• Journal of Life Science
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• v.31 no.3
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• pp.356-365
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• 2021

Recently, we sequenced the entire genome of a freshwater agar-degrading bacterium Cellvibrio sp. KY-GH-1 (KCTC13629BP) to explore genetic information encoding agarases that hydrolyze agarose into monomers 3,6-anhydro-L-galactose (L-AHG) and D-galactose. The KY-GH-1 strain appeared to possess nine β-agarase genes and two α-neoagarobiose hydrolase (α-NABH) genes in a 77-kb agarase gene cluster. Based on these genetic information, the KY-GH-1 strain-caused agarose degradation into L-AHG and D-galactose was predicted to be initiated by both endolytic GH16 and GH86 β-agarases to generate NAOS (NA4/NA6/NA8), and further processed by exolytic GH50 β-agarases to generate NA2, and then terminated by GH117 α-NABHs which degrade NA2 into L-AHG and D-galactose. More recently, by employing E. coli expression system with pET-30a vector we obtained three recombinant His-tagged GH50 family β-agarases (GH50A, GH50B, and GH50C) derived from Cellvibrio sp. KY-GH-1 to compare their enzymatic properties. GH50A β-agarase turned out to have the highest exolytic β-agarase activity among the three GH50 isozymes, catalyzing efficient NA2 production from the substrate (agarose, NAOS or AOS). Additionally, we determined that GH117A α-NABH, but not GH117B α-NABH, could potently degrade NA2 into L-AHG and D-galactose. Sequentially, we examined the enzymatic characteristics of GH50A β-agarase and GH117A α-NABH, and assessed their efficiency for NA2 production from agarose and for production of L-AHG and D-galactose from NA2, respectively. In this review, we describe the benefits of recombinant GH50A β-agarase and GH117A α-NABH originated from Cellvibrio sp. KY-GH-1, which may be useful for the enzymatic hydrolysis of agarose for mass production of L-AHG and D-galactose.

• Journal of Marine Bioscience and Biotechnology
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• v.8 no.1
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• pp.1-9
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• 2016

The hydrolysis of biomass to fermentable sugar (saccharification) and to oligosaccharide is an essential process in biotechnology including biorefinery and biofood. Various macroalgae are commercially cultivated in several Asian countries as a useful resource for food and agar production. Agar is a major component of the cell walls of red algae that can be hydrolyzed by agarase. Agarases are classified into ${\alpha}$-agarase (E.C. 3.2.1.158) and ${\beta}$-agarase (E.C. 3.2.1.81) according to the cleavage pattern and grouped in the glycoside hydrolase (GH) family (GH-16, GH-58, GH-86, GH-96, and GH-118) based on the amino acid sequences of the proteins. Agarases have been isolated from various bacteria found in seawater and marine sediments. To increase productivity of the enzyme, a research on recombinant enzymes has been done. The application of recombinant agarase can be possible in the various filed such as energy, food, cosmetics, medical and so on. This paper reviews the source, biochemical characteristics and production system of recombinant agarases for further study.

• Journal of Life Science
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• v.22 no.2
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• pp.266-280
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• 2012

Agar is a cell wall component of macro red algae that can be hydrolyzed by agarase. Agarases are classified into ${\alpha}$-agarase (E.C. 3.2.1.158) and ${\beta}$-agarase (E.C. 3.2.1.81), in accordance with their cleavage pattern, and can be grouped in the glycoside hydrolase (GH)-16, -58, -86, -96, and -118 family according to the amino acid sequences of the proteins. Many agarases and/or their genes have been detected, isolated, and recombinantly expressed from bacteria, and metagenomes have their origins in sea and terrestrial environments. Products of agarases, agarooligosaccharides and neoagarooligosaccharides, represent wide functions such as antitumor, immune stimulation, antioxidation, prebiotic, hepa-protective, antibacterial, whitening, and moisturizing effects; hence, broad applications would be possible in the food industry, cosmetics, and medical fields. In addition, agarases are also used as a tool enzyme for research. This paper reviews the sources, purifications and detection methods, and application fields of agarases. The role of agarases in agar metabolism and the function of their enzymatic products are also surveyed.

