Characterization of L-arabinose/D-galactose 1-dehydrogenase from Thermotoga maritima and its application in galactonate production.

The first hyperthermophilic L-arabinose/D-galactose 1-dehydrogenase (TmAraDH) from Thermotoga maritima was heterologously purified from Escherichia coli. It belongs to the Gfo/Idh/MocA protein family, prefers NAD+/NADP+ as a cofactor. The purified TmAraDH exhibited maximum activity toward L-arabinose at 75 °C and pH 8.0, and retained 63.7% of its activity after 24 h at 60 °C, and over 60% of its activity after holding a pH ranging from 7.0 to 9.0 for 1 h. Among all tested substrates, TmAraDH exclusively catalyzed the NAD(P)+-dependent oxidation of L-arabinose, D-galactose and D-fucose. The catalytic efficiency (kcat/Km) towards L-arabinose and D-galactose was 123.85, 179.26 min-1 mM-1 for NAD+, and 56.06, 18.19 min-1 mM-1 for NADP+, respectively. TmAraDH exhibited complete oxidative conversion in 12 h at 70 °C to D-galactonate with 5 mM D-galactose. Modelling provides structural insights into the cofactor and substrate recognition specificity. Our results suggest that TmAraDH have great potential for the conversion of L-arabinose and D-galactose to L-arabonate and D-galactonate.

Engineering Thermotoga maritima ß-glucosidase for improved alkyl glycosides synthesis by site-directed mutagenesis.

Alkyl glycosides are well-characterized nonionic surfactants, and can be prepared by transglycosylation reactions with retaining GH1 glycosidases being normally used for this purpose. The produced alkyl glycosides can also be hydrolyzed by the glycosidase, and hence, the yields of alkyl glycosides can be too low for industrial use. To improve the transglycosylation-to-hydrolysis ratio for a ß-glucosidase from Thermotoga maritima (TmBglA) for the synthesis of alkyl glycoside, six mutants (N222F, N223C, N223Q, G224A, Y295F, and F414S) were produced. N222F, N223C, N223Q, G224A improved catalytic activity, F295Y and F414S are hydrolytically crippled with p-nitrophenol-ß-d-glucopyranoside (pNPG) as substrate with an 85 and 70-fold decrease in apparent kcat, respectively; N222F shows the highest kcat/km value for pNPG. The substrate selectivity altered from pNPG to pNP-ß-d-fucoside for N222F, F295Y, and F414S and from cellubiose to gentiobiose for N222F and F414S. Using pNPG (34 mM) and hexanol 80% (vol/vol), N222F, Y295F, and F414S synthesized hexyl-ß-glycoside (HG) yields of 84.7%, 50.9%, and 54.1%, respectively, HG increased from 14.49 (TmBglA) to 22.8 mM (N222F) at 2 hr by 57.42%. However, this higher transglycosylation effect depended on that three mutants creates an environment more suited for hexanol in the active site pocket, and consequently suppressed its HG hydrolysis.

Characterization of a uronate dehydrogenase from Thermobispora bispora for production of glucaric acid from hemicellulose substrate.

