Improvement of the Transglycosylation Efficiency of a Lacto-N-Biosidase from Bifidobacterium bifidum by Protein Engineering
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals
2.2. In Silico Analyses
2.3. Protein Engineering and Recombinant Expression of LnbB in E. coli
2.4. Synthesis of LNT2 Isomers
2.5. Synthesis of LNB-Oxazoline
2.6. Screening of LnbB Variants by Enzymatic Transglycosylation with LNB-Oxazoline and Lactose
2.7. Enzymatic Transglycosylation with pNP-LNB and Lactose for Selected LnbB Variants
2.8. Determination of Melting Temperature Tm
2.9. Analysis of Transglycosylation Reactions
2.10. Preparative Purification of LNT Isomers
2.11. Structural Elucidation of LNT Isomers
2.12. Reduction
2.13. Statistics
3. Results and Discussion
3.1. Screening of LnbB Variants
3.1.1. Glycosynthase Variants: D320E, D320T, and Y419N
3.1.2. Conserved Residues: W373F, W373H, W394F, W394H, W465F, W465H, and D467N
3.1.3. Residues Conserved in LNBases Only: N259T
3.1.4. The Arg Residue at the Edge of the Active Site: G398R and ΔG398
3.2. Regioselectivity in Transglycosylation
Reaction Product Analysis
3.3. Significance of the Donor Substrate
3.4. Thermal Stability of Best Variants
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Sano, M.; Hayakawa, K.; Kato, I. An enzyme releasing lacto-N-biose from oligosaccharides. Proc. Natl. Acad. Sci. USA 1992, 89, 8512–8516. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Sano, M.; Hayakawa, K.; Kato, I. Purification and characterization of an enzyme releasing lacto-N-biose from oligosaccharides with type 1 chain. J. Biol. Chem. 1993, 268, 18560–18566. [Google Scholar] [CrossRef]
- Wada, J.; Ando, T.; Kiyohara, M.; Ashida, H.; Kitaoka, M.; Yamaguchi, M.; Kumagai, H.; Katayama, T.; Yamamoto, K. Bifidobacterium bifidum lacto-N-biosidase, a critical enzyme for the degradation of human milk oligosaccharides with a type 1 structure. Appl. Environ. Microbiol. 2008, 74, 3996–4004. [Google Scholar] [CrossRef] [Green Version]
- Lombard, V.; Golaconda Ramulu, H.; Drula, E.; Coutinho, P.M.; Henrissat, B. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 2014, 42, D490–D495. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Yamada, C.; Gotoh, A.; Sakanaka, M.; Hattie, M.; Stubbs, K.A.; Katayama-Ikegami, A.; Hirose, J.; Kurihara, S.; Arakawa, T.; Kitaoka, M.; et al. Molecular insight into evolution of symbiosis between breast-fed infants and a member of the human gut microbiome Bifidobacterium longum. Cell Chem. Biol. 2017, 24, 515–524.e5. [Google Scholar] [CrossRef] [Green Version]
- Hattie, M.; Ito, T.; Debowski, A.W.; Arakawa, T.; Katayama, T.; Yamamoto, K.; Fushinobu, S.; Stubbs, K.A. Gaining insight into the catalysis by GH20 lacto-N-biosidase using small molecule inhibitors and structural analysis. Chem. Commun. 2015, 51, 15008–15011. [Google Scholar] [CrossRef] [Green Version]
- Ito, T.; Katayama, T.; Hattie, M.; Sakurama, H.; Wada, J.; Suzuki, R.; Ashida, H.; Wakagi, T.; Yamamoto, K.; Stubbs, K.A.; et al. Crystal structures of a glycoside hydrolase family 20 lacto-N-biosidase from Bifidobacterium bifidum. J. Biol. Chem. 2013, 288, 11795–11806. [Google Scholar] [CrossRef] [Green Version]
