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author:("yun, Yu-ryan")
1.  Characterization of a Novel d-Lyxose Isomerase from Cohnella laevoribosii RI-39 sp. nov.▿  
Journal of Bacteriology  2006;189(5):1655-1663.
A newly isolated bacterium, Cohnella laevoribosii RI-39, could grow in a defined medium with l-ribose as the sole carbon source. A 21-kDa protein isomerizing l-ribose to l-ribulose, as well as d-lyxose to d-xylulose, was purified to homogeneity from this bacterium. Based on the N-terminal and internal amino acid sequences of the purified enzyme obtained by N-terminal sequencing and quantitative time of flight mass spectrometry-mass spectrometry analyses, a 549-bp gene (lyxA) encoding d-lyxose (l-ribose) isomerase was cloned and expressed in Escherichia coli. The purified endogenous enzyme and the recombinant enzyme formed homodimers that were activated by Mn2+. C. laevoribosii d-lyxose (l-ribose) isomerase (CLLI) exhibits maximal activity at pH 6.5 and 70°C in the presence of Mn2+ for d-lyxose and l-ribose, and its isoelectric point (pI) is 4.2 (calculated pI, 4.9). The enzyme is specific for d-lyxose, l-ribose, and d-mannose, with apparent Km values of 22.4 ± 1.5 mM, 121.7 ± 10.8 mM, and 34.0 ± 1.1 mM, respectively. The catalytic efficiencies (kcat/Km) of CLLI were 84.9 ± 5.8 mM−1 s−1 for d-lyxose (Vmax, 5,434.8 U mg−1), 0.2 mM−1 s−1 for l-ribose (Vmax, 75.5 ± 6.0 U mg−1), and 1.4 ± 0.1 mM−1 s−1 for d-mannose (Vmax, 131.8 ± 7.4 U mg−1). The ability of lyxA to permit E. coli cells to grow on d-lyxose and l-ribose and homology searches of other sugar-related enzymes, as well as previously described sugar isomerases, suggest that CLLI is a novel type of rare sugar isomerase.
doi:10.1128/JB.01568-06
PMCID: PMC1855708  PMID: 17189362
2.  Characterization of a Thermoacidophilic l-Arabinose Isomerase from Alicyclobacillus acidocaldarius: Role of Lys-269 in pH Optimum 
Applied and Environmental Microbiology  2005;71(12):7888-7896.
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 Mn2+, Co2+, and Mg2+ for its activity. The isoelectric point (pI) of the enzyme was about 5.0 (calculated pI of 5.5). The apparent Km values of the recombinant AAAI for l-arabinose and d-galactose were 48.0 mM (Vmax, 35.5 U/mg) and 129 mM (Vmax, 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 (kcat/Km) of each mutant at different pHs was significantly affected by an increase or decrease in Vmax. 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.
doi:10.1128/AEM.71.12.7888-7896.2005
PMCID: PMC1317409  PMID: 16332764
3.  Secretion of Recombinant Pediocin PA-1 by Bifidobacterium longum, Using the Signal Sequence for Bifidobacterial α-Amylase 
A recombinant DNA, encoding the chimeric protein of the signal sequence for bifidobacterial α-amylase mature pediocin PA-1, was introduced into Bifidobacterium longum MG1. Biologically active pediocin PA-1 was successfully secreted from the strain and showed bactericidal activity against Listeria monocytogenes and the same molecular mass as native pediocin PA-1.
doi:10.1128/AEM.71.9.5630-5632.2005
PMCID: PMC1214669  PMID: 16151166
4.  Characterization of a Thermostable l-Arabinose (d-Galactose) Isomerase from the Hyperthermophilic Eubacterium Thermotoga maritima 
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 Ni2+ 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 Km values for l-arabinose and d-galactose were 31 and 60 mM, respectively; the apparent Vmax values (at 90°C) were 41.3 U/mg (l-arabinose) and 8.9 U/mg (d-galactose), and the catalytic efficiencies (kcat/Km) of the enzyme were 74.8 mM−1 · min−1 (l-arabinose) and 8.5 mM−1 · 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 Mn2+ and/or Co2+ 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.
doi:10.1128/AEM.70.3.1397-1404.2004
PMCID: PMC368370  PMID: 15006759
5.  Efficient Library Construction by In Vivo Recombination with a Telomere-Originated Autonomously Replicating Sequence of Hansenula polymorpha 
A high frequency of transformation and an equal gene dosage between transformants are generally required for activity-based selection of mutants from a library obtained by directed evolution. An efficient library construction method was developed by using in vivo recombination in Hansenula polymorpha. Various linear sets of vectors and insert fragments were transformed and analyzed to optimize the in vivo recombination system. A telomere-originated autonomously replicating sequence (ARS) of H. polymorpha, reported as a recombination hot spot, facilitates in vivo recombination between the linear transforming DNA and chromosomes. In vivo recombination of two linear DNA fragments containing the telomeric ARS drastically increases the transforming frequency, up to 10-fold, compared to the frequency of circular plasmids. Direct integration of the one-end-recombined linear fragment into chromosomes produced transformants with single-copy gene integration, resulting in the same expression level for the reporter protein between transformants. This newly developed in vivo recombination system of H. polymorpha provides a suitable library for activity-based selection of mutants after directed evolution.
doi:10.1128/AEM.69.8.4448-4454.2003
PMCID: PMC169078  PMID: 12902228

Results 1-5 (5)