A thermostable uronate dehydrogenase Tb-UDH from Thermobispora bispora was over-expressed in Escherichia coli using the T7 polymerase expression system. The Tb-UDH was purified by metal affinity chromatography, and gave a single band on SDS-PAGE. The maximum activity on glucuronic acid was found at 60 °C and pH 7.0. The purified enzyme retained over 58% of its activity after holding a pH ranging from 7.0 to 7.5 for 1 h at 60 °C. The Km and Vmax values of the purified Tb-UDH for Glucuronic acid (GluUA) were 0.165 mM and 117.7 U mg-1, respectively, those for galacturonic acid (GalUA) were 0.115 mM and 104.2 U mg-1, respectively, and those for NAD+ were 0.120 mM and 133.3 U mg-1, respectively; the turnover number (kcat) with GluUA as a substrate was higher than that with GalUA; however, the Michaelis constant (Km) for GalUA was lower than that for GluUA. After 60 min of incubation at 50 °C, Tb-UDH exhibited a conversion ratio for glucuronic acid to the glucaric acid of 84% on chemical reagent and 81.3% on hydrolysates from breech xylans formed by xylanase and α-glucuronidase. This work shows that biocatalytic routes have great potential for the conversion of hemicellulose substrate into value-added products derived from renewable biomass. TOC GRAPHIC: (A) The structure of the xylan is described and the site of action of the xylan degrading enzyme is indicated. (B) The effect of substrate concentration on recombinant Tb-UDH activity when galacturonic acid was used as substrate. (C) SDS-PAGE analysis of E. coli BL21 (DE3) harboring pET-20b(+) and pET-20b-Tb-UDH. (D) Oxidative conversion of glucuronic acid from a beechwood xylan to glucaric acid.

Comparison of 4-hydroxynonenal-induced p53-mediated apoptosis in prostate cancer cells LNCaP and DU145.

AIM OF THE STUDY: To explore the mechanism of oxidative stress in the development of prostate cancer, here we compared 4-hydroxynonenal (4-HNE)- treated LNCaP (hormone-sensitive) and DU145 (hormone insensitive) cells with significant differences in sensitivity to androgen. MATERIAL AND METHODS: The prostate cancer cell line LNCaP and late cell line DU145 were treated with different concentrations of 4-HNE. The cell proliferation, apoptosis and mitochondrial transmembrane potential were detected at different time points, and expression of related molecules in cell proliferation and apoptosis signal pathway was analyzed by Western blot, and the over-expression of glutathione S-transferase (GSTA-4) was used to validate the changes of the effects of 4-HNE on the two kinds of cells. RESULTS: LNCaP cells showed greater antiproliferative and proapoptotic activities of HNE in a time- and dose-dependent manner corresponding to the activation of p53-mediated intrinsic apoptotic signaling, but JNK activation was not observed. In contrast, HNE-treated DU145 cells showed less apoptosis and proliferation was not inhibited; instead there was sustained activation of JNK, but activation of p53, p-p53, p21, Bax and caspase-3 was not observed. In addition, their effect of induction of apoptosis can be inhibited by overexpression of GSTA-4. CONCLUSIONS: These studies suggest that 4-HNE promotes prostate cancer cell apoptosis through the p53 signaling pathway; the differences of sensitivity to 4-HNE in LNCaP and DU145 cells may be related to the androgen sensitivity of prostate cancer cells; and the 4-HNE-induced p53-mediated apoptosis signal is regulated by GSTA-4.

Characterization of Thermotoga maritima Esterase Capable of Hydrolyzing Bis(2-hydroxyethyl) Terephthalate.

The gene-encoding carboxylesterase (TM1022) from the hyperthermophilic bacterium Thermotoga maritima (T. maritima) was cloned and expressed in Escherichia coli Top10 and BL21 (DE3). Recombinant TM1022 showed the best activity at pH 8.0 and 85 °C and retained 57% activity after 8 h cultivation at 90 °C. TM1022 exhibited good stability at pH 6.0-9.0, maintaining 53% activity after incubation at pH 10.0 and 37 °C for 6 h. The esterase TM1022 exhibited the optimum thermo-alkali stability and kcat/Km (598.57 ± 19.97 s-1mM-1) for pN-C4. TM1022 hydrolyzed poly(ethylene terephthalate) (PET) degradation intermediates, such as bis(2-hydroxyethyl) terephthalate (BHET) and mono(2-hydroxyethyl) terephthalate (MHET). The Km, kcat, and kcat/Km values for BHET were 0.82 ± 0.01 mM, 2.20 ± 0.02 s-1, and 2.67 ± 0.02 mM-1 s-1, respectively; those for MHET were 2.43 ± 0.07 mM, 0.04 ± 0.001 s-1, and 0.02 ± 0.001 mM-1 s-1, respectively. When purified TM1022 was added to the cutinase BhrPETase, hydrolysis of PET from drinking water bottle tops produced pure terephthalic acids (TPA) with 166% higher yield than those obtained after 72 h of incubation with BhrPETase alone as control. The above findings demonstrate that the esterase TM1022 from T. maritima has substantial potential for depolymerizing PET into monomers for reuse.