- Muschiol, J.; Vuillemin, M.; Meyer, A.S.; Zeuner, B. β-N-Acetylhexosaminidases for carbohydrate synthesis via trans-glycosylation. Catalysts 2020, 10, 365. [Google Scholar] [CrossRef] [Green Version]
- Castejón-Vilatersana, M.; Faijes, M.; Planas, A. Transglycosylation activity of engineered Bifidobacterium lacto-N-biosidase mutants at donor subsites for lacto-N-tetraose synthesis. Int. J. Mol. Sci. 2021, 22, 3230. [Google Scholar] [CrossRef]
- Vocadlo, D.J.; Withers, S.G. Detailed comparative analysis of the catalytic mechanisms of β-N-acetylglucosaminidases from families 3 and 20 of glycoside hydrolases. Biochemistry 2005, 44, 12809–12818. [Google Scholar] [CrossRef]
- Bojarová, P.; Bruthans, J.; Křen, V. β-N-Acetylhexosaminidases—The wizards of glycosylation. Appl. Microbiol. Biotechnol. 2019, 103, 7869–7881. [Google Scholar] [CrossRef]
- Zeuner, B.; Teze, D.; Muschiol, J.; Meyer, A.S. Synthesis of human milk oligosaccharides: Protein engineering strategies for improved enzymatic transglycosylation. Molecules 2019, 24, 2033. [Google Scholar] [CrossRef] [Green Version]
- Thurl, S.; Munzert, M.; Boehm, G.; Matthews, C.; Stahl, B. Systematic review of the concentrations of oligosaccharides in human milk. Nutr. Rev. 2017, 75, 920–933. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Murata, T.; Inukai, T.; Suzuki, M.; Yamagashi, M.; Usui, T. Facile enzymatic conversion of lactose into lacto-N-tetraose and lacto-N-neotetraose. Glycoconj. J. 1999, 16, 189–195. [Google Scholar] [CrossRef] [PubMed]
- Schmölzer, K.; Weingarten, M.; Baldenius, K.; Nidetzky, B. Lacto-N-tetraose synthesis by wild-type and glycosynthase variants of the β-N-hexosaminidase from Bifidobacterium bifidum. Org. Biomol. Chem. 2019, 17, 5661–5665. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Muschiol, J.; Meyer, A.S. A chemo-enzymatic approach for the synthesis of human milk oligosaccharide backbone structures. Z. Naturforsch. C. 2019, 74, 85–89. [Google Scholar] [CrossRef] [PubMed]
- Schmölzer, K.; Weingarten, M.; Baldenius, K.; Nidetzky, B. Glycosynthase principle transformed into biocatalytic process technology: Lacto-N-triose II production with engineered exo-hexosaminidase. ACS Catal. 2019, 9, 5503–5514. [Google Scholar] [CrossRef]
- Visnapuu, T.; Teze, D.; Kjeldsen, C.; Lie, A.; Duus, J.Ø.; André-Miral, C.; Pedersen, L.H.; Stougaard, P.; Svensson, B. Identification and characterization of a β-N-acetylhexosaminidase with a biosynthetic activity from the marine bacterium Paraglaciecola hydrolytica S66T. Int. J. Mol. Sci. 2020, 21, 417. [Google Scholar] [CrossRef] [Green Version]
- Danby, P.M.; Withers, S.G. Advances in enzymatic glycoside synthesis. ACS Chem. Biol. 2016, 11, 1784–1794. [Google Scholar] [CrossRef]
- Teze, D.; Coines, J.; Raich, L.; Kalichuk, V.; Solleux, C.; Tellier, C.; André-Miral, C.; Svensson, B.; Rovira, C. A single point mutation converts GH84 O-GlcNAc hydrolases into phosphorylases: Experimental and theoretical Evidence. J. Am. Chem. Soc. 2020, 142, 2120–2124. [Google Scholar] [CrossRef] [PubMed]
