• Journal of Veterinary Science
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• 제21권1호
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• pp.7.1-7.13
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• 2020

Aging triggers cellular and molecular alterations, including genomic instability and organ dysfunction, which increases the risk of disease in mammals. Recently, due to the markedly growing number of aging dogs in the world, as much as 49% in total number of pet dogs, it is necessary to improve and maintain their quality of life by understanding of the biological effects of aging. Therefore, the aim of this study was to determine specific biomarkers in aging dogs as a means of defining a set of hematological/biochemical biomarkers that influence the aging process. Blood samples were collected from younger (1-3 years) and older (7-10 years) dogs of middle/large size. The hematological/biochemistry analysis was performed to evaluate parameters significantly associated with age. Enzyme-linked immunosorbent assay was used to target growth hormone (GH)/insulin growth factor-1 (IGF-1), one of the main regulators of the aging process. Declining levels of total protein and increased levels of glucose in young dogs was observed regardless of their body size. Notably, a significantly high concentration of GH and IGF-1 in the younger dogs compared to the older dogs was found in middle/large-sized dogs. GH and IGF-1 were also found at significantly high levels in large-sized dogs compared to middle-sized dogs, suggesting a similar trend to that of elderly humans. Consequently, glucose, total protein, GH, and IGF-1 were identified as potential biomarkers for regulating the aging process in large/middle-sized dogs. These findings provide an invaluable insight into the mechanism of aging for the field of aging research.

• Journal of Microbiology and Biotechnology
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• 제31권10호
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• pp.1438-1445
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• 2021

A bifunctional glycoside hydrolase GH78 from the ascomycete Xylaria polymorpha (XpoGH78) possesses catalytic versatility towards both glycosides and esters, which may be advantageous for the efficient degradation of the plant cell-wall complex that contains both diverse sugar residues and esterified structures. The contribution of XpoGH78 to the conversion of lignocellulosic materials without any chemical pretreatment to release the water-soluble aromatic fragments, carbohydrates, and methanol was studied. The disintegrating effect of enzymatic lignocellulose treatment can be significantly improved by using different kinds of hydrolases and phenoloxidases. The considerable changes in low (3 kDa), medium (30 kDa), and high (> 200 kDa) aromatic fragments were observed after the treatment with XpoGH78 alone or with this potent cocktail. Synergistic conversion of rape straw also resulted in a release of 17.3 mg of total carbohydrates (e.g., arabinose, galactose, glucose, mannose, xylose) per gram of substrate after incubating for 72 h. Moreover, the treatment of rape straw with XpoGH78 led to a marginal methanol release of approximately 17 ㎍/g and improved to 270 ㎍/g by cooperation with the above accessory enzymes. In the case of beech wood conversion, the combined catalysis by XpoGH78 and laccase caused an effect comparable with that of fungal strain X. polymorpha in woody cultures concerning the liberation of aromatic lignocellulose fragments.

• Asian-Australasian Journal of Animal Sciences
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• 제29권10호
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• pp.1508-1514
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• 2016

A series of antagonists specifically targeting growth hormone receptors (GHR) in different species, such as humans, rats, bovines, and mice, have been designed; however, there are currently no antagonists that target the porcine growth hormone (GH). Therefore, in this study, we developed and characterized a porcine GHR (pGHR) antibody antagonist (denoted by AN98) via the hybridoma technique. The results from enzyme-linked immunosorbent assay, fluorescence activated cell sorter, indirect immunoinfluscent assay, and competitive receptor binding analysis showed that AN98 could specifically recognize pGHR, and further experiments indicated that AN98 could effectively inhibit pGH-induced signalling in CHO-pGHR cells and porcine hepatocytes. In addition, AN98 also inhibited GH-induced insulin-like growth factor-1 (IGF-1) secretion in porcine hepatocytes. In summary, these findings indicated that AN98, as a pGHR-specific antagonist, has potential applications in pGH-pGHR-related research on domestic pigs.

• Asian-Australasian Journal of Animal Sciences
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• 제18권5호
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• pp.741-746
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• 2005

To investigate the basic information and the possibility of ACE-inhibitory peptides for antihypertension materials, goat's caisin (CN) was hydrolyzed by various proteolytic enzymes and ACE-inhibitory peptides were separated and purified. ACE-inhibition ratios of enzymatic hydrolysates of goat's CN and various characteristics of ACE-inhibitory peptides were determined. ACE-inhibition ratios of goat's CN hydrolysates were shown the highest with 87.84% by pepsin for 48 h. By Sephadex G-25 gel chromatograms, Fraction 3 from goat's CN hydrolysates by pepsin for 48 h was confirmed the highest ACE-inhibition activity. Fraction 3 g and Fraction 3 gh from peptic hydrolysates by RP-HPLC to first and second purification were the highest in ACE-inhibition activity, respectively. The most abundant amino acid was leucine (18.83%) in Fraction 3 gh of ACE-inhibitory peptides after second purification. Amino acid sequence analysis of Fraction 3 gh of ACE-inhibitory peptides was shown that the Ala-Tyr-Phe-Tyr, Pro-Tyr-Tyr and Tyr-Leu. IC$_{50}$ calibrated in peptic hydrolysates at 48 h, Fraction 3, Fraction 3 g and Fraction 3 gh from goat's CN hydrolysates by pepsin for 48 h were 29.89, 3.07, 1.85 and 0.87 g/ml, respectively. Based on the results of this experiment, goat's CN hydrolysates by pepsin were shown to have ACE-inhibitory activity.