On the change in bacterial growth and magnetosome formation for Magnetospirillum sp. strain AMB-1 under different concentrations of reducing agents.

Magnetosome produced by Magnetospirillum AMB-1 is highly crystalline, monodisperse, bioengineerable and a better nanomagnetic material candidate for a broad range of applications, ranging from medicine, biology and electronics to aerospace engineering. Many efforts have been made to get the optimal growth conditions and improve the accumulation of magnetosome. Studies have showed that oxygen concentration is a key factor to the formation of magnetosome. The effects of various of reducing agents in different concentrations on the growth of Magnetospirillum AMB-1 and the corresponding magnetosome formation have been examined in our study. The results show that the growth of the bacterial cells and the synthesis of magnetosome are both influenced in the presence of different concentrations of reducing agents.

Fusion of Oligopeptide to the C Terminus of α-Glucuronidase from Thermotoga maritima Improves the Catalytic Efficiency for Hemicellulose Biotransformation.

Fusion protein combined the oligopeptide (HQAFFHA) with the C terminus of α-glucuronidase from Thermotoga maritima was produced in E. coli and purified for characterization and applications of glucuronic and glucaric acid production. The fusion protein with oligopeptide exhibited a 2.97-fold higher specific activity than individual protein. Their catalytic efficiency kcat/Km and kcat increased from 469.3 ± 2.6 s-1 (g mL-1)-1 and 62.4 ± 0.9 s-1 to 2209.5 ± 26.3 s-1 (g mL-1)-1 and 293.9 ± 4.9 s-1, respectively. Fusion protein had similar temperature and pH profiles to those without oligopeptide, but the thermal stability decreases and the pH stability shifts to alkaline. Using beech xylan hydrolysate as a substrate, the glucuronic acid yield of fusion enzyme increased by 9.94% compared with its parent at 65 °C pH 8.5 for 10 h, and can hydrolyze corn cob xylan with xylanase to obtain glucuronic acid, and can be combined with uronate dehydrogenase to obtain high-added value glucaric acid. Homologous modeling analysis revealed the factors contributing to the high catalytic efficiency of fusion enzyme. These results show that the peptide fusion strategy described here may be useful for improving the catalytic efficiency and stability of other industrial enzymes, and has great potential for producing high value-added products from agricultural waste.

Characterization of a novel beta-xylosidase, XylC, from Thermoanaerobacterium saccharolyticum JW/SL-YS485.

The 1,914-bp open reading frame of xylC from Thermoanaerobacterium saccharolyticum JW/SL-YS485 encodes a calculated 73-kDa ß-xylosidase, XylC, different from any glycosyl hydrolase in the database and representing a novel glycohydrolase family. Hydrolysis occurred under retention of the anomeric configuration, and transglycosylation occurred in the presence of alcohols as acceptors. With the use of vector pHsh, expression of XylC, the third ß-xylosidase in this bacterium, increased approximately 4-fold when a loop within the translational initiation region in the mRNA was removed by site-directed mutagenesis. The increased expression of xylC(m) is due to removal of a stem-loop structure without a change of the amino acid sequence of the heterologously expressed enzyme (XylC(rec)). When gel filtration was applied, purified XylC had molecular masses of 210 kDa and 265 kDa using native gradient gel electrophoresis. The protein consisted of 78-kDa subunits based on SDS gel electrophoresis and contained 6% carbohydrates. XylC and XylC(rec) exhibited maximum activity at 65°C and pH(65°C) 6.0, a 1-h half-life at 67°C, a K(m) for p-nitrophenyl-ß-D-xyloside of 28 mM, and a V(max) of 276 U/mg and retained 70% activity in the presence of 200 mM xylose, suggesting potential for industrial applications.

