Efficient chemoselective synthesis of 3,4′-dihydroxypropiophenone 3-O-β-D-glucoside by thermophilic β-glycosidase from Sulfolobus solfataricus

Summary A comparison of different systems for the -glycosidase-atalyzed synthesis of 3,4-dihydroxypropiophenone 3-O--D-glucoside is reported including various enzymatic sources and different reaction conditions. The best yield was obtained using thermophilic -glycosidase from Sulfolobus solfataricus.     

Characterization of a β-glucosidase from Sulfolobus solfataricus for isoflavone glycosides

The specific activity of a recombinant β-glucosidase from Sulfolobus solfataricus for isoflavones was: daidzin > glycitin > genistin > malonyl genistin > malonyl daidzin > malonyl glycitin. The hydrolytic activity of this enzyme for daidzin was highest at pH 5.5 and 90°C with a half-life of 18 h, a K _m of 0.5 mM, and a k _cat of 2532 s⁻¹. The enzyme converted 1 mM daidzin to 1 mM daidzein after 1 h with a molar yield of 100% and a productivity of 1 mM h⁻¹. Among β-glucosidases, that from S. solfataricus β had the highest thermostability, k _cat, k _cat/K _m, conversion yield, and productivity in the hydrolysis of daidzin.     

Biosynthesis of δ-aminolevulinic acid from glutamate by Sulfolobus solfataricus

The extremely thermophilic, obligately aerobic bacterium Sulfolobus solfataricus forms the tetrapyrrole precursor, -aminolevulinic acid (ALA), from glutamate by the tRNA-dependent five-carbon pathway. This pathway has been previously shown to occur in plants, algae, and most prokaryotes with the exception of the -group of proteobacteria (purple bacteria). An alternative mode of ALA formation by condensation of glycine and succinyl-CoA occurs in animals, yeasts, fungi, and the -proteobacteria. Sulfolobus and several other thermophilic, sulfur-dependent bacteria, have been variously placed within a subgroup of archaea (archaebacteria) named crenarchaeotes, or have been proposed to comprise a distinct prokaryotic group designated eocytes. On the basis of ribosomal structure and certain other criteria, eocytes have been proposed as predecessors of the nuclear-cytoplasmic descent line of eukaryotes. Because aplastidic eukaryotes differ from most prokaryotes in their mode of ALA formation, and in view of the proposed affiliation of eocytes to eukaryotes, it was of interest to determine how eocytes form ALA. Sulfolobus extracts were able to incorporate label from [1-¹⁴C]glutamate, but not from [2-¹⁴C]glycine, into ALA. Glutamate incorporation was abolished by preincubation of the extract with RNase. Sulfolobus extracts contained glutamate-1-semialdehyde aminotransferase activity, which is indicative of the five-carbon pathway. Growth of Sulfolobus was inhibited by gabaculine, a mechanism-based inhibitor of glutamate-1-semialdehyde aminotransferase, an enzyme of the five-carbon ALA biosynthetic pathway. These results indicate that Sulfolobus uses the five-carbon pathway for ALA formation.Abbreviations AHA 4-amino-5-hexynoic acid - ALA -aminolevulinic acid, Gabaculine, 3-amino-2,3-dihydrobenzoic acid - GSA glutamate 1-semialdehyde     

Compound K Production from Red Ginseng Extract by β-Glycosidase from Sulfolobus solfataricus Supplemented with α-L-Arabinofuranosidase from Caldicellulosiruptor saccharolyticus

