Purification and characterization of laccase from the white rot fungus Trametes versicolor

Laccase is one of the ligninolytic enzymes of white rot fungus Trametes versicolor 951022, a strain first isolated in Korea. This laccase was purified 209-fold from culture fluid with a yield of 6.2% using ethanol precipitation, DEAE-Sepharose, Phenyl-Sepharose, and Sephadex G-100 chromatography. T. versicolor 951022 excretes a single monomeric laccase showing a high specific activity of 91,443 U/mg for 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as a substrate. The enzyme has a molecular mass of approximately 97 kDa as determined by SDS-PAGE, which is larger than those of other laccases reported. It exhibits high enzyme activity over broad pH and temperature ranges with optimum activity at pH 3.0 and a temperature of 50 degrees C. The Km value of the enzyme for substrate ABTS is 12.8 micrometer and its corresponding Vmax value is 8125.4 U/mg. The specific activity and substrate affinity of this laccase are higher than those of other white rot fungi, therefore, it may be potentially useful for industrial purposes.     

Functional expression of human pyruvate carboxylase for reduced lactic acid formation of Chinese hamster ovary cells (DG44)

To investigate the effect of human pyruvate carboxylase (hPC) on lactate formation in Chinese hamster ovary (CHO) cell lines, FLAG-tagged hPC was introduced into a dihydrofolate-deficient CHO cell line (DG44). Three clones expressing high levels of hPC, determined by Western blotting using an anti-FLAG monoclonal antibody, and a control cell line were established. Immunocytochemistry revealed that a substantial amount of expressed hPC protein was localized in the mitochondria of the cells. hPC expression did not impair cell proliferation. Rather, it improved cell viability at the end of adherent batch cultures with the serum-containing medium probably because of reduced lactate formation. Compared with control cells, specific lactate production rate of the three clones was decreased by 21–39%, which was because of a decreased specific glucose uptake rate and yield of lactate from glucose. Reduced lactate formation by hPC expression was also observed in suspension fed-batch cultures using a serum-free medium. Taken together, these results demonstrate that through the expression of the hPC enzyme, lactate formation in CHO cell culture can be efficiently reduced.     

Growth of Pichia guilliermondii A9, an osmotolerant yeast, in waste brine generated from kimchi production

The possibility of culturing an osmotolerant yeast using waste brine from a kimchi factory as a substrate for the production of single cell protein was investigated. Pichia guilliermondii A9 was selected from 70 isolates of yeast demonstrating substantial growth in the waste brine. The growth of P. guilliermondii A9 in waste brine was not inhibited by NaCl concentrations of up to 10% (w/v). However, it was reduced drastically at concentrations greater than 12% (w/v). Approximately 90% of BOD was removed from the waste brine by culturing of P. guilliermondii A9 for 24 h. The maximum cell yield was 0.69 g of dry cells per liter, containing 40% of protein. When the waste brine was enriched with cabbage juice from waste cabbage, the final cell mass increased proportionally with the amount of added organic material. Salt stressed cells of P. guilliermondii A9 grown in waste brine are shown in scanning electron micrographs. In conclusion, the large amounts of waste brine generated from kimchi production could be used directly for the culture of the osmotolerant yeast P. guilliermondii A9.     

Characterization of xylanase from Lentinus edodes M290 cultured on waste mushroom logs

Extracellular enzymes from Lentinus edodes M290 on normal woods (Quercus mongolica) and waste logs from oak mushroom production were comparatively investigated. Endoglucanase, cellobiohydrolase, beta-glucosidase, and xylanase activities were higher on waste mushroom logs than on normal woods after L. edodes M290 inoculation. Xylanase activity was especially different, with a three times higher activity on waste mushroom logs. When the waste mushroom logs were used as a carbon source, a new 35 kDa protein appeared. After the purification, the optimal pH and temperature for xylanase activity were determined to be 4.0 and 50 degrees C, respectively. More than 50% of the optimal xylanase activity was retained when the temperature was increased from 20 to 60 degrees C, after a 240 min reaction. At 40 degrees C, the xylanase maintained 93% of the optimal activity, after a 240 min reaction. The purified xylanase showed a very high homology to the xylanase family 10 from Aspergillus terreus by LC/MS-MS analysis. The highest Xcorr (1.737) was obtained from the peptide KWI SQGIPIDGIG SQTHLGSGGS WTVK originated from Aspergillus terreus, indicating that the 35 kDa protein was xylanase. This protein showed low homology to a previously reported L. edodes xylanase sequence.     