Large batch production of Galactooligosaccharides using ß-glucosidase immobilized on chitosan-functionalized magnetic nanoparticle.

ß-glucosidase (BglA) immobilization from Thermotoga maritima on magnetic nanoparticles (MNPs) functionalized with chitosan (Cs) were efficiently investigated to improve lactose conversion and galactooligosaccharides (GOS) production. We used a batch method in order to improve the conversion of lactose to GOS. The efficiency and yield of immobilization were 79% and immobilized BglA was effectively recycled via a magnetic separation procedure through a batch-wise GOS with no activity lessening. Furthermore, analyses were done through screening kinetics of enzyme activity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Fourier transform infrared spectroscopy (FT-IR), and transmission electron microscopy (TEM). Proposed methodology of immobilization shows a potential application as it is stable which was proved through many methods including pH, temperature, heat treatment, storage, and kinetics of the enzyme. GOS and residual enzyme activity showed to be 28.76 and 40.44%, respectively. However, free enzyme synthesis of GOS yield was just 24% after 12 hr. This study proposed applying magnet in the immobilization process of BglA on Cs-MNPs to produce GOS as new method for immobilizing enzyme in a biostable and cost-efficient way. PRACTICAL APPLICATIONS: This paper focus on immobilization of BglA from T. maritima onto MNPs functionalized with CS to investigate their further possibility improving lactose conversion and GOS production. Interestingly, a successful immobilization of Tm-BglA on the substrates were achieved in Cs-MNPs. The obtained results from enzyme activity, SDS-PAGE, FT-IR, and TEM showed that the high binding capacity of BglA to Cs-MNPs was successfully obtained. Furthermore, the binding efficiency calculation indicated that the immobilized BglA-Cs-MNPs conserved 40.44% of its native activity at the end of its 6th repeated use. In addition, magnetic separation technique was successfully employed for reuse of the immobilized BglA for repetitive batch-wise GOS without significant loss of activity.

Immobilization of ß-Glucosidase from Thermatoga maritima on Chitin-functionalized Magnetic Nanoparticle via a Novel Thermostable Chitin-binding Domain.

Enzyme immobilization is a powerful tool not only as a protective agent against harsh reaction conditions but also for the enhancement of enzyme activity, stability, reusability, and for the improvement of enzyme properties as well. Herein, immobilization of ß-glucosidase from Thermotoga maritima (Tm-ß-Glu) on magnetic nanoparticles (MNPs) functionalized with chitin (Ch) was investigated. This technology showed a novel thermostable chitin-binding domain (Tt-ChBD), which is more desirable in a wide range of large-scale applications. This exclusive approach was fabricated to improve the Galacto-oligosaccharide (GOS) production from a cheap and abundant by-product such as lactose through a novel green synthesis route. Additionally, SDS-PAGE, enzyme activity kinetics, transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FT-IR) revealed that among the immobilization strategies for Thermotoga maritime-ß-Glucosidase thermostable chitin-binding domain (Tm-ß-Glu-Tt-ChBD) on the attractive substrate; Ch-MNPs had the highest enzyme binding capacity and GOS production ratio when compared to the native enzyme. More interestingly, a magnetic separation technique was successfully employed in recycling the immobilized Tm-ß-Glu for repetitive batch-wise GOS without significant loss or reduction of enzyme activity. This immobilization system displayed an operative stability status under various parameters, for instance, temperature, pH, thermal conditions, storage stabilities, and enzyme kinetics when compared with the native enzyme. Conclusively, the GOS yield and residual activity of the immobilized enzyme after the 10th cycles were 31.23% and 66%, respectively. Whereas the GOS yield from native enzyme synthesis was just 25% after 12 h in the first batch. This study recommends applying Tt-ChBD in the immobilization process of Tm-ß-Glu on Ch-MNPs to produce a low-cost GOS as a new eco-friendly process besides increasing the biostability and efficiency of the immobilized enzyme.