Ginsenoside compound K (C-K) is attracting a lot of interest because of its biological and pharmaceutical activities, including hepatoprotective, antitumor, anti-wrinkling, and anti-skin aging activities. C-K has been used as the principal ingredient in skin care products. For the effective application of ginseng extracts to the manufacture of cosmetics, the PPD-type ginsenosides in ginseng extracts should be converted to C-K by enzymatic conversion. For increased yield of C-K from the protopanaxadiol (PPD)-type ginsenosides in red-ginseng extract (RGE), the α-l-arabinofuranoside-hydrolyzing α-l-arabinofuranosidase from Caldicellulosiruptor saccharolyticus (CS-abf) was used along with the β-d-glucopyranoside/α-l-arabinopyranoside-hydrolyzing β-glycosidase from Sulfolobus solfataricus (SS-bgly) because SS-bgly showed very low hydrolytic activity on the α-l-arabinofuranoside linkage in ginsenosides. The optimal reaction conditions for C-K production were as follows: pH 6.0, 80°C, 2 U/mL SS-bgly, 3 U/mL CS-abf, and 7.5 g/L PPD-type ginsenosides in RGE. Under these optimized conditions, SS-bgly supplemented with CS-abf produced 4.2 g/L C-K from 7.5 g/L PPD-type ginsenosides in 12 h without other ginsenosides, with a molar yield of 100% and a productivity of 348 mg/L/h. To the best of our knowledge, this is the highest concentration and productivity of C-K from ginseng extract ever published in literature.     

An additional glucose dehydrogenase from Sulfolobus solfataricus: fine-tuning of sugar degradation?

Within the SulfoSYS (Sulfolobus Systems Biology) project, the effect of temperature on a metabolic network is investigated at the systems level. Sulfolobus solfataricus utilizes an unusual branched ED (Entner-Doudoroff) pathway for sugar degradation that is promiscuous for glucose and galactose. In the course of metabolic pathway reconstruction, a glucose dehydrogenase isoenzyme (GDH-2, SSO3204) was identified. GDH-2 exhibits high similarity to the previously characterized GDH-1 (SSO3003, 61% amino acid identity), but possesses different enzymatic properties, particularly regarding substrate specificity and catalytic efficiency. In contrast with GDH-1, which exhibits broad substrate specificity for C5 and C6 sugars, GDH-2 is absolutely specific for glucose. The comparison of kinetic parameters suggests that GDH-2 might represent the major player in glucose catabolism via the branched ED pathway, whereas GDH-1 might have a dominant role in galactose degradation via the same pathway as well as in different sugar-degradation pathways.     

Highly regioselective glucosylation of 2'-deoxynucleosides by using the crude β-glycosidase from bovine liver

An enzymatic regioselective approach for the glucosylation of a series of 2′-deoxynucleosides was described by using the crude β-glycosidase from bovine liver that is less expensive and can be simply prepared in a standard organic laboratory. With the glucosylation of 2′-deoxyuridine as a model reaction, the effects of several key factors on the enzymatic reaction were examined. The optimum enzyme dosage, buffer pH and temperature were 0.05 U/ml, 9.5 and 42 °C, respectively. The presence of alkali β-glycosidase as the main active component in the crude enzyme extract might account for the high glucosylation activity at pH 9.5. In addition, the desired 5′-O-glucosylated derivatives of 2′-deoxynucleosides were synthesized with the yields of 22-72% and exclusive 5′-regioselectivities (>99%).     

Production of trisaccharide from lactose using β-galactosidase from Bacillus circulans modified by glutaraldehyde

Summary Free amino groups of -galactosidase-1-from Bacillus circulans were partially modified using different glutaraldehyde concentrations to increase trisaccharide production from lactose. Glutaraldehyde of 0.01%–0.03% modified 15%–40% of the free amino groups of the enzyme. The maximum yield of trisaccharide increased from 6% to 12% depending upon the degree of modification with 25% conversion of 127 mM lactose. Modification of 50% of the free amino groups of the enzyme using 0.05% glutaraldehyde produced a considerable amount of tetrasaccharide along with trisaccharide even at the initial stage of the reaction.     

Characterization of a β-glycosidase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius

In cell free extracts of the thermoacidophilic gram-positive bacterium Alicyclobacillus acidocaldarius ATCC27009, we have identified β-gluco- and galactosidase activities showing a specific activity of 0.1 and 12 U/mg, respectively. The two enzymatic activities are associated with different polypeptides and we show here the functional cloning, the expression in Escherichia coli and the characterisation of the β-glucosidase (Aaβ-gly). The enzyme, which is optimally active and stable at temperatures above 65°C, belongs to glycoside hydrolase family 1 (GH1) and shows wide substrate specificity on different aryl-glycosides and cello-oligosaccharides with k _cat/K _M for 4-nitrophenyl-β-D-glucoside and cellobiose of 2,976 and 185 s⁻¹mM⁻¹, respectively. Interestingly, upstream to the β-glycosidase gene, we identified a second ORF homologous to the ATPase subunit of the bacterial ABC transporters (abc1) that is co-transcribed with the β-glycosidase gene glyB and that could be involved in the carbohydrate import. The activity of the enzyme on cello-oligosaccharides of up to five glucose units strongly indicates that the enzyme could be involved in vivo in the degradation of glucans together with endoglucanase enzymes previously described. This, together with the co-expression of the two genes, suggests a role for the glyB-abc1 cluster in A. acidocaldarius in the degradation of cellulose and hemicelluloses. Enzymes: EC 3.2.1.21; EC 3.2.1.23; EC 3.2.1.25; EC 3.2.1.38; EC 3.2.1.37 An erratum to this article can be found at     

Production of xylanase and β-xylosidase from autohydrolysis liquor of corncob using two fungal strains

Agroindustrial residues are materials often rich in cellulose and hemicellulose. The use of these substrates for the microbial production of enzymes of industrial interest is mainly due to their high availability associated with their low cost. In this work, corncob (CCs) particles decomposed to soluble compounds (liquor) were incorporated in the microbial growth medium through autohydrolysis, as a strategy to increase and undervalue xylanase and β-xylosidase production by Aspergillus terricola and Aspergillus ochraceus. The CCs autohydrolysis liquor produced at 200 °C for 5, 15, 30 or 50 min was used as the sole carbon source or associated with untreated CC. The best condition for enzyme synthesis was observed with CCs submitted to 30 min of autohydrolysis. The enzymatic production with untreated CCs plus CC liquor was higher than with birchwood xylan for both microorganisms. A. terricola produced 750 total U of xylanase (144 h cultivation) and 30 total U of β-xylosidase (96-168 h) with 0.75% untreated CCs and 6% CCs liquor, against 650 total U of xylanase and 2 total U of β-xylosidase in xylan; A. ochraceus produced 605 total U of xylanase and 56 total U of β-xylosidase (168 h cultivation) with 1% untreated CCs and 10% CCs liquor against 400 total U of xylanase and 38 total U of β-xylosidase in xylan. These results indicate that the treatment of agroindustrial wastes through autohydrolysis can be a viable strategy in the production of high levels of xylanolytic enzymes.     

An insertion element of the extremely thermophilic archaeon Sulfolobus solfataricus transposes into the endogenous β-galactosidase gene

Three phenotypically stable mutants of the extremely thermophilic archaeon Sulfolobus solfataricus have been isolated by screening for β-galactosidase negative colonies on plates with X-Gal (5-bromo-4-chloro-3-indolyl-(3-d-galactopyranoside). From one of these mutants an insertion element, designated ISC1217, was isolated and characterized. Sequence analysis of ISC1217 and of the regions adjacent to the insertion site in the β-galactosidase gene revealed features typical of a transposable element: ISC1217 contained terminal inverted repeats and was flanked by a direct repeat of 6 bp. The 1147 by sequence contained an open reading frame encoding a putative protein of 354 amino acid residues and, overlapping this, two smaller open reading frames on the opposite strand. There were approximately 8 copies of the insertion element in the S. solfataricus genome. ISC1217 did not cross-hybridize with DNA of other Sulfolobus species. All three independently isolated β-galactosidase mutants of S. solfataricus arose by transposition of ISC1217 or a related element.     

Preparation of a glycosynthase from the β-glycosidase of the Archaeon Pyrococcus horikoshii

The β-glycosidase from the hyperthermophilic Archaeon Pyrococcus horikoshii (Phoβ-gly) is a monomeric enzyme with wide substrate specificity belonging to family 1 of glycoside hydrolases classification. Inspection of the three-dimensional structure of the enzyme, recently resolved, showed that Phoβ-gly is membrane bound and that the residues putatively involved in the catalytic activity are Glu155 and Glu324 working as the general acid/base and the nucleophile of the reaction, respectively. We show here that mutation of the latter completely eliminated the activity of the enzyme and that it could be reactivated in the presence of sodium formate. Analysis of the products obtained in the presence of sodium formate buffer pH 4.0 at 75°C showed that the Glu324Gly mutant acts as a hyperthermophilic glycosynthase.     