- Umekawa, M.; Huang, W.; Li, B.; Fujita, K.; Ashida, H.; Wang, L.X.; Yamamoto, K. Mutants of Mucor hiemalis endo-β-N-acetylglucosaminidase show enhanced transglycosylation and glycosynthase-like activities. J. Biol. Chem. 2008, 283, 4469–4479. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Slámová, K.; Krejzová, J.; Marhol, P.; Kalachova, L.; Kulik, N.; Pelantová, H.; Cvačka, J.; Křen, V. Synthesis of derivatized chitooligomers using transglycosidases engineered from the fungal GH20 β-N-acetylhexosaminidase. Adv. Synth. Catal. 2015, 357, 1941–1950. [Google Scholar] [CrossRef]
- Ruzic, L.; Bolivar, J.M.; Nidetzky, B. Glycosynthase reaction meets the flow: Continuous synthesis of lacto-N-triose II by engineered β-hexosaminidase immobilized on solid support. Biotechnol. Bioeng. 2020, 117, 1597–1602. [Google Scholar] [CrossRef] [Green Version]
- Fujita, M.; Shoda, S.I.; Haneda, K.; Inazu, T.; Takegawa, K.; Yamamoto, K. A novel disaccharide substrate having 1,2-oxazoline moiety for detection of transglycosylating activity of endoglycosidases. Biochim. Biophys. Acta Gen. Subj. 2001, 1528, 9–14. [Google Scholar] [CrossRef]
- Sievers, F.; Wilm, A.; Dineen, D.; Gibson, T.J.; Karplus, K.; Li, W.; Lopez, R.; McWilliam, H.; Remmert, M.; Söding, J.; et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 2011, 7, 539. [Google Scholar] [CrossRef] [PubMed]
- Teze, D.; Zhao, J.; Wiemann, M.; Ara, K.Z.G.; Lupo, R.; Zeuner, B.; Vuillemin, M.; Rønne, M.E.; Carlström, G.; Duus, J.Ø.; et al. Rational enzyme design without structural knowledge: A sequence-based approach for efficient generation of transglycosylases. Chem. Eur. J. 2021, 27, 10323–10334. [Google Scholar] [CrossRef] [PubMed]
- Chen, X.; Jin, L.; Jiang, X.; Guo, L.; Gu, G.; Xu, L.; Lu, L.; Wang, F.; Xiao, M. Converting a β-N-acetylhexosaminidase into two trans-β-N-acetylhexosaminidases by domain-targeted mutagenesis. Appl. Microbiol. Biotechnol. 2019, 104, 661–673. [Google Scholar] [CrossRef] [PubMed]
- Jamek, S.B.; Mikkelsen, J.D.; Busk, P.K.; Meyer, A.S.; Holck, J.; Zeuner, B.; Muschiol, J. Loop protein engineering for improved transglycosylation activity of a β-N-acetylhexosaminidase. ChemBioChem 2018, 19, 1858–1865. [Google Scholar] [CrossRef]
- Madeira, F.; Park, Y.M.; Lee, J.; Buso, N.; Gur, T.; Madhusoodanan, N.; Basutkar, P.; Tivey, A.R.N.; Potter, S.C.; Finn, R.D.; et al. The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res. 2019, 47, W636–W641. [Google Scholar] [CrossRef] [Green Version]
- Barrett, K.; Hunt, C.J.; Lange, L.; Meyer, A.S. Conserved unique peptide patterns (CUPP) online platform: Peptide-based functional annotation of carbohydrate active enzymes. Nucleic Acids Res. 2020, 48, 110–115. [Google Scholar] [CrossRef]
- Laskowski, R.A.; Swindells, M.B. LigPlot+: Multiple ligand-protein interaction diagrams for drug discovery. J. Chem. Inf. Model. 2011, 51, 2778–2786. [Google Scholar] [CrossRef]
- Almagro Armenteros, J.J.; Tsirigos, K.D.; Sønderby, C.K.; Petersen, T.N.; Winther, O.; Brunak, S.; von Heijne, G.; Nielsen, H. SignalP 5.0 improves signal peptide predictions using deep neural networks. Nat. Biotechnol. 2019, 37, 420–423. [Google Scholar] [CrossRef] [PubMed]
