Improving high-degree-polymerization GOS synthesis of a marine cold-active β-galactosidase via semi-rational design
Open accessPublished version of a preprint
Abstract
Cold-active β-galactosidase has significant application value in the enzymatic synthesis of galacto-oligosaccharides (GOS); however, its low transglycosylation efficiency and poor selectivity toward high-degree polymerization (DP) products remain major challenges. In this study, a cold-active β-galactosidase (K3-β-gal) from marine Bacillus sp. K-3 was cloned and expressed. Four mutants (R252M, F261W, Y281R, and F355K) were constructed via semi-rational design, among which R252M exhibited markedly enhanced transglycosylation activity. Compared with the wild-type enzyme, R252M showed no change in optimum temperature (20 °C) or optimum pH (6.0), and retained good stability under low-temperature and weakly acidic conditions. The total GOS yield increased from 21.68% to 38.12% (a 75.83% improvement), and the DP4/DP3 ratio increased from 0.95 to 2.6, while the hydrolytic activity (k cat/k m) decreased by approximately 44%, indicating a shift in the catalytic equilibrium from hydrolysis toward transglycosylation. Molecular dynamics simulations revealed that R252M remodels the hydrogen-bond network in the active pocket, enhances protein rigidity, and reduces dynamic perturbations around the active site, which may contribute to substrate binding and transglycosylation efficiency.", This mutant holds promising potential for the green synthesis of high-DP GOS.
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
The self-check needs the paper’s full text.
Cites
Highly accurate protein structure prediction with AlphaFold
Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., Tunyasuvunakool, K., Bates, R., Žídek, A., Potapenko, A., et al. (2021) · Nature · doi:10.1038/s41586-021-03819-2 · Cited by 43,764 · cited 1× · see citing works
“Based on this, the three-dimen - sional structure of the target gene was predicted using AlphaFold2, and the predicted structure was aligned with the aforementioned homologous proteins to identify the substrate-binding pocket (Jumper et al. 2021).”
Huang, S.-Y., Grinter, S. Z., & Zou, X. (2010) · Physical Chemistry Chemical Physics · doi:10.1039/c0cp00151a · Cited by 508 · cited 1× · see citing works
“The overlap between the two energy terms is less than 30%, and their complementary use helps reduce the false positive rate (Huang et al. 2010).”
Marín-Manzano, M. C., Abecia, L., Hernández-Hernández, O., Sanz, M. L., Montilla, A., Olano, A., Rubio, L. A., Moreno, F. J., & Clemente, A. (2013) · Journal of Agricultural and Food Chemistry · doi:10.1021/jf402218z · Cited by 69 · see citing works
Flagged first, then by citations
- Authors
- Pan, L., Zhang, Y., Wang, Z., Fan, C., You, Q., Wu, J., Feng, F., & Wang, X.
- Affiliations
- Dalian University (CN)
- Corresponding author
- Wang, Xiaohui
- ORCID
- Pan, Lin
- Venue
- Archives of Microbiology
- Published
- 2026-06-30
- Volume
- 208
- Issue
- 9
- ISSN
- 0003-9276 (ISSN-L)
- 0302-8933
- 1432-072X
- OpenAlex
- W7166648798
- Cited by
- 0
- References
- 3
- FWCI
- 0.00
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Licence
- Locations
- Archives of Microbiology (publishedVersion)
- Research Square (acceptedVersion, cc-by)
- SSRN Electronic Journal (acceptedVersion)
- PubMed (publishedVersion)
- Funders
- National Natural Science Foundation of China for Young Scientists Project (31500039)
- Dalian Youth Science and Technology Star Project (2017RQ155)
- Natural Science Foundation of Liaoning Province (20180550728)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Enzyme Production and Characterization
- Physical Sciences › Chemistry › Organic Chemistry › Carbohydrate Chemistry and Synthesis
- Keywords
- galactooligosaccharides, transglycosylation, site-directed mutagenesis, marine enzymes, enzyme stability, molecular dynamics simulation, enzymatic synthesis
- MeSH terms
- Bacillus, Bacterial Proteins, beta-Galactosidase, Cold Temperature, Enzyme Stability, Galactose, Glycosylation, Hydrogen-Ion Concentration, Kinetics, Oligosaccharides, Substrate Specificity, Molecular Dynamics Simulation, Polymerization
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
