論文 - 詳細
| RRC ID | 88762 |
|---|---|
| 著者 | Vuillemin M, Lengyel M, Muschiol J, Matwiejuk M, Zhang Y, Cannac N, Molnar-Gabor D, Meyer AS, Zeuner B. |
| タイトル | Enzymatic lacto-N-biose elongation of human milk oligosaccharides with the GH136 lacto-N-biosidase LnbX engineered for improved transglycosylation. |
| ジャーナル | Enzyme Microb Technol |
| Abstract |
The lacto-N-biosidase (EC 3.2.1.140) LnbX from Bifidobacterium longum subsp. longum JCM 1217, a member of the glycoside hydrolase family 136 (GH136), was used for single-step lacto-N-biose (LNB) elongation of human milk oligosaccharides (HMOs) via disaccharide transglycosylation to expand the portfolio of HMOs available from enzymatic synthesis. We used the commercially available HMOs lacto-N-tetraose (LNT) as donor and lacto-N-neotetraose (LNnT) as acceptor for the synthesis of para-lacto-N-hexaose (pLNH). To improve the transglycosylation performance of LnbX (18 % molar yield), we designed 14 single mutants using two different strategies: 1) conservative substitution of conserved residues in the active site, and 2) shielding of the active site by large, hydrophobic residues. Protein engineering improved pLNH yield 1.5-fold as compared to the wild type (to 27 %). More than a 3-fold increase was obtained when optimizing the reaction conditions using the best variant, LnbX D416N, by an experimental design including reaction temperature, pH, donor substrate concentration, acceptor-to-donor (A/D) ratio, and enzyme concentration. Higher LNT concentrations and A/D ratios led to increased pLNH yields, and a high A/D ratio also increased the proportion of pLNH among the reaction products. The maximum molar yield of 57 % was obtained after 3 h of reaction at 100 mM LNT, 500 mM LNnT, 1 μM enzyme, 35 °C and pH 6.5. The LnbX D416N enzyme, which can use lacto-N-fucopentaose III (LNFP-III) as an alternative acceptor substrate, is active and stable at industrially relevant ranges of pH and temperature. The enzyme may thus be useful for diversification of the industrially available HMO portfolio. |
| 巻・号 | 189 |
| ページ | 110660 |
| 公開日 | 2025-9-1 |
| DOI | 10.1016/j.enzmictec.2025.110660 |
| PII | S0141-0229(25)00080-8 |
| PMID | 40328212 |
| MeSH | Bacterial Proteins* / chemistry Bacterial Proteins* / genetics Bacterial Proteins* / metabolism Bifidobacterium longum* / enzymology Bifidobacterium longum* / genetics Catalytic Domain Enzyme Stability Glycoside Hydrolases* / chemistry Glycoside Hydrolases* / genetics Glycoside Hydrolases* / metabolism Glycosylation Humans Hydrogen-Ion Concentration Kinetics Milk, Human* / chemistry Milk, Human* / metabolism Mutagenesis, Site-Directed Oligosaccharides* / metabolism Protein Engineering Recombinant Proteins / genetics Recombinant Proteins / metabolism Substrate Specificity Temperature |
| オルトメトリクス指標 |
オルトメトリクス指標項目
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| 最多言及媒体 | X(Twitter) |
| 各媒体での言及数の合計 | 1 |
| 過去6か月間でのオルトメトリクス指標の変動値 | 0.0 |
| リソース情報 | |
| 一般微生物 | JCM1217 |