Occurrence of Capnophilic Lactic Fermentation in the Hyperthermophilic Anaerobic Bacterium Thermotoga sp. Strain RQ7.

Capnophilic lactic fermentation (CLF) is an anaplerotic pathway exclusively identified in the anaerobic hyperthermophilic bacterium Thermotoga neapolitana, a member of the order Thermotogales. The CO2-activated pathway enables non-competitive synthesis of hydrogen and L-lactic acid at high yields, making it an economically attractive process for bioenergy production. In this work, we discovered and characterized CLF in Thermotoga sp. strain RQ7, a naturally competent strain, opening a new avenue for molecular investigation of the pathway. Evaluation of the fermentation products and expression analyses of key CLF-genes by RT-PCR revealed similar CLF-phenotypes between T. neapolitana and T. sp. strain RQ7, which were absent in the non-CLF-performing strain T. maritima. Key CLF enzymes, such as PFOR, HYD, LDH, RNF, and NFN, are up-regulated in the two CLF strains. Another important finding is the up-regulation of V-ATPase, which couples ATP hydrolysis to proton transport across the membranes, in the two CLF-performing strains. The fact that V-ATPase is absent in T. maritima suggested that this enzyme plays a key role in maintaining the necessary proton gradient to support high demand of reducing equivalents for simultaneous hydrogen and lactic acid synthesis in CLF.

Genus Thermotoga: A valuable home of multifunctional glycoside hydrolases (GHs) for industrial sustainability.

Nature is a dexterous and prolific chemist for cataloging a number of hostile niches that are the ideal residence of various thermophiles. Apart from having other species, these subsurface environments are considered a throne of bacterial genus Thermotoga. The genome sequence of Thermotogales encodes complex and incongruent clusters of glycoside hydrolases (GHs), which are superior to their mesophilic counterparts and play a prominent role in various applications due to their extreme intrinsic stability. They have a tremendous capacity to use a wide variety of simple and multifaceted carbohydrates through GHs, formulate fermentative hydrogen and bioethanol at extraordinary yield, and catalyze high-temperature reactions for various biotechnological applications. Nevertheless, no stringent rules exist for the thermo-stabilization of biocatalysts present in the genus Thermotoga. These enzymes endure immense attraction in fundamental aspects of how these polypeptides attain and stabilize their distinctive three-dimensional (3D) structures to accomplish their physiological roles. Moreover, numerous genome sequences from Thermotoga species have revealed a significant fraction of genes most closely related to those of archaeal species, thus firming a staunch belief of lateral gene transfer mechanism. However, the question of its magnitude is still in its infancy. In addition to GHs, this genus is a paragon of encapsulins which carry pharmacological and industrial significance in the field of life sciences. This review highlights an intricate balance between the genomic organizations, factors inducing the thermostability, and pharmacological and industrial applications of GHs isolated from genus Thermotoga.

Molecular dynamics simulation guided distal mutation of Thermotoga naphthophila ß-glucosidase for significantly enhanced synthesis of galactooligosaccharides and expanded product scope.

For efficient enzymatic production of health-beneficial galactooligosaccharides (GOSs), a glycone (-1)/aglycone (+2) subsite mutation strategy to engineer a thermophilic GH1 ß-glucosidase (Tn0602) from Thermotoga naphthophila RKU-10 was introduced. Six single mutation variants (F226G, N246G, N246E, N222F, N222Y, G224T) and two double mutants (F226GF414S, F226GF414Y) were designed. The +2-subsite variant F226G produced 136 mM galactooligosaccharide 1.2-fold more than the wild type (115 mM). More significantly, a superimposed mutation of the -1/+2 subsites F226G/F414S gave a total GOS production of 314 mM (82.16% lactose conversion), 2.7-fold higher than the total GOS production of the wild type. Furthermore, the variant F226GF414S was profiled 241 mM of trisaccharide (galß (1 â†’ 3)/(1 â†’ 4) lactose) and 73 mM tetrasaccharide (galß (1 â†’ 3)/(1 â†’ 4) galß (1 â†’ 3)/(1 â†’ 4) lactose). According to a 300-ns molecular dynamic simulation, the superimposed mutation increased GOS productivity and expanded the scope of products by changing the structural flexibility and reducing the steric hindrance of the substrate tunnel. Overall, our study successfully demonstrated that a - 1/+2 subsite mutagenesis method could be used in ß-glucosidases Tn0602 to improve enzyme productivity and expand product scope, which could be a potential route to evolve retaining glycosidases towards the desired direction.

