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Charoenwongpaiboon, Thanapon, Klaewkla, Methus, Chunsrivirot, Surasak, Wangpaiboon, Karan, Pichyangkura, Rath, Field, Robert A. 
ORCID: https://orcid.org/0000-0001-8574-0275 and Prousoontorn, Manchumas Hengsakul
(2019)
Rational re-design of Lactobacillus reuteri 121 inulosucrase for product chain length control.
RSC Advances, 9 (26).
pp. 14957-14965.
ISSN 2046-2069
Abstract
Fructooligosaccharides (FOSs) are well-known prebiotics that are widely used in the food, beverage and pharmaceutical industries. Inulosucrase (E.C. 2.4.1.9) can potentially be used to synthesise FOSs from sucrose. In this study, inulosucrase from Lactobacillus reuteri 121 was engineered by site-directed mutagenesis to change the FOS chain length. Three variants (R483F, R483Y and R483W) were designed, and their binding free energies with 1,1,1-kestopentaose (GF4) were calculated with the Rosetta software. R483F and R483Y were predicted to bind with GF4 better than the wild type, suggesting that these engineered enzymes should be able to effectively extend GF4 by one residue and produce a greater quantity of GF5 than the wild type. MALDI-TOF MS analysis showed that R483F, R483Y and R483W variants could synthesise shorter chain FOSs with a degree of polymerization (DP) up to 11, 10, and 10, respectively, while wild type produced longer FOSs and in polymeric form. Although the decrease in catalytic activity and the increase of hydrolysis/transglycosylation activity ratio was observed, the variants could effectively synthesise FOSs with the yield up to 73% of substrate. Quantitative analysis demonstrated that these variants produced a larger quantity of GF5 than wild type, which was in good agreement with the predicted binding free energy results. Our findings demonstrate the success of using aromatic amino acid residues, at position D418, to block the oligosaccharide binding track of inulosucrase in controlling product chain length.
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| Additional Information: | Funding Information: TC is thankful for scholarship supports from Science Achievement Scholarship of Thailand (SAST). Supports from the Overseas Research Experience Scholarship for Graduate Student and Faculty of Science Chulalongkorn University are acknowledged. MK gratefully acknowledged the Scholarship from the Graduate School, Chulalongkorn University to commemorate the 72nd anniversary of his Majesty King Bhumibol Adulyadej. SC was also partially supported by Structural and Computational Biology Research Unit, Department of Biochemistry, Faculty of Science, Rachadaphiseksomphot Endowment Fund, Chula-longkorn University, the Institute for the Promotion of Teaching Science and Technology (IPST) under the Research Fund for DPST Graduate with First Placement [Grant no. 07/2557] and Chulalongkorn University; Government Budget. Research at the JIC is supported by the UK BBSRC Institute Strategic Program on Molecules from Nature - Products and Pathways [BBS/E/J/ 000PR9790] and the John Innes Foundation. Publisher Copyright: © 2019 The Royal Society of Chemistry. |
| Uncontrolled Keywords: | chemistry(all),chemical engineering(all) ,/dk/atira/pure/subjectarea/asjc/1600 |
| Faculty \ School: | Faculty of Science > School of Chemistry, Pharmacy and Pharmacology |
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| Depositing User: | LivePure Connector |
| Date Deposited: | 02 Sep 2024 15:30 |
| Last Modified: | 25 Sep 2024 18:05 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/96450 |
| DOI: | 10.1039/c9ra02137j |
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