Thermostable Chicken Feather Degrading Enzymes from L-23 Isolate from Indonesia

The thermostable chicken feather degrading protease enzymes used here was extracted and partially purified from thermophilic bacteria L-23 isolated from a coastal hot spring in North Sulawesi, Indonesia. The L-23 was grown in the selective medium containing 1% chicken feather powder at 70 °C and pH 7. The cell-free culture was precipitated with ammonium sulphate at 80% saturation, followed by heating at 65 °C for 1 h before applied onto Sephadex G-100. The molecular weight of the two enzymes identified were estimated as 47 and 64 kDa. The optimum pH of the mixed enzymes preparation was 7 while the optimum temperature was 65 °C. Zymogram analysis showed that one of the enzymes was still active after being heated at 100 °C for 20 min and was also resistant towards organic solvents and SDS. The activity was enhanced by addition of 1 mM FeCl3

Expression of human β-defensin-1 in recombinant Escherichia coli and analysis of its antimicrobial spectrum

Escherichia coli BL21(DE3) was transformed with a pHCD1 plasmid harboring the human β-defensin-1 (hBD1) gene fused in frame behind a disulfide bond isomerase (DsbC), a His-tag, and an enterokinase cleavage site. After induction, the DsbC-hBD1 was expressed as a ~36 kDa soluble fusion protein in recombinant E. coli, which also inhibited host cell growth. After cell disruption, the soluble protein was easily recovered by Ni2+ affinity chromatography and cleaved by enterokinase to yield a mature hBD1 of about 4 kDa. Importantly, the mature hBD1 showed broad antimicrobial activity against Gram-positive and -negative pathogenic bacteria, including Streptococcus pneumoniae, E. coli O157:H7, and Klebsiella pneumoniae.Key words: Antimicrobial activity, Escherichia coli, human β-defensin-1, soluble expression

Characterization of a Novel d-Lyxose Isomerase from Cohnella laevoribosii RI-39 sp. nov.

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 Mn(2+). C. laevoribosii d-lyxose (l-ribose) isomerase (CLLI) exhibits maximal activity at pH 6.5 and 70°C in the presence of Mn(2+) 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 K(m) values of 22.4 ± 1.5 mM, 121.7 ± 10.8 mM, and 34.0 ± 1.1 mM, respectively. The catalytic efficiencies (k(cat)/K(m)) of CLLI were 84.9 ± 5.8 mM(−1) s(−1) for d-lyxose (V(max), 5,434.8 U mg(−1)), 0.2 mM(−1) s(−1) for l-ribose (V(max), 75.5 ± 6.0 U mg(−1)), and 1.4 ± 0.1 mM(−1) s(−1) for d-mannose (V(max), 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

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

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

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 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(−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 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