Adapted laboratory evolution of Thermotoga sp. strain RQ7 under carbon starvation.

OBJECTIVE: Adaptive laboratory evolution (ALE) is an effective approach to study the evolution behavior of bacterial cultures and to select for strains with desired metabolic features. In this study, we explored the possibility of evolving Thermotoga sp. strain RQ7 for cellulose-degrading abilities. RESULTS: Wild type RQ7 strain was subject to a series of transfers over six and half years with cellulose filter paper as the main and eventually the sole carbon source. Each transfer was accompanied with the addition of 50 µg of Caldicellulosiruptor saccharolyticus DSM 8903 genomic DNA. A total of 331 transfers were completed. No cellulose degradation was observed with the RQ7 cultures. Thirty three (33) isolates from six time points were sampled and sequenced. Nineteen (19) of the 33 isolates were unique, and the rest were duplicated clones. None of the isolates acquired C. saccharolyticus DNA, but all accumulated small-scale mutations throughout their genomes. Sequence analyses revealed 35 mutations that were preserved throughout the generations and another 15 mutations emerged near the end of the study. Many of the affected genes participate in phosphate metabolism, substrate transport, stress response, sensory transduction, and gene regulation.

Thermostable D-amino acid decarboxylases derived from Thermotoga maritima diaminopimelate decarboxylase.

Diaminopimelate decarboxylases (DAPDCs) are highly selective enzymes that catalyze the common final step in different lysine biosynthetic pathways, i.e. the conversion of meso-diaminopimelate (DAP) to L-lysine. We examined the modification of the substrate specificity of the thermostable decarboxylase from Thermotoga maritima with the aim to introduce activity with 2-aminopimelic acid (2-APA) since its decarboxylation leads to 6-aminocaproic acid (6-ACA), a building block for the synthesis of nylon-6. Structure-based mutagenesis of the distal carboxylate binding site resulted in a set of enzyme variants with new activities toward different D-amino acids. One of the mutants (E315T) had lost most of its activity toward DAP and primarily acted as a 2-APA decarboxylase. We next used computational modeling to explain the observed shift in catalytic activities of the mutants. The results suggest that predictive computational protocols can support the redesign of the catalytic properties of this class of decarboxylating PLP-dependent enzymes.

Synthesis of fucosylated oligosaccharides with α-L-fucosidase from Thermotoga maritima immobilized on Eupergit® CM.

Fucosylated oligosaccharides present in human milk perform various biological functions that benefit infants' health. These compounds can be also obtained by enzymatic synthesis. In this work, the effect of the immobilization of α-L-fucosidase from Thermotoga maritima on the synthesis of fucosylated oligosaccharides was studied, using lactose and 4-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as acceptor and donor substrates, respectively, and Eupergit® CM as an immobilization support. The enzyme was immobilized with 90% efficiency at pH 8 and ionic strength of 1.5 M. Immobilization decreased enzyme affinity for the donor substrate as shown by a 1.5-times higher KM value and a 22-times decrease of the kcat/KM ratio in comparison to the unbound enzyme. In contrast, no effect was observed on the synthesis/hydrolysis ratio (rs/rh) when α-L-fucosidase was immobilized. Also, the effect of initial concentration of substrates was studied. An increase of the acceptor concentration improved the yields of fucosylated oligosaccharides regardless enzyme immobilization. The synthesis yields of 38.9 and 40.6% were obtained using Eupergit® CM-bound or unbound enzyme, respectively, and 3.5 mM pNP-Fuc and 146 mM lactose. In conclusion, α-L-fucosidase from Thermotoga maritima was efficiently immobilized on Eupergit® CM support without affecting the synthesis of fucosylated oligosaccharides.

Kinetic modeling of hydrogen and L-lactic acid production by Thermotoga neapolitana via capnophilic lactic fermentation of starch.