Effect of ß-endoxylanase and α-arabinofuranosidase enzymatic hydrolysis on nutritional and technological properties of wheat brans.

Wheat bran (WB) was treated using xylanase and arabinofuranosidase from Thermotoga maritima and added to steamed breads on 15% flour weight basis. The antioxidant capacity and water and oil retention capacity of brans were increased while their soluble xylooligosaccharides and phenolic acids content were increased. Two enzymes treatment was found to be more effective in decreasing the resistance to extension, softening degree, water absorption and development time, and in increasing the extensibility, stability time, porosity and sensorial characteristics of the steamed breads. Two enzymes treatment had significantly (P < 0.05) greater specific volume, springiness and cohesiveness and lower crumb firmness, gumminess, chewiness than single enzyme treatment. All results highlighted that combination of xylanase and arabinofuranosidase can improve the degrees hydrolysis of WB and its soluble AX xylooligosaccharides produced, having a synergetic effect on the dough rheology and nutritional and quality characteristics of steamed bread.

Hydrolysis of soy isoflavone glycosides by recombinant beta-glucosidase from hyperthermophile Thermotoga maritima.

A recombinant Thermotoga maritima beta-glucosidase A (BglA) was purified to homogeneity for performing enzymatic hydrolysis of isoflavone glycosides from soy flour. The kinetic properties K(m), k(cat), and k(cat)/K(m) of BglA towards isoflavone glycosides, determined using high-performance liquid chromatography, confirmed the higher efficiency of BglA in hydrolyzing malonylglycosides than non-conjugated glycosides (daidzin and genistin). During hydrolysis of soy flour by BglA at 80 degrees C, the isoflavone glycosides (soluble form) were extracted from soy flour (solid state) into the solution (liquid state) in thermal condition and converted to their aglycones (insoluble form), which mostly existed in the pellet to be separated from BglA in the reaction solution. The enzymatic hydrolysis in one-step and two-step approaches yielded 0.38 and 0.35 mg genistein and daidzein per gram of soy flour, respectively. The optimum conditions for conversion of isoflavone aglycones were 100 U per gram of soy flour, substrate concentration 25% (w/v), and incubation time 3 h for 80 degrees C.

Enhanced catalytic efficiency in quercetin-4'-glucoside hydrolysis of Thermotoga maritima ß-glucosidase A by site-directed mutagenesis.

Te-BglA and Tm-BglA are glycoside hydrolase family 1 ß-glucosidases from Thermoanaerobacter ethanolicus JW200 and Thermotoga maritima, respectively, with 53% sequence identity. However, Te-BglA could more effectively hydrolyze isoflavone glucosides to their aglycones than could Tm-BglA, possibly due to the difference in amino acid residues around their glycone binding pockets. Site-directed mutagenesis was used to replace the amino acid residues of Tm-BglA with the corresponding residues of Te-BglA, generating three single mutants (F221L, N223L, and G224T), as well as the corresponding three double mutants (F221L/N223L, F221L/G224T, and N223L/G224T) and one triple mutant (F221L/N223L/G224T). The seven mutants have been purified, characterized, and compared to the wild-type Tm-BglA. The effects of the mutations on kinetics, enzyme activity, and substrate specificity were determined. All mutants showed pH-activity curves narrower on the basic side and wider on the acid side and had similar optimal pH and stability at pH 6.5-8.3. They were more stable up to 85 °C, but G224T displayed higher optimal temperature than Tm-BglA. Seven mutants indicated an obvious increase in catalytic efficiency toward p-nitrophenyl ß-D-glucopyranoside (pNPG) but an increase or not change in K(m). All mutants showed a decrease in catalytic efficiency of isoflavonoid glycosides and were not changed for F221L and lost for N223L in enzymatic hydrolysis on quercetin glucosides. Contrarily, G224T resulted in a dramatic increase conversion of Q4' (35.5%) and Q3,4' (28.6%) in accord with an increased turnover number (k(cat), 1.4×) and catalytic efficiency (k(cat)/K(m), 2.2×) as well as a decrease in K(m) (0.24) for Q4'. Modeling showed that G224T mutation at position 224 may enhance the interaction between G224T and 5-OH and 3-OH on the quercetin backbone of Q4'.