• Journal of Microbiology and Biotechnology
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• v.28 no.2
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• pp.284-292
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• 2018

A novel ${\beta}$-agarase, AgaJ5, was identified from an agar-degrading marine bacterium, Gayadomonas joobiniege G7. It belongs to the glycoside hydrolase family 86 and is composed of 805 amino acids with a 30-amino-acid signal peptide. Zymogram analysis showed that purified AgaJ5 has agarase activity. The optimum temperature and pH for AgaJ5 activity were determined to be $30^{\circ}C$ and 4.5, respectively. AgaJ5 was an acidic ${\beta}$-agarase that had strong activity at a narrow pH range of 4.5-5.5, and was a cold-adapted enzyme, retaining 40% of enzymatic activity at $10^{\circ}C$. AgaJ5 required monovalent ions such as $Na^+$ and $K^+$ for its maximum activity, but its activity was severely inhibited by several metal ions. The $K_m$ and $V_{max}$ of AgaJ5 for agarose were 8.9 mg/ml and 188.6 U/mg, respectively. Notably, thin-layer chromatography, mass spectrometry, and agarose-liquefication analyses revealed that AgaJ5 was an endo-type ${\beta}$-agarase producing neoagarohexaose as the final main product of agarose hydrolysis. Therefore, these results suggest that AgaJ5 from G. joobiniege G7 is a novel endo-type neoagarohexaose-producing ${\beta}$-agarase having specific biochemical features that may be useful for industrial applications.

• Childhood Kidney Diseases
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• v.10 no.2
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• pp.142-151
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• 2006

Purpose : Growth retardation is one of the serious problems in children with nephropathy requiring long-term steroid therapy. We observed the efficacy and safety of recombinant human growth hormone(rhGH) on the growth in children with long-term steroid therapy. Methods : We studied 60 children(male 47, female 13) with nephropathy who received rhGH(1 U/kg/week) for more than 0.5 years($1.39{\pm}1.12$). Their mean age was 11.0 years($11.17{\pm}2.62$). They received steroid therapy from January 1987 through July 2005, and the mean duration of steroid therapy was $4.32{\pm}2.97$ years. Among the patients, there were 32 nephrotic syndrome, 9 IgA nephropathy, 4 mesangial proliferative glomerulonephritis, 4 focal segmental glomerulosclerosis, 2 Henoch $Sch\ddot{o}nlein$ nephritis, 2 Alport syndrome and 7 other cases. Data were gathered on the growth parameters, such as growth velocity, height standard deviation score(SDS), IGF-1, IGFBP-3, bone mass density(BMD) and general chemistry changes. Results : Height velocity increased significantly with rhGH therapy from $3.29{\pm}1.95$ to $8.66{\pm}3.75$(cm/yr) and height SDS decreased from $-0.72{\pm}0.93$ to $-1.04{\pm}0.86$ at one year after steroid therapy but increased to $-0.55{\pm}0.96$ at one year after rhGH administration(P<0.05). BMD improved from $0.71{\pm}0.14$ to $0.79{\pm}0.15g/cm^2$(P<0.05). IGF-1 increased from $445.09{\pm}138.01$ to $506.62{\pm}181.31ng/mL$(P<0.05). IGFBP-3 decreased from $4073.75{\pm}700.78$ to $3933.61{\pm}789.25ug/L$ numerically, but there was no statistically significant difference(P=0.533). Conclusion : The administration of rhGH in the short stature patients who received long-term steroid therapy showed improvement in growth parameters such as SDS, growth velocity, and BMD without significant side-effects or changes in the biochemical parameters.

• Journal of Animal Science and Technology
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• v.44 no.6
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• pp.693-700
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• 2002

The objects of this study were to estimate gene frequencies of the bovine growth hormone(bGH), and to investigate the relationship between the bGH polymorphism and semen characteristics in Hanwoo bull. One hundred nine heads of Hanwoo bulls were used to identify bGH genotypes by the PCR-RFLP, followed by digestion with Alu I restriction enzyme. The frequencies of leucine(Leu) and Valine(Val) alleles were 0.88 and 0.12, respectively. Observed number of LL, LV and VV genotypes were 83, 25 and 1, respectively. Semen characteristics(semen volume, sperm concentration) were analyzed by GH genotypes in 25,559 ejaculates of 109 heads. Although bGH genotypes showed no significant effects on semen characteristics, those of bulls with VV genotype were tended to be lower than those of other bulls with LL or LV genotypes. And, in 1998, total sperm number(60.47${\times}$$10^8$) of VV bulls were significantly lower(P<0.05) than those(86.21${\sim}$92.22${\times}$$10^8$) of other genotypes bulls. This results provide that the VV bull in bGH locus may be worse, under the LL and LV bulls on semen characteristics. However, the number of examined VV bulls was only one and further investigations are needed to confirm the results.

• The Transactions of The Korean Institute of Electrical Engineers
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• v.63 no.10
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• pp.1455-1460
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• 2014

Wind turbine generators were designed on general regulations of wind condition. At real situations, it could be different from the design conditions. There are many control methods and definitions of transient region, because an efficient wind turbine generator control logic is the important matter in generator performance and annual energy production at real conditions. In this document, the power generation sensitivity for wind speed and turbulence intensities was defined to know the sensitive transient region. Wind conditions are applied for the ranges of 7~10m/s mean wind speed and 14~20% turbulence intensity. The sensibility of HR-D86 wind generator was increased in transient region(8~10m/s) on power curve diagram through a torque control to a pitch control. And then GH-bladed simulations was performed for performance analysis of the torque mode switching in transient region on 2MW direct drive wind generator(HR-D86) which is designed IEC class II for onshore. Through the sensitivity and performance analysis, the sensitivity for real wind condition could be the performance index for an wind generator. And the torque mode switching in transient region can increase the mean power generation on HR-D86 wind turbine generator.