This study investigated the feasibility of hydrogen (H2) and L-lactic acid production from starch under capnophilic lactic fermentation (CLF) conditions by using Thermotoga neapolitana. Batch experiments were performed in 120 mL serum bottles and a 3 L pH-controlled continuous stirred-tank reactors (CSTR) system with potato and wheat starch as the substrates. A H2 yield of 3.34 (±0.17) and 2.79 (±0.17) mol H2/mol of glucose eq. was achieved with, respectively, potato and wheat starch. In the presence of CO2, L-lactic acid production by the acetyl-CoA carboxylation was significantly higher for the potato starch (0.88 ± 0.39 mol lactic acid/mol glucose eq.) than wheat starch (0.33 ± 0.11 mol lactic acid/mol glucose eq.). A kinetic model was applied to simulate and predict the T. neapolitana metabolic profile and bioreactor performance under CLF conditions. The CLF-based starch fermentation suggests a new direction to biotransform agri-food waste into biofuels and valuable biochemicals.

A novel bifunctional aldehyde/alcohol dehydrogenase catalyzing reduction of acetyl-CoA to ethanol at temperatures up to 95 °C.

Hyperthermophilic Thermotoga spp. are excellent candidates for the biosynthesis of cellulosic ethanol producing strains because they can grow optimally at 80 °C with ability to degrade and utilize cellulosic biomass. In T. neapolitana (Tne), a putative iron-containing alcohol dehydrogenase was, for the first time, revealed to be a bifunctional aldehyde/alcohol dehydrogenase (Fe-AAdh) that catalyzed both reactions from acetyl-coenzyme A (ac-CoA) to acetaldehyde (ac-ald), and from ac-ald to ethanol, while the putative aldehyde dehydrogenase (Aldh) exhibited only CoA-independent activity that oxidizes ac-ald to acetic acid. The biochemical properties of Fe-AAdh were characterized, and bioinformatics were analyzed. Fe-AAdh exhibited the highest activities for the reductions of ac-CoA and acetaldehyde at 80-85 °C, pH 7.54, and had a 1-h half-life at about 92 °C. The Fe-AAdh gene is highly conserved in Thermotoga spp., Pyrococcus furiosus and Thermococcus kodakarensis, indicating the existence of a fermentation pathway from ac-CoA to ethanol via acetaldehyde as the intermediate in hyperthermophiles.

Immobilization of GH78 α-L-Rhamnosidase from Thermotoga petrophilea with High-Temperature-Resistant Magnetic Particles Fe3O4-SiO2-NH2-Cellu-ZIF8 and Its Application in the Production of Prunin Form Naringin.

To efficiently recycle GH78 thermostable rhamnosidase (TpeRha) and easily separate it from the reaction mixture and furtherly improve the enzyme properties, the magnetic particle Fe3O4-SiO2-NH2-Cellu-ZIF8 (FSNcZ8) was prepared by modifying Fe3O4-NH2 with tetraethyl silicate (TEOS), microcrystalline cellulose and zinc nitrate hexahydrate. FSNcZ8 displayed better magnetic stability and higher-temperature stability than unmodified Fe3O4-NH2 (FN), and it was used to adsorb and immobilize TpeRha from Thermotoga petrophilea 13995. As for properties, FSNcZ8-TpeRha showed optimal reaction temperature and pH of 90°C and 5.0, while its highest activity approached 714 U/g. In addition, FSNcZ8-TpeRha had better higher-temperature stability than FN. After incubation at 80°C for 3 h, the residual enzyme activities of FSNcZ8-TpeRha, FN-TpeRha and free enzyme were 93.5%, 63.32%, and 62.77%, respectively. The organic solvent tolerance and the monosaccharides tolerance of FSNcZ8-TpeRha, compared with free TpeRha, were greatly improved. Using naringin (1 mmol/l) as the substrate, the optimal conversion conditions were as follows: FSNcZ8-TpeRha concentration was 6 U/ml; induction temperature was 80°C; the pH was 5.5; induction time was 30 min, and the yield of products was the same as free enzyme. After repeating the reaction 10 times, the conversion of naringin remained above 80%, showing great improvement of the catalytic efficiency and repeated utilization of the immobilized α-L-rhamnosidase.

Construction and Validation of a Genome-Scale Metabolic Network of Thermotoga sp. Strain RQ7.

Thermotoga are anaerobic hyperthermophiles that have a deep lineage to the last universal ancestor and produce biological hydrogen gas accompanying cell growth. In recent years, systems-level approaches have been used to elucidate their metabolic capacities, by integrating mathematical modeling and experimental results. To assist biochemical engineering studies of T. sp. strain RQ7, this work aims at building a metabolic model of the bacterium that quantitatively simulates its metabolism at the genome scale. The constructed model, RQ7_iJG408, consists of 408 genes, 692 reactions, and 538 metabolites. Constraint-based flux balance analyses were used to simulate cell growth in both the complex and defined media. Quantitative comparison of the predicted and measured growth rates resulted in good agreements. This model serves as a foundation for an integrated biochemical description of T. sp. strain RQ7. It is a useful tool in designing growth media, identifying metabolic engineering strategies, and exploiting the physiological potentials of this biotechnologically significant organism.