• BMB Reports
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• 제40권5호
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• pp.791-800
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• 2007

Ethylene performs an important function in plant growth and development. 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS), the key enzyme involved in ethylene biosynthesis, has been the focus of most ethylene studies. Here, a cotton ACS gene referred to as Gossypium hirsutum ACS1 (GhACS1), was isolated. The full-length cDNA of GhACS1 encodes for a 476-amino acid protein which harbors seven conserved regions, 11 invariant amino acid residues, and the PLP binding active site, all of which characterize ACC synthases. Alignment analysis showed that GhACS1 shared a high degree of identity with other known ACC synthases from different species. Two introns were detected in the genomic DNA sequence, and the results of Southern blot analysis suggested that there might be a multi-gene family encoding for ACC synthase in cotton. From the phylogenetic tree constructed with 24 different kinds of ACC synthases, we determined that GhACS1 falls into group II, and was closely associated with the wound-inducible ACS of citrus. The analysis of the 5' flanking region of GhACS1 revealed a group of putative cis-acting elements. The results of expression analysis showed that GhACS1 displayed its transient expression nature after wounding, abscisic acid (ABA), and $CuCl_2$ treatments. These results indicate that GhACS1, which was transiently expressed in response to certain stimuli, may be involved in the production of ethylene for the transmission of stress signals.

• Journal of Microbiology and Biotechnology
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• 제29권2호
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• pp.235-243
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• 2019

A special eosinophilic agarase exo-type ${\beta}$-agarase gene, AgaDL6, was cloned from a marine agar-degrading bacterium, Flammeovirga sp. HQM9. The gene comprised 1,383-bp nucleotides encoding a putative agarase AgaDL6 of 461 amino acids with a calculated molecular mass of 52.8 kDa. Sequence analysis revealed a ${\beta}$-agarase domain that belongs to the glycoside hydrolase family (GH) 16 and a carbohydrate-binding module (CBM_4_9) unique to agarases. AgaDL6 was heterologously expressed in Escherichia coli BL21 (DE3). Enzyme activity analysis of the purified protein showed that the optimal temperature and pH of AgaDL6 were $50^{\circ}C$ and 3.0, respectively. AgaDL6 showed thermal stability by retaining more than 98% of activity after incubation for 2 h at $50^{\circ}C$, a feature quite different from other agarases. AgaDL6 also exhibited outstanding acid stability, retaining 100% of activity after incubation for 24 h at pH 2.0 to 5.0, a property distinct from other agarases. This is the first agarase characterized to have such high acid stability. In addition, we observed no obvious stimulation or inhibition of AgaDL6 in the presence of various metal ions and denaturants. AgaDL6 is an exo-type ${\beta}$-1,4 agarase that cleaved agarose into neoagarotetraose and neoagarohexaose as the final products. These characteristics make AgaDL6 a potentially valuable enzyme in the cosmetic, food, and pharmaceutical industries.

• Journal of Microbiology and Biotechnology
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• 제22권4호
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• pp.501-509
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• 2012

A 990 bp full-length gene (xynAHJ2) encoding a 329-residue polypeptide (XynAHJ2) with a calculated mass of 38.4 kDa was cloned from Bacillus sp. HJ2 harbored in a saline soil. XynAHJ2 showed no signal peptide, distinct amino acid stretches of glycoside hydrolase (GH) family 10 intracellular endoxylanases, and the highest amino acid sequence identity of 65.3% with the identified GH 10 intracellular mesophilic endoxylanase iM-KRICT PX1-Ps from Paenibacillus sp. HPL-001 (ACJ06666). The recombinant enzyme (rXynAHJ2) was expressed in Escherichia coli and displayed the typical characteristics of low-temperature-active enzyme (exhibiting optimum activity at $35^{\circ}C$, 62% at $20^{\circ}C$, and 38% at $10^{\circ}C$; thermolability at ${\geq}45^{\circ}C$). Compared with the reported GH 10 low-temperature-active endoxylanases, which are all extracellular, rXynAHJ2 showed low amino acid sequence identities (<45%), low homology (different phylogenetic cluster), and difference of structure (decreased amount of total accessible surface area and exposed nonpolar accessible surface area). Compared with the reported GH 10 intracellular endoxylanases, which are all mesophilic and thermophilic, rXynAHJ2 has decreased numbers of arginine residues and salt bridges, and showed resistance to $Ni^{2+}$, $Ca^{2+}$, or EDTA at 10 mM final concentration. The above mechanism of structural adaptation for low-temperature activity of intracellular endoxylanase rXynAHJ2 is different from that of GH 10 extracellular low-temperature-active endoxylanases. This is the first report of the molecular and biochemical characterizations of a novel intracellular low-temperature-active xylanase.

