• Journal of Genetic Medicine
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• v.7 no.2
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• pp.119-124
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• 2010

More than 7,000 rare Mendelian diseases have been reported, but less than half of all rare monogenic disorders has been discovered. In addition, the majority of mutations that are known to cause Mendelian disorders are located in protein-coding regions. Therefore, exome sequencing is an efficient strategy to selectively sequence the coding regions of the human genome to identify novel genes associated with rare genetic disorders. The "exome" represents all of the exons in the human genome, constituting about 1.5% of the human genome. Exome sequencing is carried out by targeted capture and intense parallel sequencing. After the first report of successful exome sequencing for the identification of causal genes and mutations in Freeman Sheldon syndrome, exome sequencing has become a standard approach to identify genes in rare Mendelian disorders. Exome sequencing is also used to search the causal genes and variants in complex diseases. The successful use of exome sequencing in Mendelian disorders and complex diseases will facilitate the development of personalized genomic medicine.

• Microbiology and Biotechnology Letters
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• v.47 no.2
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• pp.259-268
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• 2019

In this study, the extracellular metalloprotease from Serratia marcescens PPB-26 was purified to homogeneity via ethanol fractionation and DEAE-cellulose column chromatography. Thus, a 3.8-fold purification was achieved with a 20% yield and specific activity of 76.2 U/mg. The purified protease was a 50-kDa monomer whose optimum pH and temperature for activity were 7.5 and $30^{\circ}C$ respectively; however, it was found to remain active in the 5-9 pH range and up to $40^{\circ}C$ for 6 h. The protease had a half-life of 15 days at $4^{\circ}C$, an optimum reaction time of 10 min, and an optimum substrate (casein) concentration of 0.25%. Furthermore, the Michaelis constant ($K_m$) and reaction velocity ($V_{max}$) of the protease were calculated to be 0.28% and $111.11{\mu}moles/(min{\cdot}mg)^{-1}$, respectively. The protease was stable when subjected to metal ions (2 mM), showing increased activity with most (especially $CoCl_2$ and $MgSO_4$ (30.54% increase)). It was also stable when exposed to oxidizing agents, bleaching agents, and detergents (5% v/v for 60 min). It retained 93% of its activity in non-ionic detergents (Tween-20, Tween-80, and Triton X-100). Moreover, wash performance analysis in commercial detergents (Ariel and Tide) showed that not only was the protease capable of protein stain removal, but also reduced cleaning time by 80% when added to detergents. Thus, the Serratia marcescens PPB-26 metalloprotease appears to be a promising new candidate as a laundry additive in the detergent industry.

• Journal of Microbiology and Biotechnology
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• v.34 no.2
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• pp.436-456
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• 2024

Several thermostable proteases have been identified, yet only a handful have undergone the processes of cloning, comprehensive characterization, and full exploitation in various industrial applications. Our primary aim in this study was to clone a thermostable alkaline protease from a thermophilic bacterium and assess its potential for use in various industries. The research involved the amplification of the SpSKF4 protease gene, a thermostable alkaline serine protease obtained from the Geobacillus thermoglucosidasius SKF4 bacterium through polymerase chain reaction (PCR). The purified recombinant SpSKF4 protease was characterized, followed by evaluation of its possible industrial applications. The analysis of the gene sequence revealed an open reading frame (ORF) consisting of 1,206 bp, coding for a protein containing 401 amino acids. The cloned gene was expressed in Escherichia coli. The molecular weight of the enzyme was measured at 28 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The partially purified enzyme has its highest activity at a pH of 10 and a temperature of 80℃. In addition, the enzyme showed a half-life of 15 h at 80℃, and there was a 60% increase in its activity at 10 mM Ca²⁺ concentration. The activity of the protease was completely inhibited (100%) by phenylmethylsulfonyl fluoride (PMSF); however, the addition of sodium dodecyl sulfate (SDS) resulted in a 20% increase in activity. The enzyme was also stable in various organic solvents and in certain commercial detergents. Furthermore, the enzyme exhibited strong potential for industrial use, particularly as a detergent additive and for facilitating the recovery of silver from X-ray film.

• Animal cells and systems
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• v.4 no.2
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• pp.157-163
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• 2000

