Yong-Keun Chang

Identification and biochemical characterization of Sco3487 from Streptomyces coelicolor A3(2): an exo- and endo-type β-agarase-producing neoagarobiose

Streptomyces coelicolor can degrade agar, the main cell wall component of red macroalgae, for growth. To constitute a crucial carbon source for bacterial growth, the alternating α-(1,3) and β-(1,4) linkages between the 3,6-anhydro-L-galactoses and D-galactoses of agar must be hydrolyzed by α/β-agarases. In S. coelicolor, DagA was confirmed to be an endo-type β-agarase that degrades agar into neoagarotetraose and neoagarohexaose. Genomic sequencing data of S. coelicolor revealed that Sco3487, annotated as a putative hydrolase, has high similarity to the glycoside hydrolase (GH) GH50 β-agarases. Sco3487 encodes a primary translation product (88.5 kDa) of 798 amino acids, including a 45-amino-acid signal peptide. The sco3487 gene was cloned and expressed under the control of the ermE promoter in Streptomyces lividans TK24. β-Agarase activity was detected in transformant culture broth using the artificial chromogenic substrate p-nitrophenyl-β-D-galactopyranoside. Mature Sco3487 (83.9 kDa) was purified 52-fold with a yield of 66% from the culture broth. The optimum pH and temperature for Sco3487 activity were 7.0 and 40°C, respectively. The K(m) and V(max) for agarose were 4.87 mg/ml (4 × 10(-5) M) and 10.75 U/mg, respectively. Sco3487 did not require metal ions for its activity, but severe inhibition by Mn(2+) and Cu(2+) was observed. Thin-layer chromatography analysis, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and Fourier transform-nuclear magnetic resonance spectrometry of the Sco3487 hydrolysis products revealed that Sco3487 is both an exo- and endo-type β-agarase that degrades agarose, neoagarotetraose, and neoagarohexaose into neoagarobiose.

Overexpression and biochemical characterization of DagA from Streptomyces coelicolor A3(2): an endo-type β-agarase producing neoagarotetraose and neoagarohexaose

The DagA product of Streptomyces coelicolor is an agarase with a primary translation product (35xa0kDa) of 309 amino acids, including a 30-amino acid signal peptide. Although dagA expression in Streptomyces lividans under the control of its own set of promoters was previously reported, its enzymatic properties have never been elucidated. To develop an improved expression system for dagA, three types of strong promoters for the Streptomyces host were linked to dagA, and their efficiencies in DagA production were compared in S. lividans TK24. All of the transformants with dagA grew at improved rates and produced larger amounts of DagA in the modified R2YE medium containing 0.5% agar as the sole carbon source. Of the three transformants, the S. lividans TK24/pUWL201-DagA (ermE promoter) produced the highest agarase activity (A540u2009=u20094.24), and even the S. lividans TK24/pHSEV1-DagA (tipA promoter) and S. lividans TK24/pWHM3-DagA (sprT promoter) produced higher agarase activity (A540u2009=u20090.24 and 0.12, respectively) than the control (A540u2009=u20090.01) in the modified R2YE medium. The mature form of DagA protein (32xa0kDa) was successfully purified by one-step affinity column chromatography by using agarose beads with excellent yield. The purified DagA was found to exhibit maximal agarase activity at 40°C and pHxa07.0. The Km, Vmax, and Kcat values for agarose were 2.18xa0mg/ml (approximately 1.82u2009×u200910−5xa0M), 39.06xa0U/mg of protein, and 9.5u2009×u2009103/s, respectively. Thin layer chromatography (TLC) analysis, matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry, and Fourier transform nuclear magnetic resonance (FT-NMR) spectrometry of the hydrolyzed products of agarose by DagA revealed that DagA is an endo-type β-agarase that degrades agarose into neoagarotetraose and neoagarohexaose.

Microbial coal desulfurization in an airlift bioreactor by sulfur-oxidizing bacterium Thiobacillus ferrooxidans

Microbial desulfurization of a domestic anthracite coal by using an acidophilic, sulfuroxidizing bacterium, Thiobacillus ferrooxidans has been studied in an airlift slurry reactor of 12 L volume. Effects of coal slurry density and CO 2 supplement on microbial pyrite removal have been evaluated. High sulfur removal rates have been obtained even for very high coal slurry densities (up to 70% w/v). About 90 ∼ 95 % of the sulfur in the coal could be removed in 15 ∼ 20 days. The efficiency of microbial desulfurization was significantly improved with CO 2 enriched air supply for high coal slurry densities

Cloning, Expression, and Biochemical Characterization of a GH16 β-Agarase AgaH71 from Pseudoalteromonas hodoensis H7

