Te-BglA and Tm-BglA are glycoside hydrolase family 1 β-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 β-d-glucopyranoside (pNPG) but an increase or not change in Km. 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 (kcat, 1.4×) and catalytic efficiency (kcat/Km, 2.2×) as well as a decrease in Km (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′.

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.

The gene encoding esterase (CE1) from Bacillus pumilus ARA with a calculated molecular weight of 28.4 kDa was cloned, sequenced and efficiently expressed in Escherichia coli. The open reading frame of 747 nucleotides encoded a protein, which was classified as a carboxylesterase with an identity of 87 % to esterase from Bacillus subtilis 168. Recombinant CE1 was purified in a single step to electrophoretic homogeneity by IMAC (Ni2+). The enzyme displayed maximum activity toward p-nitrophenyl (pNP) acetate at 37–40 °C and pH 6.5–7.0. It was stable in the pH range from 6.5 to 8.0, and at temperature from 25 to 40 °C. Among four p-nitrophenyl esters tested, the best substrate was pNP acetate with Km and kcat values of 0.33 mM and 4.07 s−1, respectively. Amounts of 2 mM Ca2+ and Co2+ significantly increased the esterase activity to 190 and 121 %, respectively. These results suggest that CE1 has very attractive applications of increasing feed digestibility in animal nutrition in this moderate temperature range.

A novel thermostable β-glucosidase (Te-BglA) from Thermoanaerobacter ethanolicus JW200 was cloned, characterized and compared for its activity against isoflavone glycosides with two β-glucosidases (Tm-BglA, Tm-BglB) from Thermotoga maritima. Te-BglA exhibited maximum hydrolytic activity toward pNP-β-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 Km for the natural glycoside salicin and the highest relative substrate specificity (kcat/Km)(salicin)/(kcat/Km)(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 β-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.

A recombinant Thermotoga maritima β-glucosidase A (BglA) was purified to homogeneity for performing enzymatic hydrolysis of isoflavone glycosides from soy flour. The kinetic properties Km, kcat, and kcat/Km 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°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°C.

To produce aglycone isoflavones from soy flour, the β-glucosidase A gene (bglA) of Thermotoga maritima was overexpressed in Escherichia coli BL21-CodonPlus (DE3)-RIL. The Km and Vmax values of the purified BglA for pNPG were 0.43 mM and 323.6 U mg−1, respectively, and those for salicin were 9.0 mM and 183.2 U mg−1, respectively. The biochemical and kinetic characteristics of his-tagged BglA were found to be similar to those of BglA, except for the temperature stability and specific activity. Production of aglycone isoflavones from soy flour by BglA was examined by HPLC. For 3 h at 80°C, all the isoflavone glycosides approximated to the complete conversion into aglycone isoflavones, over seven times higher than that obtained from soy flour without BglA.

The trifunctional enzyme (XAR–XYN) associating the Thermoanaerobacter ethanolicus xylosidase-arabinosidase (XAR) with the Thermomyces lanuginosus xylanase (XYN) was produced in E. coli to study the effect of the physical association of the fusion partners on the enzymatic efficiency. Recombinant XAR, XYN and XAR–XYN were purified to homogeneity and characterized. The optimal pH and temperature of the XAR–XYN were found to be similar to those of the XAR and XYN, except for less temperature optimum of α-arabinosidase activity. Its pH and xylanase activity exhibited more stable than those of the XAR and XYN. Finally, the XAR–XYN was tested for degradation of oat spelt xylan and wheat bran, the XAR–XYN was found to be more facile than the corresponding free enzyme degradation of wheat bran but provided little or no advantage on purified xylan. Furthermore cooperation within a trifunctional enzyme containing linker SAGSSAAGSGSG between each partner was achieved, leading to a trifunctional enzyme with enhanced enzymatic efficiency on arabinoxylan.

Fusion proteins composed of a xylosidase–arabinosidase (Xar) from Thermoanaerobacter ethanolicus and Thermomyces lanuginosu xylanase (XynA) were construted using different peptide linkers. The aim was to create optimal linkers that were able to join the functional modules without disturbing their function. Five fusion variants with linker containing different length and ratio of Ala/Gly were produced in Escherichia coli and purified for characterization of their enzymatic and kinetic properties. The fusion proteins with linkers had similar temperature and pH profiles to those without linkers, being well in accordance with their parents. However, the fusion variant containing SAGSSAAGSGSG had greater stability for xylosidase–arabinosidase activity than other fusion variants at 65–80 °C and pH 6.6–8.2. An obvious increase in activity, which occurred in the first 60 min at 65–75 °C for xylosidase–arabinosidase, appeared for xylanase and arabinosidase at pH 6.6–7.8, and for xylosidase at pH 7.8–9.5. The reducing sugar release efficiency of fusion variants containing SAGSSAAGSGSG and SGGSSAAGSGSG were obviously higher compared with those containing other linkers. These results indicate that introducing Ala in monotonous peptides consisting of only Gly and Ser would make the best linkers for the Xar–XynA fusion protein.