Characterization of a novel thermostable chitin-binding domain and its application in immobilization of a multifunctional hemicellulase.

A novel thermostable chitin-binding domain (Tt-ChBD) of chitinase A1 from Thermoanaerobacterium thermosaccharolyticum DSM571 was cloned, characterized, and compared for its binding activity with another mesophilic chitin-binding domain (Bc-ChBD). Recombinant protein with Tt-ChBD exhibits stronger affinity to chitin than those with Bc-ChBD at temperatures from 65 °C to at least 75 °C, but not to other polysaccharides including xylan, chitosan, cellulose, and agarose. For repeated production of xylose from arabinoxylan-containing feedstocks, a best-characterized trifunctional chimeric enzyme Xar-L1-Xyn (XX) constructed in our previous work was attempted to be immobilized on chitin efficiently by genetically fusing Tt-ChBD to the N-terminal region of XX (named CXX) and the C-terminal region of XX (named XXC), respectively. The fusing position of Tt-CBD affected the affinity-binding activity to chitin. Recombinant XX, XXC, and CXX were purified to homogeneity and characterized. According to the xylosidase activities, the optimum temperature and pH profiles of the CXX and XXC both in free and immobilized form were the same as those of XX. However, the thermal and pH stabilities of the immobilized XXC and CXX were both greatly improved in the range from 70 to 90 °C and pH 4.2-8.2. The immobilized multifunctional hemicellulase exhibited high stability to producing xylose for at least 19 or 30 times in continuous operation with the achievement of 60% or 80% conversion yield at temperatures up to 65 °C. These results indicate the usefulness of Tt-ChBD as an affinity tag for the simultaneous purification and immobilization of the enzyme on chitin and the great potential applications for thermophilic enzyme immobilization at higher temperatures.

Comparison of three thermostable ß-glucosidases for application in the hydrolysis of soybean isoflavone glycosides.

A novel thermostable ß-glucosidase (Te-BglA) from Thermoanaerobacter ethanolicus JW200 was cloned, characterized and compared for its activity against isoflavone glycosides with two ß-glucosidases (Tm-BglA, Tm-BglB) from Thermotoga maritima. Te-BglA exhibited maximum hydrolytic activity toward pNP-ß-d-glucopyranoside (pNPG) at 80 °C and pH 7.0, was stable for a pH range of 4.6-7.8 and at 65 °C for 3 h, and had the lowest K(m) for the natural glycoside salicin and the highest relative substrate specificity (k(cat)/K(m))((salicin))/(k(cat)/K(m))((pNPG)) among the three enzymes. It converted isoflavone glycosides, including malonyl glycosides, in soybean flour to their aglycons more efficiently than Tm-BglA and Tm-BglB. After 3 h of incubation at 65 °C, Te-BglA produced complete hydrolysis of four isoflavone glycosides (namely, daidzin, genistin and their malonylated forms), exhibiting higher productivity of genistein and daidzein than the other two ß-glucosidases. Our results suggest that Te-BglA is preferable to Tm-BglA and Tm-BglB, but all three enzymes have great potential applications in converting isoflavone glycosides into their aglycons.