An agarase gene (agaH71) was identified from Pseudoalteromonas hodoensis, an agar utilizing marine bacterium. The nucleotide sequence revealed that AgaH71 had significant homology to glycosyl hydrolase (GH) 16 agarases. agaH71 encodes a primary translation product (32.7xa0kDa) of 290 amino acids, including a 21-amino-acid signal peptide. The entire AgaH71 was expressed in a fused protein with glutathione-S-transferase (GST) at its N-terminal (GST-AgaH71) in Escherichia coli. Purified GST-AgaH71 had an apparent molecular weight of 59xa0kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which was consistent with the calculated molecular weight (58.7xa0kDa). Agarase activity of the purified protein was confirmed by zymogram assay. GST-AgaH71 could hydrolyze p-nitrophenyl-β-d-galactopyranoside, but not p-nitrophenyl-α-d-galactopyranoside. The optimum pH and temperature for GST-AgaH71 were 6.0 and 45xa0°C, respectively. GST-AgaH71 retained more than 95 and 90xa0% of its initial activity at 40 and 45xa0°C after heat treatment for 60xa0min, respectively. The Km and Vmax for agarose were 28.33xa0mg/ml and 88.25xa0U/mg, respectively. GST-AgaH71 did not require metal ions for its activity, but severe inhibition by divalent metal ions was observed. Thin-layer chromatography (TLC) analysis, mass spectrometry, and nuclear magnetic resonance (NMR) spectrometry of the GST-AgaH71 hydrolysis products revealed that GST-AgaH71 is an endo-type β-agarase that hydrolyzes agarose into predominantly neoagarotetraose and small proportions of neoagarobiose and neoagarohexaose.

Identification and characterization of a novel β-galactosidase from Victivallis vadensis ATCC BAA-548, an anaerobic fecal bacterium

Victivallis vadensis ATCC BAA-548 is a Gram-negative, anaerobic bacterium that was isolated from a human fecal sample. From the genomic sequence of V. vadensis, one gene was found to encode agarase; however, its enzymatic properties have never been characterized. The gene encoding the putative agarase (NCBI reference number ZP_01923925) was cloned by PCR and expressed in E. coli Rosetta-gami by using the inducible T7 promoter of pET28a(+). The expressed protein with a 6×His tag at the N-terminus was named His6-VadG925 and purified as a soluble protein by Ni2+-NTA agarose affinity column chromatography. The purification of the enzyme was 26.8-fold, with a yield of 73.2% and a specific activity of 1.02 U/mg of protein. The purified His6-VadG925 produced a single band with an approximate MW of 155 kDa, which is consistent with the calculated value (154,660 Da) including the 6×His tag. Although VadG925 and many of its homologs were annotated as agarases, it did not hydrolyze agarose. Instead, purified His6-VadG925 hydrolyzed an artificial chromogenic substrate, p-nitrophenyl-β-d-galactopyranoside, but not p-nitrophenyl-α-d-galactopyranoside. The optimum pH and temperature for this β-galactosidase activity were pH 7.0 and 40°C, respectively. The Km and Vmax of His6-VadG925 towards p-nitrophenyl-β-d-galactopyranoside were 1.69 mg/ml (0.0056 M) and 30.3 U/mg, respectively. His6-VadG925 efficiently hydrolyzed lactose into glucose and galactose, which was demonstrated by TLC and mass spectroscopy. These results clearly demonstrated that VadG925 is a novel β-galactosidase that can hydrolyze lactose, which is unusual because of its low homology to validated β-galactosidases.

Gas holdup and mass transfer characteristics of carboxymethyl cellulose solutions in a bubble column with a radial gas sparger

The gas phase holdup and mass transfer characteristics of carboxymethyl cellulose (CMC) solutions in a bubble column having a radial gas sparger have been determined and a new flow regime map has been proposed. The gas holdup increases with gas velocity in the bubbly flow regime, decreases in the churn-turbulent flow regime, and increases again in the slug flow regime. The volumetric mass transfer coefficient (kLa) significantly decreases with increasing liquid viscosity. The gas holdup and kLa values in the present bubble column of CMC solutions are found to be much higher than those in bubble columns or external-loop airlift columns with a plate-type sparger. The obtained gas phase holdup (ɛg) and kLa data have been correlated with pertinent dimensionless groups in both the bubbly and the churn-turbulent flow regimes.

Alternately Stacked Langmuir-Blodgett Film of Phospholipid and ZnO as an Olfactory Sensing Membrane

Abstract Langmuir-Blodgett films of Phospholipid and ZnO were investigated as a sensing membrane to enhance the sensitivity of an olfactory sensor. The membranes were fabricated by alternate stack of dipalmitoylphophatidic acid (DPPA) and ZnO. Compared with the DPPA LB film, the alternately stacked film system showed higher sensitivity and faster response to gases. The chemical vapors used in this study were methanol, ethanol, and acetone.