• BMB Reports
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• v.41 no.1
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• pp.72-78
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• 2008

Human NeuNAc-9-P synthase is a two-domain protein with ability to synthesize both NeuNAc-9-P and KDN-9-P. Its mouse counterpart differs by only 20 out of 359 amino acids but does not produce KDN-9-P. By replacing the AFL domain of the human NeuNAc-9-P synthase which accommodates 12 of these differences, with the mouse AFL domain we examined its importance for the secondary KDN-9-P synthetic activity. The chimeric protein retained almost half of the ability of the human enzyme for KDN-9-P synthesis while the NeuNAc-9-P production was reduced to less than 10%. Data from the homology modeling and the effect of divalent ions and temperature on the enzyme activities suggest conformational differences between the human and mouse AFL domains that alter the shape of the cavity accommodating the substrates. Therefore, although the AFL domain itself does not define the ability of the human enzyme for KDN-9-P synthesis, it is important for both activities by aiding optimal positioning of the substrates.

• Journal of Microbiology and Biotechnology
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• v.9 no.6
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• pp.839-846
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• 1999

An extracellular chitinase-producing bacterial strain induced by colloidal chitin was isolated from sea sand and was identified to be a member of the genus Cytophaga. The chitinase was purified successively by 30-60% ammonium sulfate fractionation, and DEAE-Bio gel A column, Octyl-Sepharose CL-4B column, and DEAE-Bio gel A column chromatographies. The enzyme had a molecular mass of 59.75 kDa, and the amino terminal amino acid sequence was ATPNAPVISW MPTDXXLQNXS. The enzyme acted better on colloidal chitin as a substrate than on chitosan. For colloidal chitin and chitosan (Degree of Acetylation, 15-25%), $K_{cat}$ values were 0.60U/mg and 0.08U/mg, respectively. HPLC analysis of the enzymatic reaction products showed that the chitinase produced mostly N-acetyl-D-glucosarnine and di-N-acetylchitobiose. The optimum temperature and pH for the enzyme were $50^{\circ}C$ and 4.0, respectively. N-Bromosuccinimide and $Hg^{2+}$ inhibited the chitinase activity as much as 90%, and $Sb^{3+}$, diethylpyrocarbonate, and $Ag^{+}$ inhibited it by 50-70%.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.10
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• pp.1524-1528
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• 2003

The present study was conducted to evaluate the influence of dietary fiber on the activities of malic enzyme and citrate lyase involved in fatty acid metabolism in the colon epithelium of pigs. Thirty-six weaned 5 weeks old crossbred (Yorkshire${\times}$Swedish Landrace) piglets originating from twelve litters were randomly assigned to either a low fiber diet containing 10% non-starch polysaccharides (NSP), a control diet containing 14.7% NSP or a high fiber diet containing 20% NSP. The activity of malic enzyme in the colonic epithelium of pigs significantly (p<0.05) increased with age during the suckling-weaning transition. There was a tendency (p<0.10) of decreased malic enzyme activity in the colonic epithelium of pigs fed on the high fiber diet. At week 6, a lowered (p<0.01) activity of malic enzyme in pigs fed on the low fiber diet compared with that in pigs fed on the high fiber and the control diets. Nevertheless, there were no significant differences in the activity of citrate lyase observed either between pigs with different ages or between pigs fed with various diets. The current data suggest that piglets during the suckling-weaning transition have a limited capacity to synthesize fatty acids from carbohydrate derivatives in the coloncytes. In addition, lipogenesis in coloncytes was enhanced with age during the suckling-weaning transition. A tendency (p<0.10) to an increased capacity to utilize acetyl-CoA in coloncytes of pigs has been observed for the high fiber diet. Moreover, the present work indicated that dietary fiber resulted in a lowered rate of lipogenesis and a reduced activity of malic enzyme.

• BMB Reports
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• v.32 no.4
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• pp.414-418
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• 1999

A novel gene for malonyl-CoA decarboxylase was discovered in the mat operon, which encodes a set of genes involved in the malonate metabolism of Rhizobium trifolii (An and Kim, 1998). The subunit mass determined by SDS-PAGE was 53 kDa, which correspond to the deduced mass from the sequence data. The molecular mass of the native enzyme determined by field flow fractionation was 208 kDa, indicating that R. trifolii malonyl-CoA decarboxylase is homotetrameric. R. trifolii malonyl-CoA decarboxylase converted malonyl-CoA to acetyl-CoA with a specific activity of 100 unit/mg protein. Methylmalonyl-CoA was decarboxylated with a specific activity of 0.1 unit/mg protein. p-Chloromercuribenzoate inhibited this enzyme activity, suggesting that thiol group(s) is(are) essential for this enzyme catalysis. Database analysis showed that malonyl-CoA decarboxylase from R. trifolii shared 32.7% and 28.1% identity in amino acid sequence with those from goose and human, respectively, and it would be located in the cytoplasm. However, there is no sequence homology between this enzyme and that from Saccharopolyspora erythreus, suggesting that malonyl-CoA decarboxylases from human, goose, and R. trifolii are in the same class, whereas that from S. erythreus is in a different class or even a different enzyme, methylmalonyl-CoA decarboxylase. According to the homology analysis, Cys-214 among three cysteine residues in the enzyme was found in the homologous region, suggesting that the cysteine was located at or near the active site and plays a critical role in catalysis.