Production of β-galactosidase from thermophilic fungus Rhizomucor sp

A thermostable β-galactosidase was produced extracellularly by a thermophilic Rhizomucor sp, with maximum enzyme activity (0.21 U mg⁻¹) after 4 days under submerged fermentation condition (SmF). Solid state fermentation (SSF) resulted in a nine-fold increase in enzyme activity (2.04 U mg⁻¹). The temperature range for production of the enzyme was 38–55°C with maximum activity at 45°C. The optimum pH and temperature for the partially purified enzyme was 4.5 and 60°C, respectively. The enzyme retained its original activity on incubation at 60°C up to 1 h. Divalent cations like Co²⁺, Mn²⁺, Fe²⁺ and Zn²⁺ had strong inhibitory effects on the enzyme activity. The K _m and V _max for p-nitrophenyl-β- _D-galactopyranoside and o-nitrophenyl-β - _D-galactopyranoside were 0.39 mM, 0.785 mM and 232.1 mmol min⁻¹ mg⁻¹ respectively. The K _m and V _max for the natural substrate lactose were 66.66 μM and 0.20 μ mol min⁻¹ mg⁻¹. Received 10 March 1997/ Accepted in revised form 17 July 1997     

The Production of β-Fructofuranosidase from Bifidobacterium spp.

The productions of β-fructofuranosidase from Bifidohacterium longum A1, B. adolescentis G1, and four other strains of Bifidobacteria were investigated. All strains used in this study were grown in modified BL broth containing a mixture of fructooligosaccharides [1^F (1-β-D-fructofuranosyl)_n-1sucrose, GF_n (n = 2 – 5)] as the only carbon source. Hydrolyses of 1-kestose, sucrose, and inulin were detected in the extract of the cell. The highest activity on 1-kestose was detected in the extract of B. longum A1 followed by B. adolescentis G1. The other extracts weakly attacked 1-kestose. The relative activities of the extract of B. adolescentis G1 for 1-kestose, nystose, 1^F-fructosylnystose, sucrose, and inulin were 100, 82.5, 50.8, 28.3, and 15.0, respectively. The relative activities for various substrates differed from invertases (yeast β-fructofuranosidases) and exo-inulinase from Penicillium trzehinskii.     

TreX from Sulfolobus solfataricus ATCC 35092 Displays Isoamylase and 4-α-Glucanotransferase Activities

A treX in the trehalose biosynthesis gene cluster of Sulfolobus solfataricus ATCC 35092 has been reported to produce TreX, which hydrolyzes the α-1,6-branch portion of amylopectin and glycogen. TreX exhibited 4-α-D-glucan transferase activity, catalyzing the transfer of α-1,4-glucan oligosaccharides from one molecule to another in the case of linear maltooligosaccharides (G3–G7), and it produced cyclic glucans from amylopectin and amylose like 4-α-glucanotransferase. These results suggest that TreX is a novel isoamylase possessing the properties of 4-α-glucanotransferase.     

Characterization of a novel steviol-producing β-glucosidase from Penicillium decumbens and optimal production of the steviol

This study aimed to develop an economically viable enzyme for the optimal production of steviol (S) from stevioside (ST). Of 9 commercially available glycosidases tested, S-producing β-glucosidase (SPGase) was selected and purified 74-fold from Penicillium decumbens naringinase by a three-step column chromatography procedure. The 121-kDa protein was stable at pH 2.3–6.0 and at 40–60 °C. Hydrolysis of ST by SPGase produced rubusoside (R), steviolbioside (SteB), steviol mono-glucoside (SMG), and S, as determined by HPLC, HPLC-MS, and ¹H- and ¹³C-nuclear magnetic resonance. SPGase showed higher activity toward steviol mono-glucosyl ester, ST, R, and SMG than other β-linked glucobioses. The optimal conditions for S production (30 mM, 64 % yield) were 47 mM ST and 43 μl of SPGase at pH 4.0 and 55 °C. This is the first report detailing the production of S from ST hydrolysis by a novel β-glucosidase, which may be useful for the pharmaceutical and agricultural areas.     