A novel mechanism of ß-glucosidase stimulation through a monosaccharide binding-induced conformational change.

It is urgent the transition from a fossil fuel-based economy to a sustainable bioeconomy based on bioconversion technologies using renewable plant biomass feedstocks to produce high chemicals, bioplastics, and biofuels. ß-Glucosidases are key enzymes responsible for degrading the plant cell wall polymers, as they cleave glucan-based oligo- and polysaccharides to generate glucose. Monosaccharide-tolerant or -stimulated ß-glucosidases have been reported in the past decade. Here, we describe a novel mechanism of ß-glucosidase stimulation by glucose and xylose. The glycoside hydrolase 1 family ß-glucosidase from Thermotoga petrophila (TpBgl1) displays a typical glucose stimulation mechanism based on an increased Vmax and decreased Km in response to glucose. Through molecular docking and dynamics analyses, we mapped putative monosaccharide binding regions (BRs) on the surface of TpBgl1. Our results indicate that after interaction with glucose or xylose at BR1 site, an adjacent loop region assumes an extended conformation, which increases the entrance to the TpBgl1 active site, improving product formation. Biochemical assays with TpBgl1 BR1 mutants, TpBgl1D49A/Y410A and TpBgl1D49K/Y410H, resulted in decreasing and abolishing monosaccharide stimulation, respectively. These mutations also impaired the BR1 looping extension responsible for monosaccharide stimulation. This study provides a molecular basis for the rational design of ß-glucosidases for biotechnological applications.

A novel Thermotoga strain TFO isolated from a Californian petroleum reservoir phylogenetically related to Thermotoga petrophila and T. naphthophila, two thermophilic anaerobic isolates from a Japanese reservoir: Taxonomic and genomic considerations.

Hot oil reservoirs harbor diverse microbial communities, with many of them inhabiting thermophilic or hyperthermophilic fermentative Thermotogae species. A new Thermotoga sp. strain TFO was isolated from an Californian offshore oil reservoir which is phylogenetically related to thermophilic species T. petrophila RKU-1T and T. naphthophila RKU-10T, isolated from the Kubiki oil reservoir in Japan. The average nucleotide identity and DNA-DNA hybridization measures provide evidence that the novel strain TFO is closely related to T. naphthophila RKU-10T, T. petrophila RKU-1T and can not be differentiated at the species level. In the light of these results, the reclassification of T. naphthophila RKU-10 and strain TFO as heterotypic synonyms of T. petrophila is proposed. A pangenomic survey of closely related species revealed 55 TFO strain-specific proteins, many of which being linked to glycosyltransferases and mobile genetic elements such as recombinases, transposases and prophage, which can contribute to genome evolution and plasticity, promoting bacterial diversification and adaptation to environmental changes. The discovery of a TFO-specific transport system dctPQM, encoding a tripartite ATP-independent periplasmic transporter (TRAP), has to be highlighted. The presence of this TRAP system assumes that it could assist in anaerobic n-alkane degradation by addition of fumarate dicarboxylic acid, suggesting a niche-specific gene pool which correlates with the oil reservoir that T. petrophila TFO inhabits. Finally, T. naphthophila RKU-10, T. petrophila RKU-1T, T. petrophila TFO form a distinct phylogenetic lineage with different geographic origins, share the same type of ecological niche including the burial history of fields. Theses findings might support the indigenous character of this species in oil reservoirs.

Reconstructing organisms in silico: genome-scale models and their emerging applications.

Escherichia coli is considered to be the best-known microorganism given the large number of published studies detailing its genes, its genome and the biochemical functions of its molecular components. This vast literature has been systematically assembled into a reconstruction of the biochemical reaction networks that underlie E. coli's functions, a process which is now being applied to an increasing number of microorganisms. Genome-scale reconstructed networks are organized and systematized knowledge bases that have multiple uses, including conversion into computational models that interpret and predict phenotypic states and the consequences of environmental and genetic perturbations. These genome-scale models (GEMs) now enable us to develop pan-genome analyses that provide mechanistic insights, detail the selection pressures on proteome allocation and address stress phenotypes. In this Review, we first discuss the overall development of GEMs and their applications. Next, we review the evolution of the most complete GEM that has been developed to date: the E. coli GEM. Finally, we explore three emerging areas in genome-scale modelling of microbial phenotypes: collections of strain-specific models, metabolic and macromolecular expression models, and simulation of stress responses.

