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- Gulshan Kazi, Zubaida, et al.
(author)
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Glycoside hydrolases for extraction and modification of polyphenolic antioxidants
- 2013
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In: Advances in enzyme biotechnology. - New Delhi : Springer India. - 9788132210931 - 9788132210948 ; , s. 9-21
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Book chapter (peer-reviewed)abstract
- Antioxidants are important molecules that are widely used by humans, both as dietary supplements and as additives to different types of products. In this chapter, we review how flavonoids, a class of polyphenolic antioxidants that are often found in glycosylated forms in many natural resources, can be extracted and modified using glycoside hydrolases (GHs). Glycosylation is a fundamental enzymatic process in nature, affecting function of many types of molecules (glycans, proteins, lipids as well as other organic molecules such as the flavonoids). Possibilities to control glycosylation thus mean possibilities to control or modify the function of the molecule. For the flavonoids, glycosylation affect both the antioxidative power and solubility. In this chapter we overview results on in vitro deglycosylation and glycosylation of flavonoids by selected GHs. For optimal enzymatic performance, desired features include a correct specificity for the target, combined with high stability. Poor specificity towards a specific substituent is thus a major drawback for enzymes in particular applications. Efforts to develop the enzymes as conversion tools are reviewed.
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| 2. |
- Lindahl, Sofia, et al.
(author)
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Exploring the possibility of using a thermostable mutant of β-glucosidase for rapid hydrolysis of quercetin glucosides in hot water
- 2010
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In: Green Chemistry. - : The Royal Society of Chemistry. - 1463-9262 .- 1463-9270. ; 12:1, s. 159-168
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Journal article (peer-reviewed)abstract
- The antioxidant quercetin was extracted from yellow onion waste and converted to its aglycone form by a combination of subcritical water extraction and enzymatic hydrolysis. The hydrolytic step was catalysed by a double residue (N221S, P342L) mutant of the thermostable beta-glucosidase (TnBgl1A), isolated from the thermophile Thermotoga neapolitana and cloned and produced in E. coli. The activity of wt TnBgl1A was shown to be dependent on the position of the glucosylation on the quercetin backbone, favouring hydrolysis of quercetin-4'-glucoside over quercetin-3-glucoside. The mutated variant of the enzyme harboured a mutation in the +2 sub-site (N221S) and showed increased catalytic efficiency in quercetin-3-glucoside hydrolysis and also to a certain extent hydrolysis of quercetin-4'-glucoside. The mutated enzyme was used directly in yellow onion extracts, prepared by subcritical water extraction, resulting in complete hydrolysis of the glucosylated flavonoids quercetin-3,4'-diglucoside, quercetin-4'-glucoside, quercetin-3-glucoside, isorhamnetin-4'-glucoside and isorhamnetin-3,4'-diglucoside. To complete hydrolysis within five minutes, 3 mg of TnBgl1A_N221S was used per gramme of onion (dry weight). A life cycle assessment was done to compare the environmental impact of the new method with a conventional solid-liquid extraction-and-hydrolysis method utilising aqueous methanol and hydrochloric acid. Comparison of the methods showed that the new method is preferable regarding primary energy consumption and global warming potential. Another advantage of this method is that handling of toxic chemicals (methanol and HCl) is avoided. This shows that combined subcritical water extraction/enzyme hydrolysis is both a fast and sustainable method to obtain quercetin from onion waste.
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| 3. |
- Khan, Samiullah, et al.
(author)
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Aglycone specificity of Thermotoga neapolitana beta-glucosidase 1A modified by mutagenesis, leading to increased catalytic efficiency in quercetin-3-glucoside hydrolysis
- 2011
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In: BMC Biochemistry. - : Springer Science and Business Media LLC. - 1471-2091. ; 12
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Journal article (peer-reviewed)abstract
- Background: The thermostable beta-glucosidase (TnBgl1A) from Thermotoga neapolitana is a promising biocatalyst for hydrolysis of glucosylated flavonoids and can be coupled to extraction methods using pressurized hot water. Hydrolysis has however been shown to be dependent on the position of the glucosylation on the flavonoid, and e. g. quercetin-3-glucoside (Q3) was hydrolysed slowly. A set of mutants of TnBgl1A were thus created to analyse the influence on the kinetic parameters using the model substrate para-nitrophenyl-beta-D-glucopyranoside (pNPGlc), and screened for hydrolysis of Q3. Results: Structural analysis pinpointed an area in the active site pocket with non-conserved residues between specificity groups in glycoside hydrolase family 1 (GH1). Three residues in this area located on beta-strand 5 (F219, N221, and G222) close to sugar binding sub-site +2 were selected for mutagenesis and amplified in a protocol that introduced a few spontaneous mutations. Eight mutants (four triple: F219L/P165L/M278I, N221S/P165L/M278I, G222Q/P165L/M278I, G222Q/V203M/K214R, two double: F219L/K214R, N221S/P342L and two single: G222M and N221S) were produced in E. coli, and purified to apparent homogeneity. Thermostability, measured as T-m by differential scanning calorimetry (101.9 degrees C for wt), was kept in the mutated variants and significant decrease (Delta T of 5 -10 degrees C) was only observed for the triple mutants. The exchanged residue(s) in the respective mutant resulted in variations in K-M and turnover. The K-M-value was only changed in variants mutated at position 221 (N221S) and was in all cases monitored as a 2-3 x increase for pNPGlc, while the K-M decreased a corresponding extent for Q3. Turnover was only significantly changed using pNPGlc, and was decreased 2-3 x in variants mutated at position 222, while the single, double and triple mutated variants carrying a mutation at position 221 (N221S) increased turnover up to 3.5 x compared to the wild type. Modelling showed that the mutation at position 221, may alter the position of N291 resulting in increased hydrogen bonding of Q3 (at a position corresponding to the +1 subsite) which may explain the decrease in K-M for this substrate. Conclusion: These results show that residues at the +2 subsite are interesting targets for mutagenesis and mutations at these positions can directly or indirectly affect both K-M and turnover. An affinity change, leading to a decreased K-M, can be explained by an altered position of N291, while the changes in turnover are more difficult to explain and may be the result of smaller conformational changes in the active site.
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