| 1. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 2. |
- Pozzo, Tania, et al.
(författare)
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Glycosynthases from Thermotoga neapolitana beta-glucosidase 1A: A comparison of alpha-glucosyl fluoride and in situ-generated alpha-glycosyl formate donors
- 2014
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Ingår i: Journal of Molecular Catalysis B: Enzymatic. - : Elsevier BV. - 1873-3158 .- 1381-1177. ; 107, s. 132-139
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Tidskriftsartikel (refereegranskat)abstract
- TnBgl1A from the thermophile Thermotoga neapolitana is a dimeric beta-glucosidase that belongs to glycoside hydrolase family 1 (GH1), with hydrolytic activity through the retaining mechanism, and a broad substrate specificity acting on beta-1,4-, beta-1,3- and beta-1,6-linkages over a range of glyco-oligosaccharides. Three variants of the enzyme (TnBgl1A_E349G, TnBgl1A_E349A and TnBgl1A_E349S), mutated at the catalytic nucleophile, were constructed to evaluate their glycosynthase activity towards oligosaccharide synthesis. Two approaches were used for the synthesis reactions, both of which utilized 4-nitrophenyl beta-D-glucopyranoside (4NPGIc) as an acceptor molecule: the first using an alpha-glucosyl fluoride donor at low temperature (35 degrees C) in a classical glycosynthase reaction, and the second by in situ generation of the glycosyl donor with (4NPGIc), where formate served as the exogenous nucleophile under higher temperature (70 degrees C). Using the first approach, TnBgl1A_E349G and TnBgl1A_E349A synthesized disaccharides with beta-1,3-linkages in good yields (up to 61%) after long incubations (15 h). However, the GH1 glycosynthase Bg13_E383A from a mesophilic Streptomyces sp., used as reference enzyme, generated a higher yield at the same temperature with both a shorter reaction time and a lower enzyme concentration. The second approach yielded disaccharides for all three mutants with predominantly beta-1,3-linkages (up to 45%) but also beta-1,4-linkages (up to 12.5%), after 7 h reaction time. The TnBgl1A glycosynthases were also used for glycosylation of flavonoids, using the two described approaches. Quercetin-3-glycoside was tested as an acceptor molecule and the resultant product was quercetin-3,4'-diglycosides in significantly lower yields, indicating that TnBgl1A preferentially selects 4NPGIc as the acceptor. (C) 2014 Elsevier B.V. All rights reserved.
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| 3. |
- Pozzo, Tania, et al.
(författare)
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Rational design of a thermostable glycoside hydrolase from family 3 introduces β-glycosynthase activity
- 2017
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 27:2, s. 165-175
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Tidskriftsartikel (refereegranskat)abstract
- The thermostable β-glucosidase from Thermotoga neapolitana, TnBgl3B, is a monomeric three-domain representative from glycoside hydrolase family 3. By using chemical reactivation with exogenous nucleophiles in previous studies with TnBg13B, the catalytic nucleophile (D242) and corresponding acid/base residue (E458) were determined. Identifying these residues led to the attempt of converting TnBgl3B into a β-glucosynthase, where three nucleophilic variants were created (TnBgl3B_D242G, TnBgl3B_D242A, TnBgl3B_D242S) and all of them failed to exhibit glucosynthase activity. A deeper analysis of the TnBgl3B active site led to the generation of three additional variants, each of which received a single-point mutation. Two of these variants were altered at the -1 subsite (Y210F, W243F) and the third received a substitution near the binding site's aglycone region (N248R). Kinetic evaluation of these three variants revealed that W243F substitution reduced hydrolytic turnover while maintaining KM This key W243F mutation was then introduced into the original nucleophile variants and the resulting double mutants were successfully converted into β-glucosynthases that were assayed using two separate biosynthetic methods. The first reaction used an α-glucosyl fluoride donor with a 4-nitrophenyl-β-d-glucopyranoside (4NPGlc) acceptor, and the second used 4NPGlc as both the donor and acceptor in the presence of the exogenous nucleophile formate. The primary specificity observed was a β-1,3-linked disaccharide product, while a secondary β-1,4-linked disaccharide product was observed with increased incubation times. Additional analysis revealed that substituting quercetin-3-glycoside for the second reaction's acceptor molecule resulted in the successful production of quercetin-3,4'-diglycosides with yields up to 40%.
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