Purification and cloning of a thermostable xylose (glucose) isomerase with an acidic pH optimum from Thermoanaerobacterium strain JW/SL-YS 489

The araA gene encoding L-arabinose isomerase (AI) from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius was cloned, sequenced, and expressed in Escherichia coli. Analysis of the sequence revealed that the open reading frame of the araA gene consists of 1,491 bp that encodes a protein of 497 amino acid residues with a calculated molecular mass of 56,043 Da. Comparison of the deduced amino acid sequence of A. acidocaldarius AI (AAAI) with other AIs demonstrated that AAAI has 97% and 66% identities (99% and 83% similarities) to Geobacillus stearothermophilus AI (GSAI) and Bacillus halodurans AI (BHAI), respectively. The recombinant AAAI was purified to homogeneity by heat treatment, ion-exchange chromatography, and gel filtration. The purified enzyme showed maximal activity at pH 6.0 to 6.5 and 65°C under the assay conditions used, and it required divalent cations such as Mn 2؉ , Co 2؉ , and Mg 2؉ for its activity. The isoelectric point (pI) of the enzyme was about 5.0 (calculated pI of 5.5). The apparent K m values of the recombinant AAAI for L-arabinose and D-galactose were 48.0 mM (V max , 35.5 U/mg) and 129 mM (V max , 7.5 U/mg), respectively, at pH 6 and 65°C. Interestingly, although the biochemical properties of AAAI are quite similar to those of GSAI and BHAI, the three AIs from A. acidocaldarius (pH 6), G. stearothermophilus (pH 7), and B. halodurans (pH 8) exhibited different pH activity profiles. Based on alignment of the amino acid sequences of these homologous AIs, we propose that the Lys-269 residue of AAAI may be responsible for the ability of the enzyme to act at low pH. To verify the role of Lys-269, we prepared the mutants AAAI-K269E and BHAI-E268K by site-directed mutagenesis and compared their kinetic parameters with those of wild-type AIs at various pHs. The pH optima of both AAAI-K269E and BHAI-E268K were rendered by 1.0 units (pH 6 to 7 and 8 to 7, respectively) compared to the wild-type enzymes. In addition, the catalytic efficiency (k cat /K m ) of each mutant at different pHs was significantly affected by an increase or decrease in V max . From these results, we propose that the position corresponding to the Lys-269 residue of AAAI could play an important role in the determination of the pH optima of homologous AIs.

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Characterization of a Thermoacidophilic l -Arabinose Isomerase from Alicyclobacillus acidocaldarius : Role of Lys-269 in pH Optimum

Lee

Choe

et al. 2005

“…A number of thermostable xylose isomerases (XIs) (EC 5.3.1.5) have been isolated from extremophiles and have been studied extensively in connection with the manufacture of high-fructose corn syrup, because the equilibrium for the isomerization of glucose to fructose is shifted toward fructose at high temperatures (17,27,29).…”

Section: ؊1mentioning

confidence: 99%

“…It has also been classified as generally recognized as safe for use in cosmetics and drugs, and it is useful as a reduced-calorie bulking agent, as an intermediate in the synthesis of other optically active compounds, and as an additive in detergent, cosmetic, and pharmaceutical formulations. Initial quantities of D-tagatose have been produced commercially by a patented chemical process since the beginning of 2003 (2).A number of thermostable xylose isomerases (XIs) (EC 5.3.1.5) have been isolated from extremophiles and have been studied extensively in connection with the manufacture of high-fructose corn syrup, because the equilibrium for the isomerization of glucose to fructose is shifted toward fructose at high temperatures (17,27,29).Our preliminary experiments on the effect of reaction temperature on the conversion of D-galactose to D-tagatose by Thermotoga neapolitana AI showed that conversion increased as the incubation temperature was raised (12). For industrial applications of the production of D-tagatose from D-galactose, the AI should be thermostable at elevated temperatures (Ͼ50°C).…”

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confidence: 99%

Characterization of a Thermostable l -Arabinose ( d -Galactose) Isomerase from the Hyperthermophilic Eubacterium Thermotoga maritima