Enzymatic Synthesis of 2-Deoxyglycosides Using the ß-Glycosidase of the Archaeon Sulfolobus solfataricus

The synthesis of 2-deoxyglycosides and, for the first time, of 2-deoxygalactosides is reported using a thermophilic and thermostable β-glycosyl hydrolase from the archeon Sulfolobus solfataricus and glucal or galactal as donors. The yields observed with alkyl acceptors confirmed that the robustness of the biocatalyst is of great help in designing practical syntheses of pure β-anomers of 2-deoxy derivatives of 4-penten-1-ol (obtained in 80% yield at 20 fold molar excess) and 3,4-dimethoxybenzyl alcohol (obtained in 19% yield at 3.3 fold molar excess). The attachment of 2-deoxyglyco units was performed on various pyranosidic acceptors (p-nitrophenyl α-d-glucopyranoside, o-nitrophenyl 2-deoxy-N-acetyl-α-d-glucosamine and p-nitrophenyl 2-deoxy-N-acetyl-β-d-glucosamine). At low molecular excesses of the acceptors, satisfactory yields (20-40%) of chromophoric 2-deoxy di- and trisaccharides were obtained. The different regioselectivity of our enzyme with respect to mesophilic counterparts reflects the importance of biodiversity in this field for the construction of a library of different glycosidases with different specificity.     

Production of 3,6-anhydro-D-galactose from κ-carrageenan using acid catalysts

This study describes acid-catalyzed production of 3,6-anhydro-D-galactose (D-AnG) from κ-carrageenan, a sulfated polysaccharide with an alternating backbone consisting of D-AnG and D-galactose (D-Gal). We analyzed four hydrolysis products (D-AnG, 5-hydroxymethylfurfural (HMF), levulinic acid (LA), and D-Gal) and reducing sugar contents during acid hydrolysis. Acid screening was carried out using seven acid catalysts which have different acidity. The catalysts showing high D-AnG production and high selectivity were chosen for subsequent experiments. We selected four acid catalysts (HCOOH, CH₃COOH, HNO₃, and HCl), and studied the effects of catalyst acidity, hydrolysis temperature T, and reaction time t on the production of D-AnG and other hydrolysis products. The optimal condition for maximum production of D-AnG by κ-carrageenan hydrolysis was T = 100°C and t = 30 min using 0.2 M HCl. Under this condition, 2.81 g/L D-AnG (33.5% of theoretical maximum) could be obtained from 2% (w/v) κ-carrageenan. In general, the maximum values of D-AnG, D-Gal, and the sum of two by-products (HMF and LA) increased with the acidity of catalysts. However, HNO3 was an exception in that the maximum production levels of HMF and LA were unusually low compared with other acid catalysts. D-AnG was successfully purified from acid hydrolysates using silica gel chromatography and the product was nearly 100% pure. This effective D-AnG production could facilitate future studies on the conversion of D-AnG to biofuels and biochemicals.     

Production and characterization of β-glucosidases from different Aspergillus strains

-D-Glucosidase enzymes (-D-glucoside glucohydrolase, EC 3.2.1.21) from different Aspergillus strains (Aspergillus phoenicis, A. niger and A. carbonarius) were examined with respect to the enzyme production of the different strains using different carbon sources and to the effect of the pH and temperature on the enzyme activity and stability. An efficient and rapid purification procedure was used for purifying the enzymes. Kinetic experiments were carried out using p-nitrophenyl -D-glucopyranoside (pNPG) and cellobiose as substrates. Two different fermentation methods were employed in which the carbon source was glucose or wheat bran. Aspergillus carbonarius proved to be the less effective strain in -glucosidase production. Aspergillus phoenicis produced the highest amount of -glucosidase on glucose as carbon source however on wheat bran A. niger was the best enzyme producer. Each Aspergillus strain produced one single acidic -glucosidase with pI values in the range of pH 3.52–4.2. There was no significant difference considering the effect of the pH and temperature on the activity and stability among the enzymes from different origins. The enzymes examined have only -glucosidase activity. The kinetic parameters showed that all enzymes hydrolysed pNPG with higher efficiency than cellobiose. This shows that hydrophobic interaction plays an important role in substrate binding. The kinetic parameters demonstrated that there was no significant difference among the enzymes from different origins in hydrolysing pNPG and cellobiose as the substrates.