The cell agglutination agent, phytohemagglutinin-L, improves the efficiency of somatic nuclear transfer cloning in cattle (Bos taurus)

One of the several factors that contribute to the low efficiency of mammalian somatic cloning is poor fusion between the small somatic donor cell and the large recipient oocyte. This study was designed to test phytohemagglutinin (PHA) agglutination activity on fusion rate, and subsequent developmental potential of cloned bovine embryos. The toxicity of PHA was established by examining its effects on the development of parthenogenetic bovine oocytes treated with different doses (Experiment 1), and for different durations (Experiment 2). The effective dose and duration of PHA treatment (150 microg/mL, 20 min incubation) was selected and used to compare membrane fusion efficiency and embryo development following somatic cell nuclear transfer (Experiment 3). Cloning with somatic donor fibroblasts versus cumulus cells was also compared, both with and without PHA treatment (150 microg/mL, 20 min). Fusion rate of nuclear donor fibroblasts, after phytohemagglutinin treatment, was increased from 33 to 61% (P < 0.05), and from 59 to 88% (P < 0.05) with cumulus cell nuclear donors. The nuclear transfer (NT) efficiency per oocyte used was improved following PHA treatment, for both fibroblast (13% versus 22%) as well as cumulus cells (17% versus 34%; P < 0.05). The cloned embryos, both with and without PHA treatment, were subjected to vitrification and embryo transfer testing, and resulted in similar survival (approximately 90% hatching) and pregnancy rates (17-25%). Three calves were born following vitrification and embryo transfer of these embryos; two from the PHA-treated group, and one from non-PHA control group. We concluded that PHA treatment significantly improved the fusion efficiency of somatic NT in cattle, and therefore, increased the development of cloned blastocysts. Furthermore, within a determined range of dose and duration, PHA had no detrimental effect on embryo survival post-vitrification, nor on pregnancy or calving rates following embryo transfer.     

Distal NF-kB binding motif functions as an enhancer for nontypeable H. influenzae-induced DEFB4 regulation in epithelial cells

Among the antimicrobial molecules produced by epithelial cells, DEFB4 is inducible in response to proinflammatory signals such as cytokines and bacterial molecules. Nontypeable Haemophilus influenzae (NTHi) is an important human pathogen that exacerbates chronic obstructive pulmonary disease in adult and causes otitis media and sinusitis in children. Previously, we have demonstrated that DEFB4 effectively kills NTHi and is induced by NTHi via TLR2 signaling. The 5′-flanking region of DEFB4 contains several NF-κB binding motifs, but their NTHi-specific activity remains unclear. In this study, we aimed to elucidate molecular mechanism involved in DEFB4 regulation, focusing on the role of the distal NF-κB binding motif of DEFB4 responding to NTHi. Here, we show that the human middle ear epithelial cells up-regulate DEFB4 expression in response to NTHi via NF-κB activation mediated by IκKα/β−IκBα signaling. Deletion of the distal NF-κB binding motif led to a significant reduction in NTHi-induced DEFB4 up-regulation. A heterologous construct containing the distal NF-κB binding motif was found to increase the promoter activity in response to NTHi, indicating a NTHi-responding enhancer activity of the distal NF-κB binding motif. Furthermore, electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that the p65 domain of NF-κB binds to the distal NF-κB binding motif in response to NTHi. Taken together, our results suggest that NTHi-induced binding of p65 NF-κB to the distal NF-κB binding motif of DEFB4 enhances NTHi-induced DEFB4 regulation in epithelial cells.     

Simulation of sequential batch reactor (SBR) operation for simultaneous removal of nitrogen and phosphorus

Modeling of the operation of sequential batch reactor (SBR) was performed to find out optimum design parameters for simultaneous removal of nitrogen and phosphorus in a small-scale wastewater treatment plant. The models were set up with material balances on SBR operation and Monod kinetics. The model parameters were obtained to best fit the experimental results in a small scale SBR. The models were useful in optimizing hydraulic retention time (HRT) and successfully simulated operations of SBR in a larger scale. Especially the model predicted well the reactions occurring in the filling period as well as the effect of dilution, and evaluated the performance of SBR process under diverse operating conditions.