Lee

Jang

Choe

et al. 2004

128

116

The araA gene encoding L-arabinose isomerase (AI) from the hyperthermophilic bacterium Thermotoga maritima was cloned and overexpressed in Escherichia coli as a fusion protein containing a C-terminal hexahistidine sequence. This gene encodes a 497-amino-acid protein with a calculated molecular weight of 56,658. The recombinant enzyme was purified to homogeneity by heat precipitation followed by Ni 2؉ affinity chromatography. The native enzyme was estimated by gel filtration chromatography to be a homotetramer with a molecular mass of 232 kDa. The purified recombinant enzyme had an isoelectric point of 5.7 and exhibited maximal activity at 90°C and pH 7.5 under the assay conditions used. Its apparent K m values for L-arabinose and D-galactose were 31 and 60 mM, respectively; the apparent V max values (at 90°C) were 41.3 U/mg (L-arabinose) and 8.9 U/mg (D-galactose), and the catalytic efficiencies (k cat /K m ) of the enzyme were 74.8 mM ؊1⅐ min ؊1 (L-arabinose) and 8.5 mM ؊1 ⅐ min ؊1 (D-galactose). Although the T. maritima AI exhibited high levels of amino acid sequence similarity (>70%) to other heat-labile mesophilic AIs, it had greater thermostability and higher catalytic efficiency than its mesophilic counterparts at elevated temperatures. In addition, it was more thermostable in the presence of Mn 2؉ and/or Co 2؉ than in the absence of these ions. The enzyme carried out the isomerization of D-galactose to D-tagatose with a conversion yield of 56% for 6 h at 80°C.Of the eubacteria whose genomes have been sequenced to date, Thermotoga maritima has the highest percentage of genes that are most similar to archaeal genes (20). This bacterium is also of evolutionary importance, because small-subunit rRNA phylogeny has shown that it is one of the deepest and most slowly evolving lineages of the eubacteria. Thus, it could be useful for elucidating the evolutionary relationship between thermophilic eubacteria and archaea. In addition, it metabolizes many simple and complex carbohydrates, including glucose, sucrose, starch, cellulose, and xylan (9). Thermostable enzymes from T. maritima involved in carbohydrate metabolism are very attractive for industrial applications (4,15,24).Almost 7% of the predicted coding sequences in the T. maritima genome are involved in metabolism of simple and complex sugars (20). Several genes encode proteins involved in arabinose metabolism. Among them, the araA gene encoding L-arabinose isomerase (AI) (EC 5.3.1.4), which catalyzes the conversion of L-arabinose to L-ribulose, not only is important for pentose sugar isomerization in vivo but also is very attractive for use in the bioconversion of D-galactose into D-tagatose in vitro (5, 25) (Fig. 1).The ketohexose D-tagatose has a sweetness value (92%) equivalent to that of sucrose but is poorly digested (18,32). This compound has been found to be a safe low-calorie sweetener in food products and is classified as a generally recognized as safe substance in the United States. It has also been classified as generally recognized as safe for u...

“…Species of Thermoanaerobacterium are widely distributed and can efficiently utilize xylan, a potentially inexpensive substrate for industrial fermentation (15). Several enzymes from Thermoanaerobacterium saccharolyticum strain JW/SL-YS485 have been isolated and characterized, and various genes have been cloned and sequenced (10,(12)(13)(14). Due to lack of developed genetic systems for any member belonging to this group of microorganisms, T. saccharolyticum JW/SL-YS485 was chosen as a model organism for the development of genetic tools (16).…”

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confidence: 99%

Advances in Development of a Genetic System for Thermoanaerobacterium spp.: Expression of Genes Encoding Hydrolytic Enzymes, Development of a Second Shuttle Vector, and Integration of Genes into the Chromosome

Mai

Wiegel

2000

Despite recent success in transforming various thermophilic gram-type-positive anaerobes with plasmid DNA, use of shuttle vectors for the expression of genes other than antibiotic resistance markers has not previously been described. We constructed new vectors in order to express heterologous hydrolytic enzymes in our model system, Thermoanaerobacterium saccharolyticum JW/SL-YS485. Transformed Thermoanaerobacterium expressed active enzyme, indicating that this system may function as an alternate expression host, especially for genes with a thermophilic origin. To develop further the genetic system for T. saccharolyticum JW/SL-YS485, two improved Escherichia coli-Thermoanaerobacterium shuttle vectors, pRKM1 and pRUKM, were constructed. Furthermore, the kanamycin resistance cassette alone and the kanamycin resistance cassette plus the cellobiohydrolase gene (cbhA) from Clostridium thermocellum JW20 were integrated into the xylanase gene (xynA) region of the Thermoanaerobacterium chromosome via homologous recombination using pUC-based suicide vectors pUXK and pUXKC.