- Krogh, A.; Larsson, B.; Von Heijne, G.; Sonnhammer, E.L.L. Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes. J. Mol. Biol. 2001, 305, 567–580. [Google Scholar] [CrossRef] [Green Version]
- Hederos, M.; Dekany, G.; Demko, S.; Kovács, I.; Bajza, I. Manufacture of Lacto-N-Tetraose. WO2012155916A1, 22 November 2012. [Google Scholar]
- Dekany, G.; Agoston, K.; Bajza, I.; Boutet, J.; Bojstrup, M.; Fanefjord, M.; Pérez, I.F.; Hederos, M.; Horvath, F.; Kovács-Pénzes, P.; et al. Synthesis of 2’-O-Fucosyllactose. WO2010115934A1, 14 October 2010. [Google Scholar]
- Pérez Figueroa, I.; Horvath, F.; Dekany, G.; Agoston, K.; Agoston, A.; Bajza, I.; Boutet, J.; Hederos, M.; Kovács-Pénzes, P.; Kröger, L.; et al. Production of 6’-O-Sialyllactose and Intermediates. WO2011100979A1, 25 August 2011. [Google Scholar]
- Kovács, I.; Bajza, I.; Hederos, M.; Dekany, G.; Demko, S.; Khanzhin, N. Synthesis of HMO Core Structures. WO2013044928A1, 4 April 2013. [Google Scholar]
- Matsuda, H.; Ishihara, H.; Tejima, S. Chemical modification of lactose. XII. Preparation of O-(2-acetamido-2-deoxy-beta-d-glucopyranosyl)-(1-6)-o-beta-d-galactopyranolsyl-(1-4)-d-glucopyranose (6’-N-acetylglucosaminyllactose). Chem. Pharm. Bull. 1979, 27, 2564–2569. [Google Scholar] [CrossRef]
- Noguchi, M.; Tanaka, T.; Gyakushi, H.; Kobayashi, A.; Shoda, S. Efficient synthesis of sugar oxazolines from inprotected N-acetyl-2-amino sugars by using chloroformamidinium reagent in water. J. Org. Chem. 2009, 74, 2210–2212. [Google Scholar] [CrossRef]
- Zeuner, B.; Muschiol, J.; Holck, J.; Lezyk, M.; Gedde, M.R.; Jers, C.; Mikkelsen, J.D.; Meyer, A.S. Substrate specificity and transfucosylation activity of GH29 α-L-fucosidases for enzymatic production of human milk oligosaccharides. New Biotechnol. 2018, 41, 34–45. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Ito, Y.; Sasaki, T. Cloning and characterization of the gene encoding a novel β-galactosidase from Bacillus circulans. Biosci. Biotechnol. Biochem. 1997, 61, 1270–1276. [Google Scholar] [CrossRef] [Green Version]
- Fujimoto, H.; Miyasato, M.; Ito, Y.; Sasaki, T.; Ajisaka, K. Purification and properties of recombinant β-galactosidases from Bacillus circulans. Glycoconj. J. 1998, 15, 155–160. [Google Scholar] [CrossRef]
- Bao, Y.; Chen, C.; Newburg, D.S. Quantification of neutral human milk oligosaccharides by graphitic carbon high-performance liquid chromatography with tandem mass spectrometry. Anal. Biochem. 2013, 433, 28–35. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Teze, D.; Daligault, F.; Ferrières, V.; Sanejouand, Y.H.; Tellier, C. Semi-rational approach for converting a GH36 α-glycosidase into an α-transglycosidase. Glycobiology 2015, 25, 420–427. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Dennis, R.J.; Taylor, E.J.; MacAuley, M.S.; Stubbs, K.A.; Turkenburg, J.P.; Hart, S.J.; Black, G.N.; Vocadlo, D.J.; Davies, G.J. Structure and mechanism of a bacterial β-glucosaminidase having O-GlcNAcase activity. Nat. Struct. Mol. Biol. 2006, 13, 365–371. [Google Scholar] [CrossRef] [PubMed]
- Mark, B.L.; Vocadlo, D.J.; Knapp, S.; Triggs-Raine, B.L.; Withers, S.G.; James, M.N.G. Crystallographic evidence for substrate-assisted catalysis in a bacterial β-hexosaminidase. J. Biol. Chem. 2001, 276, 10330–10337. [Google Scholar] [CrossRef] [Green Version]