• Journal of Microbiology and Biotechnology
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• 제20권10호
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• pp.1415-1423
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• 2010

An extracellular xylanase was purified to homogeneity by sequential chromatography of Fomitopsis pinicola culture supernatants on a DEAE-Sepharose column, a gel filtration column, and then on a MonoQ column with fast protein liquid chromatography. The relative molecular mass of the F. pinicola xylanase was determined to be 58 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by size-exclusion chromatography, indicating that the enzyme is a monomer. The hydrolytic activity of the xylanase had a pH optimum of 4.5 and a temperature optimum of $70^{\circ}C$. The enzyme showed a $t_{1/2}$ value of 33 h at $70^{\circ}C$ and catalytic efficiency ($k_{cat}=77.4\;s^{-1}$, $k_{cat}/K_m$=22.7 mg/ml/s) for oatspelt xylan. Its internal amino acid sequences showed a significant homology with hydrolases from glycoside hydrolase (GH) family 10, indicating that the F. pinicola xylanase is a member of GH family 10.

• Journal of Microbiology and Biotechnology
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• 제19권9호
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• pp.987-996
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• 2009

Aspergillus niger GH1 previously isolated and identified by our group as a wild tannase producer was grown under solid-state (SSC) and submerged culture (SmC) conditions to select the enzyme production system. For tannase purification, extracellular tannase was produced under SSC using polyurethane foam as the inert support. Tannase was purified to apparent homogeneity by ultrafiltration, anion-exchange chromatography, and gel filtration that led to a purified enzyme with a specific activity of 238.14 IU/mg protein with a final yield of 0.3% and a purification fold of 46. Three bands were found on the SDS-PAG with molecular masses of 50, 75, and 100 kDa. PI of 3.5 and 7.1% N-glycosylation were noted. Temperature and pH optima were 600e and 6.0 [methyl 3,4,5-trihydroxybenzoate (MTB) as substrate], respectively. Tannase was found with a $K_M$ value of $0.41{\times}10^{-4}M$ and the value of $V_{max}$ was $11.03{\mu}$moL/min at $60^{\circ}C$ for MTB. Effects of several metal salts, solvents, surfactants, and typical enzyme inhibitors on tannase activity were evaluated to establish the novelty of the enzyme. Finally, the tannase from A. niger GH1 was significantly inhibited by PMSF (phenylmethylsulfonyl fluoride), and therefore, it is possible to consider the presence of a serine or cysteine residue in the catalytic site.

• 미생물학회지
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• 제52권4호
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• pp.463-470
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• 2016

Xylan의 효소적 가수분해는 고부가가치 기능성 물질 또는 바이오에너지 생산을 위한 발효성 당을 얻는 가장 유용한 방법 중 하나이다. endo-${\beta}$-Xylanase는 xylan 주사슬 내부의 ${\beta}$-1,4-결합을 가수분해하여 xylobiose, xylotriose를 포함한 다양한 XOS를 생산하는 핵심 효소이다. 이들 효소 중에서 glucuronoxylanase GH30은 methylglucuronic acid가 측쇄에 수식된 xylan에 특이적으로 작용한다. 본 연구에서는 Paenibacillus amylolyticus KCTC 3005에서 유래한 2종의 xylan 가수분해효소(PaXN_10과 PaGuXN_30) 유전자를 클로닝하고, Escherichia coli에서 각각 발현시켰다. PaXN_10 (38.7 kDa)은 ${\beta}$-xylanase GH10 계열, PaGuXN_30 (58.5 kDa)은 glucuronoxylanase GH30에 해당하는 효소이며, $50^{\circ}C$와 pH 7.0에서 최대 활성을 나타내었다. 가수분해 특성 연구를 통해 P. amylolyticus가 목질계 glucuronoxylan을 분해하는 효소 시스템을 제안하였다. 세포 외로 분비되는 PaGuXN_30은 glucuroxylan을 가수분해하여 methylglucuronic acid 측쇄를 가지는 다양한 aldouronic acid mixtures를 생성하며, 이러한 분해산물은 세포 내로 이동하여 PaXN_GH10에 의해 xylose, xylobiose와 같은 저분자 XOS로 분해되어 세포 내 대사경로에 이용될 수 있다. 또한 이들 효소의 가수분해특성을 이용하여 다양한 탄수화물 소재 생산이 가능할 것으로 기대한다.