Abstract
BackgroundThe thermostable β-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-β-D-glucopyranoside (pNPGlc), and screened for hydrolysis of Q3.ResultsStructural 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 β-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 Tm by differential scanning calorimetry (101.9°C for wt), was kept in the mutated variants and significant decrease (ΔT of 5 - 10°C) was only observed for the triple mutants. The exchanged residue(s) in the respective mutant resulted in variations in KM and turnover. The KM-value was only changed in variants mutated at position 221 (N221S) and was in all cases monitored as a 2-3 × increase for pNPGlc, while the KM decreased a corresponding extent for Q3.Turnover was only significantly changed using pNPGlc, and was decreased 2-3 × 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 × 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 KM for this substrate.ConclusionThese results show that residues at the +2 subsite are interesting targets for mutagenesis and mutations at these positions can directly or indirectly affect both KM and turnover. An affinity change, leading to a decreased KM, 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.
Highlights
The thermostable b-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
In a previous work we have shown that the oligosaccharide hydrolysing glycoside hydrolase family 1 (GH1) b-glucosidase TnBgl1A, from the hyperthermophile Thermotoga neapolitana, efficiently hydrolyses quercetin-4’-glucosides (Q4’) [3]
A few members of the family are, commercially available, and many different analytical assays have utilized b-glucosidase from almond, which has been classified under GH1 [26], and which is often available in heterogeneous preparations. b-Glucosidase A from Thermotoga neapolitana, TnBgl1A, was chosen for this work as an interesting candidate based on previous promising results in biocatalytic conversion of quercetin glucosides to quercetin and carbohydrates in yellow onion extract, extracted using a pressurized hot water extraction technique [3]
Summary
The thermostable b-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 been shown to be dependent on the position of the glucosylation on the flavonoid, and e.g. quercetin-3-glucoside (Q3) was hydrolysed slowly. Carbohydrates are essential components of biomass, which is estimated to be produced in a quantity of about 60 Gt/year [1] and contain an array of structural and storage polysaccharides To utilize these raw materials, microorganisms produce a wide variety of carbohydrate hydrolysing and modifying glycoside hydrolases. GHs hydrolyse certain flavonoid glycosides, dependent on the aglycone moiety, type of sugar and linkage [3,10] and some glucosidases classified under GH family 1 (GH1) [11] are flavonoid-hydrolysing enzymes. In a previous work we have shown that the oligosaccharide hydrolysing GH1 b-glucosidase TnBgl1A, from the hyperthermophile Thermotoga neapolitana, efficiently hydrolyses quercetin-4’-glucosides (Q4’) [3]. Q4’ and quercetin-3,4’diglucoside (Q3,4’) are the most abundant species in yellow onion [12], and to obtain the quercetin aglycone both Q4’ and Q3 species must be hydrolysed
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