• Microbiology and Biotechnology Letters
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• v.25 no.4
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• pp.414-419
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• 1997

In order to develop an enzyme-linked immunosorbent assay (ELISA) for deoxynivalenol (DON) in com, we produced a specific monocl- onal antibody and established ELISA conditions. After the spleen cells from mice immunized with DON-bovine serum albumin conjugate were fused with S$_{p}$2/0 myeloma cells, a hybridoma cell 3G7 producing anti-DON antibody was screened by ELISA. From the standard curve of competitive direct ELISA (cdELISA) using 3G7 monoclonal antibody and DON-HRP conjugate, the detection range of DON showed 3-3,000 ng/ml (ppb). The monoclonal antibody showed some cross-reactivities against DON analogues such as 15 acetyl-DON (110%), nivalenol (5.0%), 3 acetyl-DON (1.7%), fusarenon-x (0.72%), and T-2 (0.59%). When the cdELISA was applied to the spiked coms after extracting with 60% methanol and diluting 5- fold with washing buffer, the assay recoveries of DON were 313, 163, 106, and 88.9% (av., 168%) in the levels of 200, 600, 2,000, and 6,000ng/g, respectively. For the quantitation of DON in coms, 30 samples kept under two different storage conditions of cold and room temperature were assayed by cdELISA. The mean detection concentrations were 595 (detection range, 0-2,750) and 2,448 (detection range, 0-4,500) ppb, respectively.

• Clinical and Experimental Pediatrics
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• v.59 no.sup1
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• pp.37-40
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• 2016

Mucopolysaccharidosis type III (MPS III) is a rare genetic disorder caused by lysosomal storage of heparan sulfate. MPS IIIB results from a deficiency in the enzyme alpha-N-acetyl-D-glucosaminidase (NAGLU). Affected patients begin showing behavioral changes, progressive profound mental retardation, and severe disability from the age of 2 to 6 years. We report a patient with MPS IIIB with a long-term follow-up duration. He showed normal development until 3 years. Subsequently, he presented behavioral changes, sleep disturbance, and progressive motor dysfunction. He had been hospitalized owing to recurrent pneumonia and epilepsy with severe cognitive dysfunction. The patient had compound heterozygous c.1444C>T (p.R482W) and c.1675G>T (p.D559Y) variants of NAGLU. Considering that individuals with MPS IIIB have less prominent facial features and skeletal changes, evaluation of long-term clinical course is important for diagnosis. Although no effective therapies for MPS IIIB have been developed yet, early and accurate diagnosis can provide important information for family planning in families at risk of the disorder.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.6
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• pp.744-751
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• 2011

Meat production and quality traits in beef cattle are largely affected by genetic factors. Acetyl-Coenzyme A carboxylase-${\alpha}$ (ACACA) plays a key role in the regulation and metabolism of fatty acid biosynthesis in mammalian animals. The gene encoding ACACA enzyme was chosen as a candidate gene for carcass and meat traits. In this study, we investigated effects of single nucleotide polymorphisms (SNPs) in the ACACA gene on beef carcass and meat traits in Hanwoo (Korean cattle) populations. We have sequenced a fragment of intron I region of the Hanwoo ACACA gene and identified two SNPs. Genotyping of the two SNP markers (g.2344T>C and g.2447C>A) was carried out using PCR-SSCP analysis in 309 Hanwoo steers to evaluate their association with carcass and meat production traits. The g.2344C SNP marker showed a significant increasing effect on LW (p = 0.009) and CW (p = 0.017). Animals with the CC genotype had higher CW and LW compared with TT and TC genotypes (p<0.05). The g.2447A SNP marker was associated with higher MC (p = 0.019). Animals with the AA genotype had higher MC than animals with CC and CA genotypes (p<0.05). Although the degree of linkage disequilibrium (LD) was not strong between g.2344T>C and g.2447C>A in the LD analysis, four major haplotype classes were formed with two SNP information within the ACACA gene. We constructed haplotypes using the HaploView software package program and analyzed association between haplotypes and carcass traits. The haplotype of ACACA gene significantly affected the LW (p = 0.027), CW (p = 0.041) and MC (p = 0.036). The effect of h1 haplotype on LW and CW was larger than that of h3 haplotype. Animals with the h1 haplotype also had greater MC than did animals with h2 haplotype. Consequently, the ACACA gene could be useful as a DNA marker for meat production traits such as carcass yield and meat contents in Hanwoo.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.11
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• pp.1576-1584
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• 2016