High-resolution structure of a modular hyperthermostable endo-ß-1,4-mannanase from Thermotoga petrophila: The ancillary immunoglobulin-like module is a thermostabilizing domain.

The endo-ß-1,4-mannanase from the hyperthermostable bacterium Thermotoga petrophila (TpMan) is an enzyme that catalyzes the hydrolysis of mannan and heteromannan polysaccharides. Of the three domains that comprise TpMan, the N-terminal GH5 catalytic domain and the C-terminal carbohydrate-binding domain are connected through a central ancillary domain of unknown structure and function. In this study, we report the partial crystal structure of the TpMan at 1.45 Å resolution, so far, the first modular hyperthermostable endo-ß-1,4-mannanase structure determined. The structure exhibits two domains, a (ß/α)8-barrel GH5 catalytic domain connected via a linker to the central domain with an immunoglobulin-like ß-sandwich fold formed of seven ß-strands. Functional analysis showed that whereas the immunoglobulin-like domain does not have the carbohydrate-binding function, it stacks on the GH5 catalytic domain acting as a thermostabilizing domain and allowing operation at hyperthermophilic conditions. The carbohydrate-binding domain is absent in the crystal structure most likely due to its high flexibility around the immunoglobulin-like domain which may act also as a pivot. These results represent new structural and functional information useful on biotechnological applications for biofuel and food industries.

Overexpression and characterization of TnCel12B, a hyperthermophilic GH12 endo-1,4-ß-glucanase cloned from Thermotoga naphthophila RKU-10T.

A putative cellulolytic gene (825 bp) from Thermotoga naphthophila RKU-10T was overexpressed as an active soluble endo-1,4-ß-glucanase (TnCel12B), belongs to glycoside hydrolase family 12 (GH12), in a mesophilic expression host. Heterologous expression and engineered bacterial cell mass was improved through specific strategies (induction and cultivation). Hence, intracellular activity of TnCel12B was enhanced in ZYBM9 modified medium (pH 7.0) by 8.38 and 6.25 fold with lactose (200 mM) and IPTG (0.5 mM) induction, respectively; and 6.95 fold was increased in ZYP-5052 auto-inducing medium after 8 h incubation at 26 °C (200 rev min-1). Purified TnCel12B with a molecular weight of ~32 kDa, was optimally active at 90 °C and pH 6.0; and exhibited prodigious stability over a wide range of temperature (50-85 °C) and pH (5.0-9.0) for 8 h TnCel12B displayed great resistance towards different chemical modulators, though activity was improved by Mg2+, Zn2+, Pb2+ and Ca2+. Purified TnCel12B had affinity with various substrates but peak activity was observed toward barley ß-glucan (1664 U mg-1) and carboxymethyl cellulose (736 U mg-1). The values of Km, Vmax, kcat, and kcatKm-1 were found to be 4.63 mg mL-1, 916 µmol mg-1min-1, 1326.7 s-1 and 286.54 mL mg-1 s-1, respectively using CMC substrate. All noteworthy features of TnCel12B make it an appropriate industrial candidate for bioethanol production and various other potential applications.

Effect of Cultivation Parameters on Fermentation and Hydrogen Production in the Phylum Thermotogae.

The phylum Thermotogae is composed of a single class (Thermotogae), 4 orders (Thermotogales, Kosmotogales, Petrotogales, Mesoaciditogales), 5 families (Thermatogaceae, Fervidobacteriaceae, Kosmotogaceae, Petrotogaceae, Mesoaciditogaceae), and 13 genera. They have been isolated from extremely hot environments whose characteristics are reflected in the metabolic and phenotypic properties of the Thermotogae species. The metabolic versatility of Thermotogae members leads to a pool of high value-added products with application potentials in many industry fields. The low risk of contamination associated with their extreme culture conditions has made most species of the phylum attractive candidates in biotechnological processes. Almost all members of the phylum, especially those in the order Thermotogales, can produce bio-hydrogen from a variety of simple and complex sugars with yields close to the theoretical Thauer limit of 4 mol H2/mol consumed glucose. Acetate, lactate, and L-alanine are the major organic end products. Thermotagae fermentation processes are influenced by various factors, such as hydrogen partial pressure, agitation, gas sparging, culture/headspace ratio, inoculum, pH, temperature, nitrogen sources, sulfur sources, inorganic compounds, metal ions, etc. Optimization of these parameters will help to fully unleash the biotechnological potentials of Thermotogae and promote their applications in industry. This article gives an overview of how these operational parameters could impact Thermotogae fermentation in terms of sugar consumption, hydrogen yields, and organic acids production.