- Zakariassen, H.; Hansen, M.C.; Jøranli, M.; Eijsink, V.G.H.; Sørlie, M. Chitinases and construction of a hypertransglycosylating mutant. Biochemistry 2011, 50, 5693–5703. [Google Scholar] [CrossRef]
- Jamek, S.B.; Nyffenegger, C.; Muschiol, J.; Holck, J.; Meyer, A.S.; Mikkelsen, J.D. Characterization of two novel bacterial type A exo-chitobiose hydrolases having C-terminal 5/12-type carbohydrate-binding modules. Appl. Microbiol. Biotechnol. 2017, 101, 4533–4546. [Google Scholar] [CrossRef] [Green Version]
- Křen, V.; Rajnochová, E.; Huňková, Z.; Dvořáková, J.; Sedmera, P. Unusual nonreducing sugar GlcNAc-beta(1-1)Man-beta formation by beta-N-acetylhexosaminidase from Aspergillus oryzae. Tetrahedron Lett. 1998, 39, 9777–9780. [Google Scholar] [CrossRef]
- Singh, S.; Scigelova, M.; Vic, G.; Crout, D.H.G. Glycosidase-catalysed oligosaccharide synthesis of di-, tri- and tetra-saccharides using the N-acetylhexosaminidase from Aspergillus oryzae and the beta-galactosidase from Bacillus circulans. J. Chem. Soc. Perkin Trans. 1 1996, 1921–1926. [Google Scholar] [CrossRef]
- Garcia-Oliva, C.; Hoyos, P.; Petrásková, L.; Kulik, N.; Pelantová, H.; Cabanillas, A.H.; Rumbero, Á.; Křen, V.; Hernáiz, M.J.; Bojarová, P. Acceptor specificity of β-N-Acetylhexosaminidase from Talaromyces flavus: A rational explanation. Int. J. Mol. Sci. 2019, 20, 6181. [Google Scholar] [CrossRef] [Green Version]
- Murata, T.; Tashiro, A.; Itoh, T.; Usui, T. Enzymic synthesis of 3’-O- and 6’-O-N-acetylglucosaminyl-N-acetyllactosaminide glycosides catalyzed by β-N-acetyl-D-hexosaminidase from Nocardia orientalis. Biochim. Biophys. Acta Gen. Subj. 1997, 1335, 326–334. [Google Scholar] [CrossRef]
- Nilsson, K.G.I. Enzymic synthesis of HexNAc-containing glycosides. Carbohydr. Res. 1990, 204, 79–83. [Google Scholar] [CrossRef]
- Coulier, L.; Timmermans, J.; Richard, B.; Van Den Dool, R.; Haaksman, I.; Klarenbeek, B.; Slaghek, T.; Van Dongen, W. In-depth characterization of prebiotic galactooligosaccharides by a combination of analytical techniques. J. Agric. Food Chem. 2009, 57, 8488–8495. [Google Scholar] [CrossRef]
- Zeuner, B.; Jers, C.; Mikkelsen, J.D.; Meyer, A.S. Methods for improving enzymatic trans-glycosylation for synthesis of human milk oligosaccharide biomimetics. J. Agric. Food Chem. 2014, 62, 9615–9631. [Google Scholar] [CrossRef] [PubMed]
Variant | Max. LNT [μM] | Max. TG [μM] | Time of Max. Yield [h] | % LNT Hydrolyzed after 24 h | % LNT of Total TG |
---|---|---|---|---|---|
WT | 495 ± 14 e | 854 ± 7 g | 0.25 | 89 ± 0.2% | 58 ± 1% e |
N259T | 754 ± 32 cd | 1226 ± 26 e | 3 | 66 ± 1% | 62 ± 1% d |
D320E | 185 ± 9 f | 685 ± 51 h | 24 | - | 27 ± 1% i |
D320T | 303 ± 5 f | 798 ± 2 gh | 24 | - | 38 ± 1% h |
W373F | 839 ± 14 c | 1192 ± 14 e | 0.25 | 92 ± 2% | 70 ± 0.3% ab |
W373H | 696 ± 30 d | 1020 ± 42 f | 0.25 | 90 ± 2% | 68 ± 0.1% b |
W394F | 1279 ± 17 a | 1865 ± 39 b | 3 | 26 ± 2% | 69 ± 1% b |
W394H | 1189 ± 6 a | 1655 ± 9 c | 3 | 6 ± 6% * | 72 ± 0.01% a |
G398R | 750 ± 106 cd | 1183 ± 145 e | 0.25 | 91 ± 3% | 63 ± 1% cd |