Acetyl xylan esterase (AXE), which hydrolyzes the ester linkages of the naturally acetylated xylan and thus known to have an important role for hemicellulose degradation, was isolated from the anaerobic rumen fungus Neocallimastix frontatlis PMA02, heterologously expressed in Escherichi coli (E.coli) and characterized. The full-length cDNA encoding NfAXE1 was 1,494 bp, of which 978 bp constituted an open reading frame. The estimated molecular weight of NfAXE1 was 36.5 kDa with 326 amino acid residues, and the calculated isoelectric point was 4.54. The secondary protein structure was predicted to consist of nine ${\alpha}$-helixes and 12 ${\beta}$-strands. The enzyme expressed in E.coli had the highest activity at $40^{\circ}C$ and pH 8. The purified recombinant NfAXE1 had a specific activity of 100.1 U/mg when p-nitrophenyl acetate (p-NA) was used as a substrate at $40^{\circ}C$, optimum temperature. The amount of liberated acetic acids were the highest and the lowest when p-NA and acetylated birchwood xylan were used as substrates, respectively. The amount of xylose released from acetylated birchwod xylan was increased by 1.4 fold when NfAXE1 was mixed with xylanase in a reaction cocktail, implying a synergistic effect of NfAXE1 with xylanase on hemicellulose degradation.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.3
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• pp.379-385
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• 2011

Ferulic acid esterase (FAE) and acetyl esterase (AE) cleave feruloyl groups substituted at the 5'-OH group of arabinosyl residues and acetyl groups substituted at O-2/O-3 of the xylan backbone, respectively, of arabinoxylans in the cell wall of grasses. In this study, the enzyme profiles of FAE, AE and polysaccharide hydrolases of the anaerobic rumen fungus Neocallimastix sp. YQ1 grown on Chinese wildrye grass hay (CW) or alfalfa hay (AH) were investigated by two $2{\times}4$ factorial experiments, each in 10-day pure cultures. The treatments consisted of two glucose levels ($G^+$: glucose at 1.0 g/L, $G^-$: no glucose) and four N sources (N1: 1.0 g/L yeast extract, 1.0 g/L tryptone and 0.5 g/L $(NH_4)_2SO_4$; N2: 2.8 g/L yeast extract and 0.5 g/L $(NH_4)_2SO_4$; N3: 1.6 g/L tryptone and 0.5 g/L $(NH_4)_2SO_4$; N4: 1.4 g/L tryptone and 1.7 g/L yeast extract) in defined media. The optimal combinations of glucose level and N source for the fungus on CW, instead of AH, were $G^-N4$ and $G^-N3$ for maximum production of FAE and AE, respectively. Xylanase activity peaked on day 4 and day 6 for the fungus grown on CW and AH, respectively. The activities of esterases were positively correlated with those of xylanase and carboxymethyl cellulase. The fungus grown on CW exhibited a greater volatile fatty acid production than on AH with a greater release of ferulic acid from plant cell wall.

• Korean Journal of Environmental Biology
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• v.29 no.4
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• pp.258-264
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• 2011

Glutathione (GSH) has important roles in cellular defense against oxidative stress, 1) direct scavenging of reactive oxygen species (ROS), and 2) coenzyme of ROS scavenging enzyme like glutathione peroxidases (GPx). GSH peroxidase reduces free hydrogen peroxide to water using 2GSH. $N$-acetyl-L-cysteine (NAC), one of the antioxidants, is used as a precursor for intracellular GSH. In this study, relation of GSH, NAC, and GSH peroxidase was investigated through transcriptional expression of $GPX1$ and $GPX2$, which are GSH peroxidase encoding genes, in yeast cells treated with 0 mM to 20 mM of NAC or in combination with 100 Gy gamma-rays. The transcriptional expression of $GPX1$ and $GPX2$ was induced by NAC and 100 Gy gamma-rays. The gene expression of both GSH peroxidases was decreased with increasing concentrations of NAC in irradiated yeast cells. These results suggest that elevation of intracellular GSH by NAC and oxidative stress and ROS generated from gamma-rays induces expression of GSH peroxidase genes, and that NAC can protect the yeast cells against ROS generated from gamma-rays through direct scavenging of ROS and transcriptional activation of GSH peroxidase.