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.

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 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.