AI Article Synopsis
- Fructooligosaccharides (FOSs) are prebiotics useful in various industries, and inulosucrase can be engineered to synthesize different FOS chain lengths from sucrose.
- Three engineered variants of inulosucrase (R483F, R483Y, R483W) showed improved binding to a specific substrate, leading to higher production of shorter chain FOSs (GF5) compared to the wild type.
- Even though the engineered variants displayed reduced overall catalytic activity, they could still achieve FOS synthesis yields of up to 73% from the substrate, confirming the effectiveness of the modifications.
Article 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 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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| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9064246 | PMC |
| http://dx.doi.org/10.1039/c9ra02137j | DOI Listing |
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