources