BACKGROUND
glycoside hydrolases  are enzymes that hydrolyse glycosidic bonds between two or more carbohydrates, or between a carbohydrate and a non-carbohydrate moiety.
carbohydrates are essential components of biomass, which is estimated to be produced in a quantity of about  <dig> gt/year  <cit>  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.
these enzymes can also be used as specific catalysts in industrial applications, e.g.
in the food and feed industries, the paper and pulp, starch and textile industries, and in newly emerging sustainable processes  <cit>  taking advantage of their specificity in selective preparations of carbohydrate-containing raw materials.
antioxidants are bioactive compounds that have received great interest due to their potential as health beneficial agents.
the action of antioxidants is to counteract oxidative stress imposed by reactive oxygen species shown to play a crucial role in the pathophysiology associated with neoplasia, atherosclerosis and neurodegenerative diseases  <cit> .
polyphenolic compounds show a wide range of antioxidant activities, and are thought to exert protective effects against the diseases specified above  <cit> .
flavonoids are polyphenolic compounds that are important antioxidative constituents of fruits and vegetables, but the type of compound is different in different sources.
fruits and vegetables rich in anthocyanins  show highest antioxidant activities, followed by those rich in flavonones  or flavonols , while hydroxycinnamate-rich fruits  exhibit lower antioxidant activities  <cit> .
flavonols are generally not found as free aglycones , but rather as complex conjugates with sugar residues .
ghs hydrolyse certain flavonoid glycosides, dependent on the aglycone moiety, type of sugar and linkage  <cit>  and some glucosidases classified under gh family  <dig>   <cit>  are flavonoid-hydrolysing enzymes.
in a previous work we have shown that the oligosaccharide hydrolysing gh <dig> β-glucosidase tnbgl1a, from the hyperthermophile thermotoga neapolitana, efficiently hydrolyses quercetin-4'-glucosides   <cit> .
q4' and quercetin- <dig> '-diglucoside  are the most abundant species in yellow onion  <cit> , and to obtain the quercetin aglycone both q4' and q <dig> species must be hydrolysed.
most commonly quercetin and its glycosides have been extracted from yellow onion by simple liquid/solid extraction techniques  combined with chemically  catalyzed hydrolysis reaction of the extracted quercetin glucosides  <cit> .
we have instead utilized pressurized hot water to extract the quercetin species from yellow onion, followed by biocatalytic conversion of the quercetin glucosides to quercetin and carbohydrates  <cit> .
in this system, use of enzymes with high thermostability is essential, and the enzyme tnbgl1a from the hyperthermophile t. neapolitana is from this perspective a suitable biocatalyst  <cit> .
it was also shown that the q4' was much more efficiently hydrolysed than the q <dig>
in this investigation, the β-glucosidase tnbgl1a, was mutated to investigate the influence of mutations on the enzyme kinetics ), thermostability, and selective hydrolysis of glucose at two positions  on the aglycone quercetin.
we have utilized a strategy to design mutants based on bioinformatics and structural analysis, with an amplification protocol that allowed spontaneous mutations, in order to find residues that influence specificity of the enzyme.
methods
chemicals
all chemicals were of pro-analysis grade from merck eurolabs  unless otherwise stated.
cloning of tnbgl1a
the gene encoding bgl1a was pcr-amplified from genomic thermotoga neapolitana  dna as described by turner et al  <cit> .
primers  were designed to amplify the coding sequence of bgl1a  from the sequence deposited at the ncbi server  <cit>  under the accession number af <dig>  the gene was inserted in vector pet-22b  under control of the t7/lac promoter and incorporating the c-terminal hexa-histidine tag  <cit> .
the resulting plasmids were transformed into e. coli nova blue cells  and screened by colony pcr using the t <dig> forward and t <dig> reverse primers  and taq dna polymerase.
positive clones were transformed into the e. coli expression host tuner  .
the complete gene was sequenced at mwg biotech .
athe restriction sites for cloning are underlined.
binduced changes are shown in bold.
mutagenesis
mutagenesis was performed in order to introduce the following designed changes: f219l, n221s, and g222m/q, respectively.
taq polymerase, which lacks proofreading,  was used  to allow introduction of a few random additional mutations.
standard concentrations of mgcl <dig>  and dntps  were used.
in a first pcr , a mutated gene fragment encoding the c-terminal part of the enzyme was constructed using forward mismatched primers  together with the reverse gene specific primer  <dig>  encoding the c-terminal sequence of bgl1a.
the n221s mutants were obtained by amplifying the full length gene in a second pcr  using the product of the first pcr as a reverse "megaprimer" together with the forward primer  <dig> matching the start of the gene.
mutations at position  <dig> , and  <dig>  were constructed using overlap extension pcr of the mutated fragment and an overlapping gene fragment encoding the n-terminal part of bgl1a.
reverse primers  together with the forward gene specific primer  <dig> were used to create the overlapping fragments that were pcr-amplified  with the wild-type gene as a template.
overlap extension pcr reaction was then run in two steps.
firstly, extension without template at: 94°c  <dig> min;  <dig> cycles: 94°c  <dig> min,  <dig> °c  <dig> min, 72°c  <dig> s, 72°c  <dig> min and the ramp between the annealing and extension changed from the default 3°c/s to 5°c/s.
secondly, amplification at standard conditions, using the gene-specific forward and reverse primers , and the product of the overlap extension pcr as template.
all mutated genes  were purified with qiaexii gel extraction kit  after gel separation.
both insert and vector were digested and ligated as described under the cloning section.
resulting plasmids were transformed into e. coli nova blue cells  and screened by colony pcr using the t <dig> forward and t <dig> reverse primers .
selected mutant clones were fully sequenced at mwg biotech.
homology modelling and ligand binding
a tnbgl1a homology model was constructed utilizing the schrodinger  <dig> software suite  <cit> .
energy minimizations were performed with macromodel, utilizing the opls- <dig> force field and the gb/sa model for water solvation.
molecular dynamics was performed with desmond, utilizing default settings.
to find homologes to the tnbgl1a sequence, a blast search was performed on proteins with x-ray diffraction data in the pdb data base.
the amino acid sequence of the gh <dig> β-glucosidase from thermotoga maritima provided the highest similarity, 90% identity, and 97% positives.
the pdb structure 2wc <dig> of bgla from t. maritima with 3-imino-2-thio--castanospermine as ligand, had the highest resolution of these x-ray crystallographic models , and was used as template to build a 3d model of tnbgl1a .
the protein preparation wizard was utilized to add hydrogens, assign charges, optimize hydrogen bond networks, and to analyze the quality of the homology model.
the rmsd  with the template was  <dig>  Å after optimisation.
in the region of the active site, a cis-bond between w <dig> and s <dig> was found, however this cis-amide is present in all crystals of t. maritima β-glucosidase.
furthermore, energy minimization showed the cis conformer to have lower energy than the corresponding trans amide bond.
a low energy model of β-d-cellotetraose was constructed  <cit>  and placed in the homology model so that the β-d-glucose at the non-reducing end superimposed with the six membered rings of the t. maritima bgla ligands in the pdb structures 2wc <dig>  3cmj, 1qox, 1e4i, and 1bga.
the glycosidic linkage torsion angle ϕ of this sugar was adjusted from -121° to 26° to make cellotetraose fit in the ligand binding pocket.
the resulting structure was energy minimized to yield a cellotetraose binding pose that is in agreement with the proposed mechanism of family  <dig> β-glucosidases  <cit> .
the binding pose was verified as stable in  <dig> ns molecular dynamics simulation.
a conformational search was performed on q <dig>  and the lowest energy conformer was placed in the homology model using the same method as for cellotetraose.
the resulting structure was energy minimized, and the binding pose, which was in agreement with the proposed mechanism of family  <dig> β-glucosidases, was verified with a  <dig> ns molecular dynamics simulation.
figures of docking results were prepared within the schrodinger  <dig> software suite, including the program pymol.
expression and purification
the mutant and wild-type enzymes were produced in  <dig>  l batch cultivations at 37°c, ph  <dig>  using a defined medium  <cit>  with  <dig> μg ml- <dig> ampicillin and a dissolved oxygen tension  above 40%.
expression was induced at od <dig> nm =  <dig>  by the addition of  <dig> - <dig> mm isopropyl-beta-d-thiogalactopyranoside , and continued for  <dig> h. production levels were analysed by sds-page  in samples  withdrawn hourly after induction.
the cells were pelleted, resuspended in  <dig> μl  <dig> mm citrate-phosphate buffer, ph  <dig> , ultrasonicated for  <dig> ×  <dig> s with a up400s equipped with a  <dig> mm titanium probe  using a sound intensity of 60% and a cycle of  <dig>  and thereafter centrifuged for  <dig> min at  <dig>  <dig> × g to separate soluble proteins from insoluble proteins and cell debris.
the cells were harvested, separated from the cultivation medium by centrifugation , and dissolved in binding buffer .
the ice-chilled cell suspension was lysed by sonication for  <dig> ×  <dig> min using a  <dig> mm titanium probe sound intensity of 60% and a cycle of  <dig>  , centrifuged , heat treated  and again centrifuged.
the supernatant was passed through a  <dig>  μm minisart high-flow filter  and purified on an Äkta prime system  by immobilized metal ion affinity chromatography using copper as a ligand as described elsewhere  <cit> .
the fractions containing the purified protein were pooled and dialyzed against  <dig> mm citrate phosphate buffer, ph  <dig> , overnight using a spectra/por dialysis membrane with a  <dig> da molecular weigh cut-off .
the dialysed protein fractions were stored at 4°c until use.
protein analysis
the purity of each mutant and wild-type enzyme was analysed by sds-page according to  <cit> .
expression levels were also analysed by sds-page after separating insoluble and soluble proteins.
total protein concentration was estimated at  <dig> nm by the bca method  using bovine serum albumin  as standard.
differential scanning calorimetry
dsc analysis was made on a microcal differential scanning calorimeter  with the cell volume of  <dig>  ml.
the samples  were concentrated to  <dig> mg ml- <dig> using vivaspin  centrifuging tubes with a mwco of  <dig>  da and were degassed before the scans.
the samples were scanned at a rate of 1°c/min in the temperature range of 25-110°c.
enzyme activity on pnpglc, q <dig> and q4'
enzyme activity, and kinetic parameters  were determined at 80°c, ph  <dig>  using pnpglc  as substrate in  <dig> mm citrate phosphate buffer, on a shimadzu uv- <dig> visible spectrophotometer .
a volume of  <dig> μl of pnpglc  was preheated for  <dig> min, where after  <dig> μl of the enzyme solution  was added.
absorbance at  <dig> nm was measured and plotted by the shimadzu uv probe  <dig>  software as a function of time during  <dig> min.
the extinction coefficient of pnp  under these experimental conditions was determined as ε 80°c,  <dig> nm=  <dig>  ×  <dig> ml mmol-1cm- <dig>  the kinetic parameters were determined by applying the wilkinson non-linear regression method using enzpack .
the effect of glucose on activity in this system was evaluated by adding  <dig> mm glucose to the stock solution of  <dig> mm pnpglc and the kinetic values km and kcat were determined as above.
enzyme activity of selected mutants of tnbgl1a were screened for two quercetin glucosides , quercetin-3-glucoside   and quercetin-4'-glucoside   at 90°c.
<dig> nmol samples of q <dig> or q4' dissolved in methanol  were evaporated and  <dig>  ml of  <dig> mm citrate phosphate buffer, ph  <dig> , was added and the vials were heated at 90°c until substrate was dissolved.
a  <dig> μl fraction was collected and added to  <dig> μl of mobile phase composed of methanol/water  and  <dig>  m formic acid.
the reaction was started by adding  <dig> pmol of enzyme and  <dig> min after addition of enzyme,  <dig> μl fractions were collected and added to  <dig> μl of mobile phase.
samples were analyzed by hplc with uv detection .
the conditions and methodolology for the kinetic measurement of q <dig> and q4' was published in lindahl et al  <cit> .
in summary, 33- <dig> nmol q <dig> and q4' were dissolved per ml citrate-phosphate buffer ph  <dig> .
for q <dig> hydrolysis  <dig> pmol wt and  <dig> pmol mutant n221s/p342l were used, and for q4' hydrolysis  <dig> pmol wt and  <dig> pmol mutant n221s/p342l were used.
hplc analysis
hplc-uv analysis was performed using the chromatographic system ultimate  <dig> from dionex .
an agilent zorbax sb-c <dig> column  was used for isocratic separation with a methanol:water  and  <dig>  m formic acid mobile phase at a flow rate of  <dig>  ml min- <dig>  the injection volume was  <dig> μl and detection was accomplished at  <dig> nm.
quantification of quercetin and glycosides was performed using a five-point calibration curve of a quercetin dihydrate standard  and q <dig> and q4' standards at concentrations between  <dig>  and  <dig> μg ml- <dig>  each vial taken to analysis had a total volume of  <dig> μl.
RESULTS
the different members of glycoside hydrolase family  <dig> , catalyse hydrolysis of a glucose molecule from a number of different substrates, including some hydrophobic substrates linked to glucose.
only a few members of the family are, however, commercially available, and many different analytical assays have thus utilized β-glucosidase from almond, which has been classified under gh <dig>  <cit> , and which is often available in heterogeneous preparations.
β-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  <cit> .
this enzyme is thermostable, and hence suitable for application in hot water.
moreover, this enzyme belongs to a gh family with diverse substrate specificity , with many gene sequences available allowing comparison, and with necessary structural information available, including three-dimensional  structures of the closely related enzyme tmgh <dig> from thermotoga maritima  <cit> , allowing homology modelling of the 3d structure.
structural considerations and mutation strategy
as noted elsewhere  <cit> , the deduced amino acid sequence encoded by the bgl1a-gene used in this work has one change in primary sequence  compared to the deposited sequence .
sequence alignments revealed v at position  <dig> to be conserved among several members of gh family  <dig>  and the obtained sequence is hereafter referred to as wild type  and designated tnbgl1a.
a molecular model of t. neapolitana tnbgl1a was generated based on the 3d structure of β-glucosidase bgla from t. maritima   <cit> .
tmgh <dig> provided the highest similarity: 90% identity, and 97% positives.
the homology detection structure prediction server hhpred  <cit>  also confirmed tmgh <dig> as the best template.
the overall structure presented in the tnbgl1a model is the typical  <dig> barrel fold characteristic of gh <dig> .
the active site is a deep channel  narrowed at the bottom with a wider cleft at the entrance.
two conservative motifs tlnep and iteng are situated opposite to each other inside the active site, more precisely at the ends of strands β <dig> and β <dig> containing the acid/base  and the nucleophile  residues respectively.
superimposition of tmgh <dig> in complex with 2-deoxy-2-fluoro-β-d-glucopyranoside   and tnbgl1a allowed identification of the residues forming the glycone binding site  .
interacting residues included conservative residues forming hydrogen bonds  and hydrophobic interactions .
cellotetraose was modelled into the tnbgl1a model to analyse putative interactions at the + <dig> and + <dig> subsites.
the β-d-glucopyranoside at the non-reducing end  was placed by superimposing the six membered rings of the ligand  in  <dig> gh <dig> structures .
the sugars in the cellotetraose were positioned in a dynamically stable position in agreement with the proposed mechanism of family  <dig> β-glucosidases  <cit> .
the same procedure was repeated for the glucosylated quercetin to see its position in relation to the sugar binding subsites.
both q <dig>  and q4' could be fitted in positions relevant for hydrolysis, while the double glucoside  could not be accommodated with the 3-glucoside positioned at the - <dig> subsite .
this explains why hydrolysis at the 4'-position precedes hydrolysis of glucose bound at the 3-position in the double glucoside  <cit> .
the tnbgl1a aglycone + <dig> subsite is formed by the hydrophilic residues  and mainly by aromatic and hydrophobic residues .
a comparison with structure determined plant enzymes from gh <dig>  showed these enzymes to also display aromatic and hydrophobic residues at this subsite but generally with longer hydrophobic residue side chains at the corresponding positions  <cit> .
inspection of residues surrounding the + <dig> subsite showed a non-conserved region at the "floor" of the active site, more precisely at the end of β-strand  <dig>  .
the nonconserved nature of these residues were shown by analysing a multiple sequence alignment as well as by superimposition of known structures.
five more hydrophobic interactions including two aromatic residues  were also found.
superimposition of 3d structures of gh <dig> enzymes with varying substrate specificities , paenibacillus polymyxa , homo sapiens , oryza sativa japonica  and zea mays ) confirmed the variable area of β-strand  <dig> in the vicinity of the substrate pocket binding as well as variability in the loops.
in general, thermophilic β-glucosidases presented shorter loops and more compact overall structures compared to plant counterparts, in line with previous results  <cit> .
the variability at the end of β-strand  <dig> was also corroborated by the multiple sequence alignment of the tnbgl1a sequence  with sequences of oligosaccharide, flavonoid-, and isoflavonoid hydrolysing gh <dig> enzymes as well as gh <dig> enzymes active on other bulky phenol-containing substrates like e.g.
the alkaloids strictosidine and raucaffricine .
the residues at position  <dig>   <dig> and  <dig> were targeted for mutagenesis because of the sequence variation between specificity groups at these sites combined with their location close to the cellotetraose + <dig> sugar residue.
the changes   were chosen based on residues found in enzymes hydrolysing the bulky phenol-containing substrates .
in the case of g <dig> two mutations were designed, one with an hydrophobic  and another one with hydrophilic  residue.
changing a g for m was made to increase hydrophobicity at the entrance of the active site but may exclude water molecules reducing cleavage of the glucosidic linkage in hydrolysis reactions.
the g222q mutation could instead result in substrate interactions via hydrogen bonds .
the f219l and n221s mutations were selected based on residues found at corresponding positions in the enzymes specific for large and bulky substrates, and predicted to generate space for better substrate accommodation.
the residues were mutated in a protocol that introduced a few spontaneous mutations during the amplification procedure.
sequencing of the obtained genes showed that the designed mutations were obtained in all cases, and that one or two spontaneous mutation were present in six clones.
the selected clones included four genes with triple mutations: f219l/p165l/m278i, n221s/p165l/m278i, g222q/p165l/m278i, g222q/v203m/k214r, two with double mutations: f219l/k214r, n221s/p342l, and two with the single mutation g222m and n221s.
the spontaneous mutations  in principle involved conserved changes , replacing proline with a hydrophobic residue in two cases , and a charged basic residue in one case .
four of the five spontaneous mutations were located at the surface of the enzyme, opposite the active site.
replacement of proline with leucine at position  <dig>  located next to the catalytic acid/base was found in three of the four triple mutants, along with a surface located hydrophobic residue mutation .
expression and purification
the wild-type and mutated variants of t. neapolitana β-glucosidase 1a were produced in escherichia coli tuner as described by turner et al  <cit> .
the expression level was analysed by sds-page and showed all enzymes to have very similar production patterns , leaving less than half of the produced protein in a soluble active form, despite use of inducer tuning .
all mutated enzymes were screened for activity and found to hydrolyse pnpglc .
purification was accomplished by a two step protocol, including a heat treatment  followed by immobilised metal ion affinity chromatography  utilizing the c-terminal his-tag, which yielded a purity at or above 90% in all cases .
thermostability
thermostability of all enzyme variants was evaluated by differential scanning calorimetry .
a single transition peak was observed in all cases, which during unfolding resulted in aggregation .
a repeated scan confirmed the denaturation to be irreversible in all cases .
all enzyme variants kept unfolding temperatures above 90°c making them suitable as biocatalysts in applications requiring high thermostability.
as expected, thermostability decreased with increasing number of mutations, and the triple mutants showed a decrease in the apparent unfolding temperature  ranging from  <dig> - 10°c .
the single and double mutants did not change unfolding temperature to any large extent.
kinetic parameters in pnpglc hydrolysis
the kinetic parameters for hydrolysis were monitored using the model substrate pnpglc at ph  <dig>  .
the ph was set based on previously published data taking into account the ph-range for the highest kcat/km determined for the homologous t. maritima enzyme   <cit>  combined with the three point screening of tnbgl1a  for q4' hydrolysis  <cit> .
the turnover number as well as the km values showed changes in some variants.
the mutation at position  <dig>  resulted in a significant reduction in the turnover number  both for the single g222m mutant and the triple mutants including the change g222q as compared to the wt.
only a minor reduction in catalytic efficiency , was however seen, as the variations in km in most cases counteracted the change in turnover.
the triple mutant f219l/p165l/m278i showed a similar reduction in turnover, but this is likely an effect of added mutations, as the double mutant carrying the f219l change  showed parameters more similar to the wild type.
variants carrying p165l, located next to the catalytic acid/base, showed in all cases a lower turnover than other variants carrying changes in either f <dig>  n <dig> or g <dig>  but lacking the change at position  <dig> .
all three mutants carrying the n221s change instead showed an increased turnover .
the increase in turnover was however combined with a significant increase in km for all three mutants  which displayed  <dig>  ×,  <dig>  × and  <dig>  × increases in km, respectively.
as the increased km counteracted the increase in turnover only a minor change in catalytic efficiency was observed.
the relatively more pronounced effect on the km however raised an interest for trying these variants in deglycosylation reactions of the quercetin glucosides, as glucose at the 3-position generally seems more difficult to hydrolyse with the gh <dig> enzymes .
*data from lindahl et al  <cit> .
for pnpglc hydrolysis measurements were made at ph  <dig> , 80°c, and for the quercetin glucoside hydrolysis  at ph  <dig> , 90°c.
previous work by lindahl et al, and yernool et al  <cit> , have shown that glucose is acting as an activator in the wt enzyme.
to assure that this effect is maintained after mutation, the kinetics of the wt and n221s/p342l variant in pnpglc hydrolysis were determined in presence of  <dig> mm glucose.
this resulted in an increase of the turnover  but no significant change in the km value  leading to an increased catalytic efficiency in presence of glucose .
the activating effect of glucose is hence maintained to the same extent in the mutated enzyme, and no product inhibition upon glucose release is expected.
quercetin-glucoside hydrolysis
although hydrolysis of different quercetin-glucosides by enzymes from gh <dig> has been reported  <cit> , hydrolysis of the q <dig> glucoside appears to be more unusual.
in the case of tnbgl1a wt, it has been shown that hydrolysis of q <dig> is possible but slow  <cit> .
this motivated screening of obtained mutants in q <dig> hydrolysis, to monitor improvements in the hydrolysis of this substrate using a fixed concentration of enzyme and substrate .
of the positions selected at the + <dig> site the mutation n221s led to the highest increase in conversion  of q <dig> to q .
repeated trials with the n221s single mutant, showed that the second mutation p342l in the double mutant, had no major role in this increase .
the g222m mutation also led to increased q3-conversion .
these improvements may be a result of improved substrate accomodation, and indeed the modelling of q <dig> in the n221s variant showed that an additional hydrogen bonding to the substrate  can occur via the backbone carbonyl of n <dig> as a consequence of interaction changes caused by the mutation .
increased hydrophobicity could improve interactions between the substrate and enzyme, and may be the case for g222m which likely has its sidechain pointing into the catalytic cleft.
for q4' all enzyme variants completely hydrolysed the substrate within the reaction time of  <dig> min.
the  mutant resulted in the highest conversion of q <dig> and was selected for determination of the kinetic parameters for conversion of the q4' and q <dig> glucosides to q .
largest improvement was due to a decrease in km using both q4'and q <dig>  and this was especially pronounced using the q <dig> substrate , which can be explained by the added substrate interaction .
discussion
the thermostable β-glucosidase  from t. neapolitana has been used as a biocatalyst for conversion of quercetin-glucosides to quercetin  <cit>  extracted using a hot water extraction method, shown to be beneficial from an environmental perspective  <cit> .
it was shown that the enzyme completely converted the q4' species to quercetin, and that also the glucoside connected to the 3-position of the flavonoid backbone  was converted, although with lower efficiency.
this shows that the accessibility to the active site differs between enzymes in gh <dig>  as previous work on flavonoid hydrolysing enzymes in gh <dig>  have shown that enzymes capable of converting the 4'-glucoside ) are not capable of hydrolysing the flavonoid 3-glucosides  <cit> .
the tnbgl1a enzyme is thus an interesting target to study by molecular modelling, as well as by site-directed mutagenesis to analyse how changes of amino acids affect the kinetic parameters and the conversion of the quercetin-3-glucoside.
interactions with inhibitors in the - <dig> subsite of the homologous t. maritima enzyme have been extensively studied  <cit> , and have shown this site to be important for the selection of the sugar to be hydrolysed.
in a study on a sulfolobus solfataricus representative of gh <dig> by corbett et al,  <cit>  it was for example shown that mutagenesis of substrate interacting residues in the - <dig> site led to a shift in affinity towards xylose, or from glucose to mannose, dependent on the residue chosen.
here we are instead focusing on interactions closer to the entrance of the active site, and close to the + <dig> site, which has not been targeted to the same extent.
comparison of the structure of human cbg, which cannot hydrolyse the flavonoid 3-glucoside  <cit>  with the model of tnbgl1a, shows a difference in the shape of the active site .
it can be clearly seen that hcbg forms a regular oval shaped pocket, while the corresponding pocket in tnbgl1a  has a wider conformation, which may facilitate accomodation of the 3-linked substrate.
the active site entrance in gh <dig> is formed by four extended loops  <cit> .
these loops  have been defined as being responsible for the overall shape of the aglycone binding pocket, and differences in the conformation of one or more of these loops would likely change the overall shape of the entrance  <cit> .
in hcbg, loops b  and d  are short, which is claimed to result in a small entrance to the pocket.
comparison with tnbgl1a show that loop length of loop b is the same as in hcbg, and despite low sequence similarity in this area both loop a and loop b superimpose very well between the two enzymes.
loop d is however longer in tnbgl1a, in accordance with the suggestion that this may contribute to a wider active site entrance of this enzyme .
however, parts of loop c do not superimpose between the two enzymes and in tnbgl1a this loop is significantly shorter than in the human enzyme .
it is actually close to this shorter loop c that we see a significant widening of the tnbgl1a active site compared to hcbg, indicating that the longer loop in the human enzyme is closing the active site entrance.
moreover, the mutations introduced at the + <dig> site are located close to loop c in the structure and may aid in a further widening of the active site entrance simplifying binding of q <dig>
in addition to interactions at the + <dig> subsite, the modelling of q <dig> into the active site of tnbgl1a showed that + <dig> subsite binding was also affected as a consequence of changes in the interactions between residues.
the side chain of residue n <dig> selected here, which upon mutagenesis led to a decrease in km for q <dig> as well as increased catalytic efficiency, is not pointing towards the catalytic cleft in our model.
a close look at the binding interactions of n <dig> in presence of q <dig> shows that this residue is interacting with residues in neighbouring strands of the protein  , and n <dig> may thus have importance for the shape in that part of the active site.
the introduced s <dig> leads to loss of the interaction with n <dig>  but also a change in the position of the backbone carbonyl of the residue allowing hydrogen bonding to the 5-oh of q <dig> .
such a bond would explain the observed decrease in km.
calculation of the ΔΔg ) for q <dig> corresponded to a free energy change of  <dig> kj/mol.
a hydrogen bond interaction to an uncharged amino acid is in the range 2- <dig> kj/mol  <cit>  showing that the change in km is likely the result of an affinity increase.
changes in turnover are more difficult to explain, and may be caused by conformational changes caused by indirect changes in interactions between residues.
other explanations include changes in the position of the side chain of the neighbouring residue  pointing into the catalytic site.
a similar position of the sidechain is found for the corresponding residue in the homologous human glucosidase .
the hcbg residue f <dig> was shown by mutagenesis to affect the aglycone specificity  <cit> .
mutagenesis of the neighboring residue  in tnbgl1a may change the position of n <dig> in the active site, or lead to changes in the local environment that promotes an affinity change.
mutation of f <dig> , preceding n <dig>  did however not lead to any corresponding or significant changes in affinity or turnover.
all spontaneous mutations, except one, involved residues located at the surface of the enzyme.
only p165l of the spontaneous mutations, is located in the active site next to the catalytic acid/base.
this change from proline to leucine should introduce more flexibility.
the effect of this change on the activity is, based on the activity data, however not completely clear.
it may lead to a minor decrease in activity compared to clones where this mutation is lacking, but its effect appears to be small.
the additional surface located mutations, appear to mostly affect stability , but it is only in triple mutants that the transition temperature is affected to any large extent .
CONCLUSIONS
in conclusion, the mutation study done in this work shows that relatively small residue variations in the enzyme, made at or close to the + <dig> site, may modify the interactions in the active site, leading to increased substrate interactions as well as conformational changes that allow increased hydrolysis of a sterically differently attached glucose on the quercetin backbone.
in addition, effects on the turnover of the introduced mutations were often counteracted by a change in km, leading to smaller differences in catalytic efficiency, than in the separated km and kcat parameters.
list of abbreviations
dsc: differential scanning calorimetry; g2f: 2-deoxy-2-fluoro-beta-d-glucoside; gh: glycoside hydrolase; hcbg: human cytosolic beta-glucosidase; hplc: high pressure liquid chromatography; km: the michaelis constant; kcat: turnover number; pcr: polymerase chain reaction; pnpglc: para-nitrophenyl-beta-d-glucopyranoside; pnp: para-nitrophenol; tnbgl1a: thermotoga neapolitana beta-glucosidase a from glycoside hydrolase family 1; q: quercetin; q3: quercetin-3-glucoside; q4': quercetin-4'-glucoside; sds-page: sodium dodecyl sulfate polyacrylamide gel electrophoresis; 3d: three dimensional
authors' contributions
sk: took part in the construction and sequence analysis of mutants, took part in enzyme production, purification and kinetics, and in dsc-data collection.
took part in writing the manuscript and commenting on the manuscript.
tp: took part in constructing mutants.
did the homology modelling and structure analysis together with as, took part in writing the manuscript and commenting on the manuscript.
mm: took part in the construction and sequence analysis of mutants, in the enzyme production and purification, and in dsc-data collection.
took part in writing the manuscript.
sl: did the hydrolysis and hplc-analysis of quercetin and quercetin-3-glucosides, took part in writing and commenting on the manuscript.
as: did the major part of the modelling , and wrote some parts related to this work in the manuscript.
ct: took part in the interpretation of hplc-data.
took part in writing and commenting on the manuscript
enk: selected enzyme and mutation strategy.
took part in the bioinformatics analysis and the dsc-data collection.
took part in overall analysis and interpretation of results.
did the overall layout of manuscript, took part in writing and commenting on the manuscript.
all authors read and approved the final manuscript.

 glycoside hydrolases are enzymes that hydrolyse glycosidic bonds between two or more carbohydrates, or between a carbohydrate and a non-carbohydrate moiety. carbohydrates are essential components of biomass, which is estimated to be produced in a quantity of about gt/year 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. these enzymes can also be used as specific catalysts in industrial applications, e.g. in the food and feed industries, the paper and pulp, starch and textile industries, and in newly emerging sustainable processes taking advantage of their specificity in selective preparations of carbohydrate-containing raw materials. antioxidants are bioactive compounds that have received great interest due to their potential as health beneficial agents. the action of antioxidants is to counteract oxidative stress imposed by reactive oxygen species shown to play a crucial role in the pathophysiology associated with neoplasia, atherosclerosis and neurodegenerative diseases. polyphenolic compounds show a wide range of antioxidant activities, and are thought to exert protective effects against the diseases specified above. flavonoids are polyphenolic compounds that are important antioxidative constituents of fruits and vegetables, but the type of compound is different in different sources. fruits and vegetables rich in anthocyanins show highest antioxidant activities, followed by those rich in flavonones or flavonols, while hydroxycinnamate-rich fruits exhibit lower antioxidant activities. flavonols are generally not found as free aglycones, but rather as complex conjugates with sugar residues. ghs hydrolyse certain flavonoid glycosides, dependent on the aglycone moiety, type of sugar and linkage and some glucosidases classified under gh family are flavonoid-hydrolysing enzymes. in a previous work we have shown that the oligosaccharide hydrolysing gh β-glucosidase tnbgl1a, from the hyperthermophile thermotoga neapolitana, efficiently hydrolyses quercetin-4'-glucosides. q4' and quercetin-'-diglucoside are the most abundant species in yellow onion, and to obtain the quercetin aglycone both q4' and q species must be hydrolysed. most commonly quercetin and its glycosides have been extracted from yellow onion by simple liquid/solid extraction techniques combined with chemically catalyzed hydrolysis reaction of the extracted quercetin glucosides. we have instead utilized pressurized hot water to extract the quercetin species from yellow onion, followed by biocatalytic conversion of the quercetin glucosides to quercetin and carbohydrates. in this system, use of enzymes with high thermostability is essential, and the enzyme tnbgl1a from the hyperthermophile t. neapolitana is from this perspective a suitable biocatalyst. it was also shown that the q4' was much more efficiently hydrolysed than the q in this investigation, the β-glucosidase tnbgl1a, was mutated to investigate the influence of mutations on the enzyme kinetics ), thermostability, and selective hydrolysis of glucose at two positions on the aglycone quercetin. we have utilized a strategy to design mutants based on bioinformatics and structural analysis, with an amplification protocol that allowed spontaneous mutations, in order to find residues that influence specificity of the enzyme. methods chemicals all chemicals were of pro-analysis grade from merck eurolabs unless otherwise stated. cloning of tnbgl1a the gene encoding bgl1a was pcr-amplified from genomic thermotoga neapolitana dna as described by turner et al. primers were designed to amplify the coding sequence of bgl1a from the sequence deposited at the ncbi server under the accession number af the gene was inserted in vector pet-22b under control of the t7/lac promoter and incorporating the c-terminal hexa-histidine tag. the resulting plasmids were transformed into e. coli nova blue cells and screened by colony pcr using the t forward and t reverse primers and taq dna polymerase. positive clones were transformed into the e. coli expression host tuner. the complete gene was sequenced at mwg biotech. athe restriction sites for cloning are underlined. binduced changes are shown in bold. mutagenesis mutagenesis was performed in order to introduce the following designed changes: f219l, n221s, and g222m/q, respectively. taq polymerase, which lacks proofreading, was used to allow introduction of a few random additional mutations. standard concentrations of mgcl and dntps were used. in a first pcr, a mutated gene fragment encoding the c-terminal part of the enzyme was constructed using forward mismatched primers together with the reverse gene specific primer encoding the c-terminal sequence of bgl1a. the n221s mutants were obtained by amplifying the full length gene in a second pcr using the product of the first pcr as a reverse "megaprimer" together with the forward primer matching the start of the gene. mutations at position, and were constructed using overlap extension pcr of the mutated fragment and an overlapping gene fragment encoding the n-terminal part of bgl1a. reverse primers together with the forward gene specific primer were used to create the overlapping fragments that were pcr-amplified with the wild-type gene as a template. overlap extension pcr reaction was then run in two steps. firstly, extension without template at: 94°c min; cycles: 94°c min, °c min, 72°c s, 72°c min and the ramp between the annealing and extension changed from the default 3°c/s to 5°c/s. secondly, amplification at standard conditions, using the gene-specific forward and reverse primers, and the product of the overlap extension pcr as template. all mutated genes were purified with qiaexii gel extraction kit after gel separation. both insert and vector were digested and ligated as described under the cloning section. resulting plasmids were transformed into e. coli nova blue cells and screened by colony pcr using the t forward and t reverse primers. selected mutant clones were fully sequenced at mwg biotech. homology modelling and ligand binding a tnbgl1a homology model was constructed utilizing the schrodinger software suite. energy minimizations were performed with macromodel, utilizing the opls- force field and the gb/sa model for water solvation. molecular dynamics was performed with desmond, utilizing default settings. to find homologes to the tnbgl1a sequence, a blast search was performed on proteins with x-ray diffraction data in the pdb data base. the amino acid sequence of the gh β-glucosidase from thermotoga maritima provided the highest similarity, 90% identity, and 97% positives. the pdb structure 2wc of bgla from t. maritima with 3-imino-2-thio--castanospermine as ligand, had the highest resolution of these x-ray crystallographic models, and was used as template to build a 3d model of tnbgl1a. the protein preparation wizard was utilized to add hydrogens, assign charges, optimize hydrogen bond networks, and to analyze the quality of the homology model. the rmsd with the template was Å after optimisation. in the region of the active site, a cis-bond between w and s was found, however this cis-amide is present in all crystals of t. maritima β-glucosidase. furthermore, energy minimization showed the cis conformer to have lower energy than the corresponding trans amide bond. a low energy model of β-d-cellotetraose was constructed and placed in the homology model so that the β-d-glucose at the non-reducing end superimposed with the six membered rings of the t. maritima bgla ligands in the pdb structures 2wc 3cmj, 1qox, 1e4i, and 1bga. the glycosidic linkage torsion angle ϕ of this sugar was adjusted from -121° to 26° to make cellotetraose fit in the ligand binding pocket. the resulting structure was energy minimized to yield a cellotetraose binding pose that is in agreement with the proposed mechanism of family β-glucosidases. the binding pose was verified as stable in ns molecular dynamics simulation. a conformational search was performed on q and the lowest energy conformer was placed in the homology model using the same method as for cellotetraose. the resulting structure was energy minimized, and the binding pose, which was in agreement with the proposed mechanism of family β-glucosidases, was verified with a ns molecular dynamics simulation. figures of docking results were prepared within the schrodinger software suite, including the program pymol. expression and purification the mutant and wild-type enzymes were produced in l batch cultivations at 37°c, ph using a defined medium with μg ml- ampicillin and a dissolved oxygen tension above 40%. expression was induced at od nm = by the addition of - mm isopropyl-beta-d-thiogalactopyranoside, and continued for h. production levels were analysed by sds-page in samples withdrawn hourly after induction. the cells were pelleted, resuspended in μl mm citrate-phosphate buffer, ph, ultrasonicated for × s with a up400s equipped with a mm titanium probe using a sound intensity of 60% and a cycle of and thereafter centrifuged for min at × g to separate soluble proteins from insoluble proteins and cell debris. the cells were harvested, separated from the cultivation medium by centrifugation, and dissolved in binding buffer. the ice-chilled cell suspension was lysed by sonication for × min using a mm titanium probe sound intensity of 60% and a cycle of, centrifuged, heat treated and again centrifuged. the supernatant was passed through a μm minisart high-flow filter and purified on an Äkta prime system by immobilized metal ion affinity chromatography using copper as a ligand as described elsewhere. the fractions containing the purified protein were pooled and dialyzed against mm citrate phosphate buffer, ph, overnight using a spectra/por dialysis membrane with a da molecular weigh cut-off. the dialysed protein fractions were stored at 4°c until use. protein analysis the purity of each mutant and wild-type enzyme was analysed by sds-page according to. expression levels were also analysed by sds-page after separating insoluble and soluble proteins. total protein concentration was estimated at nm by the bca method using bovine serum albumin as standard. differential scanning calorimetry dsc analysis was made on a microcal differential scanning calorimeter with the cell volume of ml. the samples were concentrated to mg ml- using vivaspin centrifuging tubes with a mwco of da and were degassed before the scans. the samples were scanned at a rate of 1°c/min in the temperature range of 25-110°c. enzyme activity on pnpglc, q and q4' enzyme activity, and kinetic parameters were determined at 80°c, ph using pnpglc as substrate in mm citrate phosphate buffer, on a shimadzu uv- visible spectrophotometer. a volume of μl of pnpglc was preheated for min, where after μl of the enzyme solution was added. absorbance at nm was measured and plotted by the shimadzu uv probe software as a function of time during min. the extinction coefficient of pnp under these experimental conditions was determined as ε 80°c, nm= × ml mmol-1cm- the kinetic parameters were determined by applying the wilkinson non-linear regression method using enzpack. the effect of glucose on activity in this system was evaluated by adding mm glucose to the stock solution of mm pnpglc and the kinetic values km and kcat were determined as above. enzyme activity of selected mutants of tnbgl1a were screened for two quercetin glucosides, quercetin-3-glucoside and quercetin-4'-glucoside at 90°c. nmol samples of q or q4' dissolved in methanol were evaporated and ml of mm citrate phosphate buffer, ph, was added and the vials were heated at 90°c until substrate was dissolved. a μl fraction was collected and added to μl of mobile phase composed of methanol/water and m formic acid. the reaction was started by adding pmol of enzyme and min after addition of enzyme, μl fractions were collected and added to μl of mobile phase. samples were analyzed by hplc with uv detection. the conditions and methodolology for the kinetic measurement of q and q4' was published in lindahl et al. in summary, 33- nmol q and q4' were dissolved per ml citrate-phosphate buffer ph. for q hydrolysis pmol wt and pmol mutant n221s/p342l were used, and for q4' hydrolysis pmol wt and pmol mutant n221s/p342l were used. hplc analysis hplc-uv analysis was performed using the chromatographic system ultimate from dionex. an agilent zorbax sb-c column was used for isocratic separation with a methanol:water and m formic acid mobile phase at a flow rate of ml min- the injection volume was μl and detection was accomplished at nm. quantification of quercetin and glycosides was performed using a five-point calibration curve of a quercetin dihydrate standard and q and q4' standards at concentrations between and μg ml- each vial taken to analysis had a total volume of μl.  the different members of glycoside hydrolase family, catalyse hydrolysis of a glucose molecule from a number of different substrates, including some hydrophobic substrates linked to glucose. only a few members of the family are, however, commercially available, and many different analytical assays have thus utilized β-glucosidase from almond, which has been classified under gh, and which is often available in heterogeneous preparations. β-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. this enzyme is thermostable, and hence suitable for application in hot water. moreover, this enzyme belongs to a gh family with diverse substrate specificity, with many gene sequences available allowing comparison, and with necessary structural information available, including three-dimensional structures of the closely related enzyme tmgh from thermotoga maritima, allowing homology modelling of the 3d structure. structural considerations and mutation strategy as noted elsewhere, the deduced amino acid sequence encoded by the bgl1a-gene used in this work has one change in primary sequence compared to the deposited sequence. sequence alignments revealed v at position to be conserved among several members of gh family and the obtained sequence is hereafter referred to as wild type and designated tnbgl1a. a molecular model of t. neapolitana tnbgl1a was generated based on the 3d structure of β-glucosidase bgla from t. maritima. tmgh provided the highest similarity: 90% identity, and 97% positives. the homology detection structure prediction server hhpred also confirmed tmgh as the best template. the overall structure presented in the tnbgl1a model is the typical barrel fold characteristic of gh. the active site is a deep channel narrowed at the bottom with a wider cleft at the entrance. two conservative motifs tlnep and iteng are situated opposite to each other inside the active site, more precisely at the ends of strands β and β containing the acid/base and the nucleophile residues respectively. superimposition of tmgh in complex with 2-deoxy-2-fluoro-β-d-glucopyranoside and tnbgl1a allowed identification of the residues forming the glycone binding site. interacting residues included conservative residues forming hydrogen bonds and hydrophobic interactions. cellotetraose was modelled into the tnbgl1a model to analyse putative interactions at the + and + subsites. the β-d-glucopyranoside at the non-reducing end was placed by superimposing the six membered rings of the ligand in gh structures. the sugars in the cellotetraose were positioned in a dynamically stable position in agreement with the proposed mechanism of family β-glucosidases. the same procedure was repeated for the glucosylated quercetin to see its position in relation to the sugar binding subsites. both q and q4' could be fitted in positions relevant for hydrolysis, while the double glucoside could not be accommodated with the 3-glucoside positioned at the - subsite. this explains why hydrolysis at the 4'-position precedes hydrolysis of glucose bound at the 3-position in the double glucoside. the tnbgl1a aglycone + subsite is formed by the hydrophilic residues and mainly by aromatic and hydrophobic residues. a comparison with structure determined plant enzymes from gh showed these enzymes to also display aromatic and hydrophobic residues at this subsite but generally with longer hydrophobic residue side chains at the corresponding positions. inspection of residues surrounding the + subsite showed a non-conserved region at the "floor" of the active site, more precisely at the end of β-strand. the nonconserved nature of these residues were shown by analysing a multiple sequence alignment as well as by superimposition of known structures. five more hydrophobic interactions including two aromatic residues were also found. superimposition of 3d structures of gh enzymes with varying substrate specificities, paenibacillus polymyxa, homo sapiens, oryza sativa japonica and zea mays ) confirmed the variable area of β-strand in the vicinity of the substrate pocket binding as well as variability in the loops. in general, thermophilic β-glucosidases presented shorter loops and more compact overall structures compared to plant counterparts, in line with previous results. the variability at the end of β-strand was also corroborated by the multiple sequence alignment of the tnbgl1a sequence with sequences of oligosaccharide, flavonoid-, and isoflavonoid hydrolysing gh enzymes as well as gh enzymes active on other bulky phenol-containing substrates like e.g. the alkaloids strictosidine and raucaffricine. the residues at position and were targeted for mutagenesis because of the sequence variation between specificity groups at these sites combined with their location close to the cellotetraose + sugar residue. the changes were chosen based on residues found in enzymes hydrolysing the bulky phenol-containing substrates. in the case of g two mutations were designed, one with an hydrophobic and another one with hydrophilic residue. changing a g for m was made to increase hydrophobicity at the entrance of the active site but may exclude water molecules reducing cleavage of the glucosidic linkage in hydrolysis reactions. the g222q mutation could instead result in substrate interactions via hydrogen bonds. the f219l and n221s mutations were selected based on residues found at corresponding positions in the enzymes specific for large and bulky substrates, and predicted to generate space for better substrate accommodation. the residues were mutated in a protocol that introduced a few spontaneous mutations during the amplification procedure. sequencing of the obtained genes showed that the designed mutations were obtained in all cases, and that one or two spontaneous mutation were present in six clones. the selected clones included four genes with triple mutations: f219l/p165l/m278i, n221s/p165l/m278i, g222q/p165l/m278i, g222q/v203m/k214r, two with double mutations: f219l/k214r, n221s/p342l, and two with the single mutation g222m and n221s. the spontaneous mutations in principle involved conserved changes, replacing proline with a hydrophobic residue in two cases, and a charged basic residue in one case. four of the five spontaneous mutations were located at the surface of the enzyme, opposite the active site. replacement of proline with leucine at position located next to the catalytic acid/base was found in three of the four triple mutants, along with a surface located hydrophobic residue mutation. expression and purification the wild-type and mutated variants of t. neapolitana β-glucosidase 1a were produced in escherichia coli tuner as described by turner et al. the expression level was analysed by sds-page and showed all enzymes to have very similar production patterns, leaving less than half of the produced protein in a soluble active form, despite use of inducer tuning. all mutated enzymes were screened for activity and found to hydrolyse pnpglc. purification was accomplished by a two step protocol, including a heat treatment followed by immobilised metal ion affinity chromatography utilizing the c-terminal his-tag, which yielded a purity at or above 90% in all cases. thermostability thermostability of all enzyme variants was evaluated by differential scanning calorimetry. a single transition peak was observed in all cases, which during unfolding resulted in aggregation. a repeated scan confirmed the denaturation to be irreversible in all cases. all enzyme variants kept unfolding temperatures above 90°c making them suitable as biocatalysts in applications requiring high thermostability. as expected, thermostability decreased with increasing number of mutations, and the triple mutants showed a decrease in the apparent unfolding temperature ranging from - 10°c. the single and double mutants did not change unfolding temperature to any large extent. kinetic parameters in pnpglc hydrolysis the kinetic parameters for hydrolysis were monitored using the model substrate pnpglc at ph. the ph was set based on previously published data taking into account the ph-range for the highest kcat/km determined for the homologous t. maritima enzyme combined with the three point screening of tnbgl1a for q4' hydrolysis. the turnover number as well as the km values showed changes in some variants. the mutation at position resulted in a significant reduction in the turnover number both for the single g222m mutant and the triple mutants including the change g222q as compared to the wt. only a minor reduction in catalytic efficiency, was however seen, as the variations in km in most cases counteracted the change in turnover. the triple mutant f219l/p165l/m278i showed a similar reduction in turnover, but this is likely an effect of added mutations, as the double mutant carrying the f219l change showed parameters more similar to the wild type. variants carrying p165l, located next to the catalytic acid/base, showed in all cases a lower turnover than other variants carrying changes in either f n or g but lacking the change at position. all three mutants carrying the n221s change instead showed an increased turnover. the increase in turnover was however combined with a significant increase in km for all three mutants which displayed ×, × and × increases in km, respectively. as the increased km counteracted the increase in turnover only a minor change in catalytic efficiency was observed. the relatively more pronounced effect on the km however raised an interest for trying these variants in deglycosylation reactions of the quercetin glucosides, as glucose at the 3-position generally seems more difficult to hydrolyse with the gh enzymes. *data from lindahl et al. for pnpglc hydrolysis measurements were made at ph, 80°c, and for the quercetin glucoside hydrolysis at ph, 90°c. previous work by lindahl et al, and yernool et al, have shown that glucose is acting as an activator in the wt enzyme. to assure that this effect is maintained after mutation, the kinetics of the wt and n221s/p342l variant in pnpglc hydrolysis were determined in presence of mm glucose. this resulted in an increase of the turnover but no significant change in the km value leading to an increased catalytic efficiency in presence of glucose. the activating effect of glucose is hence maintained to the same extent in the mutated enzyme, and no product inhibition upon glucose release is expected. quercetin-glucoside hydrolysis although hydrolysis of different quercetin-glucosides by enzymes from gh has been reported, hydrolysis of the q glucoside appears to be more unusual. in the case of tnbgl1a wt, it has been shown that hydrolysis of q is possible but slow. this motivated screening of obtained mutants in q hydrolysis, to monitor improvements in the hydrolysis of this substrate using a fixed concentration of enzyme and substrate. of the positions selected at the + site the mutation n221s led to the highest increase in conversion of q to q. repeated trials with the n221s single mutant, showed that the second mutation p342l in the double mutant, had no major role in this increase. the g222m mutation also led to increased q3-conversion. these improvements may be a result of improved substrate accomodation, and indeed the modelling of q in the n221s variant showed that an additional hydrogen bonding to the substrate can occur via the backbone carbonyl of n as a consequence of interaction changes caused by the mutation. increased hydrophobicity could improve interactions between the substrate and enzyme, and may be the case for g222m which likely has its sidechain pointing into the catalytic cleft. for q4' all enzyme variants completely hydrolysed the substrate within the reaction time of min. the mutant resulted in the highest conversion of q and was selected for determination of the kinetic parameters for conversion of the q4' and q glucosides to q. largest improvement was due to a decrease in km using both q4'and q and this was especially pronounced using the q substrate, which can be explained by the added substrate interaction. discussion the thermostable β-glucosidase from t. neapolitana has been used as a biocatalyst for conversion of quercetin-glucosides to quercetin extracted using a hot water extraction method, shown to be beneficial from an environmental perspective. it was shown that the enzyme completely converted the q4' species to quercetin, and that also the glucoside connected to the 3-position of the flavonoid backbone was converted, although with lower efficiency. this shows that the accessibility to the active site differs between enzymes in gh as previous work on flavonoid hydrolysing enzymes in gh have shown that enzymes capable of converting the 4'-glucoside ) are not capable of hydrolysing the flavonoid 3-glucosides. the tnbgl1a enzyme is thus an interesting target to study by molecular modelling, as well as by site-directed mutagenesis to analyse how changes of amino acids affect the kinetic parameters and the conversion of the quercetin-3-glucoside. interactions with inhibitors in the - subsite of the homologous t. maritima enzyme have been extensively studied, and have shown this site to be important for the selection of the sugar to be hydrolysed. in a study on a sulfolobus solfataricus representative of gh by corbett et al, it was for example shown that mutagenesis of substrate interacting residues in the - site led to a shift in affinity towards xylose, or from glucose to mannose, dependent on the residue chosen. here we are instead focusing on interactions closer to the entrance of the active site, and close to the + site, which has not been targeted to the same extent. comparison of the structure of human cbg, which cannot hydrolyse the flavonoid 3-glucoside with the model of tnbgl1a, shows a difference in the shape of the active site. it can be clearly seen that hcbg forms a regular oval shaped pocket, while the corresponding pocket in tnbgl1a has a wider conformation, which may facilitate accomodation of the 3-linked substrate. the active site entrance in gh is formed by four extended loops. these loops have been defined as being responsible for the overall shape of the aglycone binding pocket, and differences in the conformation of one or more of these loops would likely change the overall shape of the entrance. in hcbg, loops b and d are short, which is claimed to result in a small entrance to the pocket. comparison with tnbgl1a show that loop length of loop b is the same as in hcbg, and despite low sequence similarity in this area both loop a and loop b superimpose very well between the two enzymes. loop d is however longer in tnbgl1a, in accordance with the suggestion that this may contribute to a wider active site entrance of this enzyme. however, parts of loop c do not superimpose between the two enzymes and in tnbgl1a this loop is significantly shorter than in the human enzyme. it is actually close to this shorter loop c that we see a significant widening of the tnbgl1a active site compared to hcbg, indicating that the longer loop in the human enzyme is closing the active site entrance. moreover, the mutations introduced at the + site are located close to loop c in the structure and may aid in a further widening of the active site entrance simplifying binding of q in addition to interactions at the + subsite, the modelling of q into the active site of tnbgl1a showed that + subsite binding was also affected as a consequence of changes in the interactions between residues. the side chain of residue n selected here, which upon mutagenesis led to a decrease in km for q as well as increased catalytic efficiency, is not pointing towards the catalytic cleft in our model. a close look at the binding interactions of n in presence of q shows that this residue is interacting with residues in neighbouring strands of the protein, and n may thus have importance for the shape in that part of the active site. the introduced s leads to loss of the interaction with n but also a change in the position of the backbone carbonyl of the residue allowing hydrogen bonding to the 5-oh of q. such a bond would explain the observed decrease in km. calculation of the ΔΔg ) for q corresponded to a free energy change of kj/mol. a hydrogen bond interaction to an uncharged amino acid is in the range 2- kj/mol showing that the change in km is likely the result of an affinity increase. changes in turnover are more difficult to explain, and may be caused by conformational changes caused by indirect changes in interactions between residues. other explanations include changes in the position of the side chain of the neighbouring residue pointing into the catalytic site. a similar position of the sidechain is found for the corresponding residue in the homologous human glucosidase. the hcbg residue f was shown by mutagenesis to affect the aglycone specificity. mutagenesis of the neighboring residue in tnbgl1a may change the position of n in the active site, or lead to changes in the local environment that promotes an affinity change. mutation of f, preceding n did however not lead to any corresponding or significant changes in affinity or turnover. all spontaneous mutations, except one, involved residues located at the surface of the enzyme. only p165l of the spontaneous mutations, is located in the active site next to the catalytic acid/base. this change from proline to leucine should introduce more flexibility. the effect of this change on the activity is, based on the activity data, however not completely clear. it may lead to a minor decrease in activity compared to clones where this mutation is lacking, but its effect appears to be small. the additional surface located mutations, appear to mostly affect stability, but it is only in triple mutants that the transition temperature is affected to any large extent.  in conclusion, the mutation study done in this work shows that relatively small residue variations in the enzyme, made at or close to the + site, may modify the interactions in the active site, leading to increased substrate interactions as well as conformational changes that allow increased hydrolysis of a sterically differently attached glucose on the quercetin backbone. in addition, effects on the turnover of the introduced mutations were often counteracted by a change in km, leading to smaller differences in catalytic efficiency, than in the separated km and kcat parameters. list of abbreviations dsc: differential scanning calorimetry; g2f: 2-deoxy-2-fluoro-beta-d-glucoside; gh: glycoside hydrolase; hcbg: human cytosolic beta-glucosidase; hplc: high pressure liquid chromatography; km: the michaelis constant; kcat: turnover number; pcr: polymerase chain reaction; pnpglc: para-nitrophenyl-beta-d-glucopyranoside; pnp: para-nitrophenol; tnbgl1a: thermotoga neapolitana beta-glucosidase a from glycoside hydrolase family 1; q: quercetin; q3: quercetin-3-glucoside; q4': quercetin-4'-glucoside; sds-page: sodium dodecyl sulfate polyacrylamide gel electrophoresis; 3d: three dimensional authors' contributions sk: took part in the construction and sequence analysis of mutants, took part in enzyme production, purification and kinetics, and in dsc-data collection. took part in writing the manuscript and commenting on the manuscript. tp: took part in constructing mutants. did the homology modelling and structure analysis together with as, took part in writing the manuscript and commenting on the manuscript. mm: took part in the construction and sequence analysis of mutants, in the enzyme production and purification, and in dsc-data collection. took part in writing the manuscript. sl: did the hydrolysis and hplc-analysis of quercetin and quercetin-3-glucosides, took part in writing and commenting on the manuscript. as: did the major part of the modelling, and wrote some parts related to this work in the manuscript. ct: took part in the interpretation of hplc-data. took part in writing and commenting on the manuscript enk: selected enzyme and mutation strategy. took part in the bioinformatics analysis and the dsc-data collection. took part in overall analysis and interpretation of results. did the overall layout of manuscript, took part in writing and commenting on the manuscript. all authors read and approved the final manuscript. 
thermostability, measured as tm by differential scanning calorimetry, was kept in the mutated variants and significant decrease was only observed for the triple mutants. turnover was only significantly changed using pnpglc, and was decreased 2- × in variants mutated at position while the single, double and triple mutated variants carrying a mutation at position increased turnover up to × compared to the wild type. structural analysis pinpointed an area in the active site pocket with non-conserved residues between specificity groups in glycoside hydrolase family. an affinity change, leading to a decreased km, can be explained by an altered position of n while the changes in turnover are more difficult to explain and may be the result of smaller conformational changes in the active site. the thermostable β-glucosidase from thermotoga neapolitana is a promising biocatalyst for hydrolysis of glucosylated flavonoids and can be coupled to extraction methods using pressurized hot water. these results show that residues at the + subsite are interesting targets for mutagenesis and mutations at these positions can directly or indirectly affect both km and turnover. 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, and screened for hydrolysis of q three residues in this area located on β-strand close to sugar binding sub-site + were selected for mutagenesis and amplified in a protocol that introduced a few spontaneous mutations.
 the thermostable β-glucosidase 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 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, and screened for hydrolysis of q  structural analysis pinpointed an area in the active site pocket with non-conserved residues between specificity groups in glycoside hydrolase family. three residues in this area located on β-strand close to sugar binding sub-site + were selected for mutagenesis and amplified in a protocol that introduced a few spontaneous mutations. eight mutants were produced in e. coli, and purified to apparent homogeneity. thermostability, measured as tm by differential scanning calorimetry, was kept in the mutated variants and significant decrease was only observed for the triple mutants. the exchanged residue in the respective mutant resulted in variations in km and turnover. the km-value was only changed in variants mutated at position and was in all cases monitored as a 2- × increase for pnpglc, while the km decreased a corresponding extent for q turnover was only significantly changed using pnpglc, and was decreased 2- × in variants mutated at position while the single, double and triple mutated variants carrying a mutation at position increased turnover up to × compared to the wild type. modelling showed that the mutation at position may alter the position of n resulting in increased hydrogen bonding of q which may explain the decrease in km for this substrate.  these results show that residues at the + 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 n while the changes in turnover are more difficult to explain and may be the result of smaller conformational changes in the active site. 