• Journal of Microbiology and Biotechnology
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• v.19 no.3
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• pp.257-264
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• 2009

A $\beta$-agarase gene, agaB34, was functionally cloned from the genomic DNA of a marine bacterium, Agarivorans albus YKW-34. The open reading frame of agaB34 consisted of 1,362 bp encoding 453 amino acids. The deduced amino acid sequence, consisting of a typical N-terminal signal peptide followed by a catalytic domain of glycoside hydrolase family 16 (GH-16) and a carbohydrate-binding module (CBM), showed 37-86% identity to those of agarases belonging to family GH-16. The recombinant enzyme (rAgaB34) with a molecular mass of 49 kDa was produced extracellularly using Escherichia coli $DH5{\alpha}$ as a host. The purified rAgaB34 was a $\beta$-agarase yielding neoagarotetraose (NA4) as the main product. It acted on neoagarohexaose to produce NA4 and neoagarobiose, but it could not further degrade NA4. The maximal activity of rAgaB34 was observed at $30^{\circ}C$ and pH 7.0. It was stable over pH 5.0-9.0 and at temperatures up to $50^{\circ}C$. Its specific activity and $k_{cat}/K_m$ value for agarose were 242 U/mg and $1.7{\times}10^6/sM$, respectively. The activity of rAgaB34 was not affected by metal ions commonly existing in seawater. It was resistant to chelating reagents (EDTA, EGTA), reducing reagents (DTT, $\beta$-mercaptoethanol), and denaturing reagents (SDS and urea). The E. coli cell harboring the pUC18-derived agarase expression vector was able to efficiently excrete agarase into the culture medium. Hence, this expression system might be used to express secretory proteins.

• KSBB Journal
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• v.15 no.1
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• pp.42-48
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• 2000

Urokinase (UK), a thrombolytic enzyme used to clear catheters obstructed by blood clots, can be also used industrially in the recombinant protein purification system to cleave a fusion protein linked with a certain fragment of GST. We have immobilized UK by covalent attachment to activated Sepharose 6B-Cl gels and evaluated its performance to cleave a fusion protein of hGH and GST. The Sepharose gels were activated by etherification with glycidol (2,3-epoxypropanol) and further oxidized with periodate resulting in glyceryl-Sepharose gels. After the activation treatment, surface density of the aldehyde groups was 7-30 $\mu$mol-aldehde/mL-gel. Immobilization yield was higher than 99% at high pH (10.5), and the immobilized UK maintained ca. 80% specific activity of the soluble UK. In a column reaction the cleavage yield heavily depended on the feed rate, and it was nearly 86% of that from soluble UK. And the immobilized UK was successfully regenerated by unfolding and refolding with 6M GuHCl. After cleavaging reaction, the monomeric hGH was purified by using expanded bed adsorption chromatography.

• Journal of Microbiology and Biotechnology
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• v.32 no.4
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• pp.447-457
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• 2022

Notoginsenoside R1 and ginsenoside Rg1 are the main active ingredients of Panax notoginseng, exhibiting anti-fatigue, anti-tumor, anti-inflammatory, and other activities. In a previous study, a GH39 β-xylosidase Xln-DT was responsible for the bioconversion of saponin, a natural active substance with a xylose group, with high selectivity for cleaving the outer xylose moiety of notoginsenoside R1 at the C-6 position, producing ginsenoside Rg1 with potent anti-fatigue activity. The optimal bioconversion temperature, pH, and enzyme dosage were obtained by optimizing the transformation conditions. Under optimal conditions (pH 6.0, 75℃, enzyme dosage 1.0 U/ml), 1.0 g/l of notoginsenoside R1 was converted into 0.86 g/l of ginsenoside Rg1 within 30 min, with a molar conversion rate of approximately 100%. Furthermore, the in vivo anti-fatigue activity of notoginsenoside R1 and ginsenoside Rg1 were compared using a suitable rat model. Compared with the control group, the forced swimming time to exhaustion was prolonged in mice by 17.3% in the Rg1 high group (20 mg/kg·d). Additionally, the levels of hepatic glycogen (69.9-83.3% increase) and muscle glycogen (36.9-93.6% increase) were increased. In the Rg1 group, hemoglobin levels were also distinctly increased by treatment concentrations. Our findings indicate that treatment with ginsenoside Rg1 enhances the anti-fatigue effects. In this study, we reveal a GH39 β-xylosidase displaying excellent hydrolytic activity to produce ginsenoside Rg1 in the pharmaceutical and food industries.