ΔG398 | 720 ± 122 d | 1169 ± 134 ef | 0.25 | 91 ± 2% | 61 ± 3% d |
Y419N | 1285 ± 5 a | 2388 ± 12 a | 3 | 14 ± 6% * | 54 ± 0.1% f |
W465F | 1007 ± 9 b | 1467 ± 8 d | 0.25 | 93 ± 2% | 69 ± 0.3% b |
W465H | 1235 ± 7 a | 1930 ± 24 b | 3 | 8 ± 4% * | 64 ± 0.4% c |
D467N | 536 ± 3 e | 1125 ± 1 ef | 3 | 15 ± 0.4% | 48 ± 0.2% g |
LNT Formation | Total LNT Isomer (TG) Formation | ||||||
---|---|---|---|---|---|---|---|
Variant | Time of Max. Yield [h] | Max. LNT [µM] | Max. LNT Yield | % LNT Hydrolyzed after 24 h | Max. TG [µM] | Max. TG Yield | % Total TG Hydrolyzed after 24 h |
WT | 0.08 | 7 ± 0.3 f | 0.3% f | 55 ± 8% | 29 ± 3 d | 1.2% d | 52 ± 7% |
W394F | 2 | 164 ± 0.05a | 6.6% a | 97 ± 0.1% | 285 ± 5 a | 11% a | 91 ± 0.3% |
W394H | 2 | 103 ± 3 b | 4.1% b | 6 ± 18% * | 286 ± 5 a | 11% a | 43 ± 10% |
Y419N | 0.25 | 76 ± 3 d | 3.0% d | 95 ± 1% | 171 ± 3 b | 6.8% b | 86 ± 2% |
W465F | 0.08 | 46 ± 0.04 e | 1.8% e | 93 ± 0.5% | 94 ± 0.7 c | 3.7% c | 86 ± 2% |
W465H | 24 | 92 ± 0.7 c | 3.7% c | - | 175 ± 3 b | 7.0% b | - |
Reaction Conditions | LNT Formation | Total LNT Isomer (TG) Formation | ||||
---|---|---|---|---|---|---|
pNP-LNB [mM] | Lactose [mM] | A/D Ratio | Max. LNT [µM] | Max. LNT Yield | Max. TG [µM] | Max. TG Yield |
2.5 | 200 | 80 | 107 ± 14 e (2 h) | 4% b | 225 ± 26 e (2 h) | 9% c |
2.5 | 400 | 160 | 188 ± 22 de (2 h) | 8% a | 396 ± 25 d (2 h) | 16% a |
5 | 200 | 40 | 247 ± 11 cd (2 h) | 5% b | 539 ± 25 c (2 h) | 11% b |
5 | 400 | 80 | 342 ± 12 bc (2 h) | 7% a | 765 ± 28 b (2 h) | 15% a |
10 | 200 | 20 | 389 ± 41 b (24 h) | 4% b | 759 ± 23 b (2 h) | 8% c |
10 | 400 | 40 | 746 ± 81 a (24 h) | 7% a | 1224 ± 65 a (2 h) | 12% b |
Enzyme | Tm [°C] |
---|---|
WT | 64.4 ± 0.01 a |
W394F | 58.0 ± 0.06 e |
W394H | 59.4 ± 0.12 d |
Y419N | 60.6 ± 0.03 c |
W465F | 63.0 ± 0.04 b |
W465H | 62.9 ± 0.01 b |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Vuillemin, M.; Holck, J.; Matwiejuk, M.; Moreno Prieto, E.S.; Muschiol, J.; Molnar-Gabor, D.; Meyer, A.S.; Zeuner, B. Improvement of the Transglycosylation Efficiency of a Lacto-N-Biosidase from Bifidobacterium bifidum by Protein Engineering. Appl. Sci. 2021, 11, 11493. https://doi.org/10.3390/app112311493
Vuillemin M, Holck J, Matwiejuk M, Moreno Prieto ES, Muschiol J, Molnar-Gabor D, Meyer AS, Zeuner B. Improvement of the Transglycosylation Efficiency of a Lacto-N-Biosidase from Bifidobacterium bifidum by Protein Engineering. Applied Sciences. 2021; 11(23):11493. https://doi.org/10.3390/app112311493
Chicago/Turabian StyleVuillemin, Marlene, Jesper Holck, Martin Matwiejuk, Eduardo S. Moreno Prieto, Jan Muschiol, Dora Molnar-Gabor, Anne S. Meyer, and Birgitte Zeuner. 2021. "Improvement of the Transglycosylation Efficiency of a Lacto-N-Biosidase from Bifidobacterium bifidum by Protein Engineering" Applied Sciences 11, no. 23: 11493. https://doi.org/10.3390/app112311493
APA StyleVuillemin, M., Holck, J., Matwiejuk, M., Moreno Prieto, E. S., Muschiol, J., Molnar-Gabor, D., Meyer, A. S., & Zeuner, B. (2021). Improvement of the Transglycosylation Efficiency of a Lacto-N-Biosidase from Bifidobacterium bifidum by Protein Engineering. Applied Sciences, 11(23), 11493. https://doi.org/10.3390/app112311493