Effect of Heterologous Expression of Chemotaxis Proteins from Genus Thermotoga on the Growth Kinetics of Escherichia coli Cells.

In the process of optimization of heterologous expression of thermostable chemotaxis proteins CheW and CheY as industrially useful polypeptides, their direct influence on the cell growth kinetics and morphology of Escherichia coli was observed. CheW and CheY of bacteria of the genus Thermotoga, being expressed in recombinant form in E. coli cells, are involved in the corresponding signal pathways of the mesophilic microorganisms. The effects of such involvement in the metabolism of "host" cells are extremely diverse: from rapid aging of the culture to elongation of the stationary growth phase. We also discuss the mechanisms of the influence of the heterologous chemotaxis proteins on cells, their positive and negative effects, as well as potential applications in industry and biomedicine.

Cloning, Purification, and Characterization of Recombinant Thermostable ß-Xylanase Tnap_0700 from Thermotoga naphthophila.

The gene of a ß-xylanase (Tnap_0700) was cloned from a hyperthermophilic Thermotoga naphthophila strain ATCC BAA-489 and expressed in Escherichia coli BL21 (DE3) via pET-21a (+) as an expression vector. The growth steps were upgraded for highest ß-xylanase expression via several factors, for example, IPTG concentration, time of induction, pH, and temperature. The pH and temperature optima for the extreme expression of ß-xylanase were 7.0 pH and 37 °C, correspondingly. Recombinant enzyme purified by heat treatment process, then later by immobilized metal ion affinity chromatography. Molecular mass of the purified ß-xylanase was 38 kDa observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at room temperature for 30 days. It exhibited high stability over wide series of temperature 50-90 °C and pH 4.0-9.0 upon the addition of 1 mM Ca+2 and reduced in the existence of Cu+2 and EDTA. The addition of about 10-30% different organic solvents have no considerable effect on enzyme. However, SDSF and urea acting as an inhibitor leads to decrease in the enzyme activity. The ß-xylanase enzyme was active to hydrolyze xylan from beechwood forming xylose. Thermostable ß-xylanase causes the breakdown of complex carbohydrates into monosaccharide components. This thermostable ß-xylanase revealed remarkable properties, which make it an encouraging candidate for various industrial applications especially in the alteration of renewable biomaterials into ethanol production, and biofuels from lignocellulosics has acknowledged much devotion subsequently in the last decade.

Cloning, overexpression and characterization of a thermostable ß-xylosidase from Thermotoga petrophila and cooperated transformation of ginsenoside extract to ginsenoside 20(S)-Rg3 with a ß-glucosidase.

A thermostable ß-xylosidase gene Tpexyl3 from Thermotoga petrophila DSM 13,995 was cloned and overexpressed by Escherichia coli. Recombinant Tpexyl3 was purified, and its molecular weight was approximately 86.7 kDa. Its optimal activity was exhibited at pH 6.0 and 90 °C. It had broad specificity to xylopyranosyl, arabinopyranosyl, arabinofuranosyl and glucopyranosyl moieties. The ß-xylosidase activity of the recombinant Tpexyl3 was 6.81 U/mL in the LB medium and 151.4 U/mL in a 7.5 L bio-reactor. It was applied to transform ginsenoside extract into the pharmacologically active minor ginsenoside 20(S)-Rg3, which was combined with thermostable ß-glucosidase Tpebgl3. After transforming under optimal condition, the 20 g/L of ginsenoside extract was transformed into 6.28 g/L of Rg3 within 90 min, with a corresponding molar conversion of 95.0% and Rg3 productivity of 1793.49 mg/L/h, respectively. This study is the highest report of a GH3 family glycosidase with arabinopyranosidase activity and also the first report on the high substrate concentration bioconversion of ginsenoside extract to ginsenoside 20(S)-Rg3 by using two thermostable glycosidases.