High-level expression of an alpha-L-arabinofuranosidase from Thermotoga maritima in Escherichia coli for the production of xylobiose from xylan.

To efficiently produce xylobiose from xylan, high-level expression of an alpha-L-arabinofuranosidase gene from Thermotoga maritima was carried out in Escherichia coli. A 1.5-kb DNA fragment, coding for an alpha-L-arabinofuranosidase of T. maritima, was inserted into plasmid pET-20b without the pelB signal sequence leader, and produced pET-20b-araA1 with 8 nt spacing between ATG and Shine-Dalgarno sequence. A maximum activity of 12 U mg(-1) was obtained from cellular extract of E. coli BL21-CodonPlus (DE3)-RIL harboring pET-20b-araA1. The over-expressed alpha-L-arabinofuranosidase was purified 13-fold with a 94% yield from the cellular extract of E. coli by a simple heat treatment. Production of xylooligosaccharides from corncob xylan by endoxylanase and alpha-L-arabinofuranosidase was examined by TLC and HPLC: xylobiose was the major product from xylan at 90 degrees C and its proportion in the xylan hydrolyzates increased with the reaction time. Hydrolysis with in the xylanase absence of alpha-L-arabinofuranosidase gave only half this yield.

Expression and characterization of a thermostable beta-xylosidase from the hyperthermophile, Thermotoga maritima.

A thermostable beta-xylosidase from a hyperthermophilic bacterium, Thermotoga maritima, was over-expressed in Escherichia coli using the T7 polymerase expression system. The expressed beta-xylosidase was purified in two steps, heat treatment and immobilized metal affinity chromatography, and gave a single band on SDS-PAGE. The maximum activity on p-nitrophenyl beta-D-xylopyranoside was at 90 degrees C and pH 6.1. The purified enzyme had a half-life of over 22-min at 95 degrees C, and retained over 57% of its activity after holding a pH ranging from 5.4 to 8.5 for 1 h at 80 degrees C. Among all tested substrates, the purified enzyme had specific activities of 275, 50 and 29 U mg(-1) on pNPX, pNPAF, and pNPG, respectively. The apparent Michaelis constant of the beta-xylosidase was 0.13 mM for p NPX with a V (max) of 280 U mg(-1). When the purified beta-xylosidase was added to xylanase, corncob xylan was hydrolized completely to xylose.

[Expression and purification of thermostable alpha-glucuronidase from Thermotoga maritima].

The xylanolytic enzymes found in Thermotoga maritima showed extremely high thermostability and considerable potential in industrial application. Yet expression level of the genes encoding these enzymes was very low. The alpha-glucuronidase gene aguA from T. maritima ATCC 43589 was cloned and expressed in several E. coli strains with different vector. The alpha-glucuronidase was overexpressed in E. coli BL21-CodonPlus(DE3)-RIL with plasmid pET-28a(+), and made up about 20% of the total proteins present in the intracellular soluble fraction. The results proved the assumption that rare codons for arginine (AGA/AGG) and isoleucine (AUA) affect the expression of aguA gene from hyperthermophilic bacterium T. maritima in E. coli. Purification procedure included two steps, heat treatment and immobilized metal affinity chromatography, and over 13.5mg of pure enzyme was obrained from 1L of induced culture. The purified enzyme showed a single band on SDS polyacrylamide gel electrophoresis with a purification of 5.1 fold, and a yield of 55.1%. The optimum activity of recombinant alpha-glucuronidase was found at pH 6.0 and 85 degrees C, the enzyme retained 70% of its activity after 1 h of incubation at 85 degrees C. The induction conditions for expression of recombinant strain BL21-CodonPlus(DE3)-RIL/pET-28a-aguA were studied on induction time and duration by IPTG. The results showed that the activity of thermostable alpha-glucuronidase reach the maximum in 5-hour after inducted at the exponential phase (OD600 of 0.7 - 0.8).