Yeast pheromone a-factor is a 13-amino acid peptide hormone that is synthesized as a part of a larger precursor, prepro-$\alpha$-factor, consisting of a signal peptide and a proregion of 64 amino acids. The carboxy-terminal half of the precursor contains four tandem copies of mature $\alpha$-factor. To investigate the molecular basis of intracellular sorting, proteolytic processing, and storage of the peptide hormone, yeast prepro-$\alpha$-factor precursors were heterologously expressed in rat pituitary $GH_3 cells. When cells harboring the precursor were metabolically labeled, a species of approximately 27 kD appeared inside the cells. Digestion with peptide: N-glycosidase F (PNG-F) shifted the molecular mass to a 19 kD, suggesting that the 27 kD protein was the glycosylated form as in yeast cells. The nascent polypeptide is efficiently targeted to the ER in the $GH_3 cells, where it undergoes cleavage of its signal peptide and core glycosylation to generate glycosylated pro-a-factor. To look at the post ER intracellular processing, the pulse-labelled cells were chased up to 2 hrs. The nascent propeptides disappeared from the cells at a half life of 30 min and only 10-25% of the newly synthesized, unprocessed precursors were stored intracellularly after the 2 h chase. However, about 20% of the pulse-labeled pro-$\alpha$-factor precursors were secreted into the medium in the pro-hormone form. With increasing chase time, the intracellular level of propeptide decreased, but the amount of secreted propeptide could not account for the disappearance of intracellular propeptide completely. This disappearance was insensitive to lysosomotropic agents, but was inhibited at $16^{circ}C or 20^{\circ}C$, suggesting that the turnover of the precursors was not occurring in the secretory pathway to trans Golgi network (TGN) or dependent on acidic compartments. From these results, it is concluded that a pan of these heterologous precursors may be processed at its paired dibasic sites by prohormone processing enzymes located in TGN/secretpry vesicles producing small peptides, and that the residual unprocessed precursors may be secreted into the medium rather than degraded intracellularly.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.327-333
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• 2005

Dihydroxy-acid dehydratase (DHAD, 2,3-dihydroxy-acid hydrolyase, EC 4.2.1.9) is one of the key enzymes involved in the biosynthetic pathway of the branched chain amino acid isoleucine and valine. Although the enzyme have been purified and characterized in various mesophiles including bacteria and eukarya, the biochemical properties of DHAD has bee not yet reported from hyperthermophilic archaea. In this study, we cloned, expressed, and purified a DHAD homologue from the thermoacidophilic archaeon Sulfolobus solfataricus P2, which grows optimally at $80\;^{\circ}C$ and pH 3, in E. coli. Characterization of the recombinant S. solfataricus DHAD (rSso_DHAD) revealed that it is the dimeric protein with a subunit molecular weight of 64,000 Da in native structure. rDHAD showed the highest activity toward 2,3-dihydroxyisovaleric acid among 17 aldonic acid substrates Interestingly, this enzyme also displayed 50 % activities toward some pentonic acids and hexonic acids when compared with the activity of this enzyme to the natural substrate. Moreover, rSso_DHAD indicated relatively higher activity toward D-gluconate than any other hexonic acids tested in substrates. $K_m$ and $V_{max}$ values of rSso_DHAD were calculated as $0.54\;{\pm}\;0.04\;mM$ toward 2,3dihydroxyisovalerate and $2.42\;{\pm}\;0.19\;mM$ toward D-gluconate, and as $21.6\;{\pm}\;0.4\;U/mg$ toward 2,3-dihydroxyisovalerate and $13.8\;{\pm}\;0.4\;U/mg$ toward D-gluconate, respectively. In the study for biochemical properties, the enzyme shows maximal activity between $70^{\circ}C$ and $80^{\circ}C$, and the pH range of pH 7.5 to 8.5. The half life time at $80^{\circ}C$ was 30 min. A divalent metal ion, $Mn^{2+}$, was only powerful activators, whereas other metal ions made the enzyme activity reduced. $Hg^{2+}$, organic mercury, and EDTA also strongly inhibited enzyme activities. Particularly, the rSso_DHAD activity was very stable under aerobic condition although the counterparts reported from mesophiles had been deactivated by oxygen.

• The Korean Journal of Mycology
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• v.32 no.2
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• pp.112-118
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• 2004

Laccase produced by Trametes hirsuta S1 isolated from Korea was partially purified and characterized using ultrafiltration, anion exchange chromatography and affinity chromatography. The laccase was produced as the predominant extracellular enzyme during primary metabolism. Neither lignin peroxidase nor veratryl alcohol oxidase (VAO) were detected in the culture fluid. Addition of 2,5-xylidine enhanced 4-fold laccase production. Purified laccase was a single polypeptide having a molecular mass of approximately 66 kDa, as determined by SDS-polyacrylamide gel electrophoresis, and carbohydrate content of 12%. $K_{m}\;and\;V_{max}$ values for laccase with ABTS [2,2-azinobis (3-ethylbenzthiazoline 6-sulfonic acid)] as a substrate (Lineweaver-Burk plot) was determined to $51.2\;{\mu}M\;and\;56.8\;{\mu}mole$, respectively. The optimal pH for laccase activity was found to be 3.0. The enzyme was very stable for 1 hour at $50^{\circ}C$. Half-life ($t_{1/2}$) of the enzyme was about 20 min at $70^{\circ}C$. Spectroscopic analysis of purified enzyme indicated that the enzyme was typical of copper-containing protein. Substrate specificity and inhibitor studies for laccase also indicated to be a typical fungal laccase. The N-terminal amino acid sequence of the T. hirsuta S1 laccase showed 100% of homology to those of laccase from C. hirsutus.

• Journal of Life Science
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• v.17 no.10
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• pp.1341-1346
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• 2007

An intracellular ${\beta}-xylosidase$ from Paenibacillus sp. DG-22 was purified to homogeneity by ion-exchange, hydrophobic interaction and gel-filtration chromatography. The molecular weight of the enzyme was measured to be 156,000 by gel filtration and 80,000 by SDS-PAGE, indicating that the enzyme consisted of two identical subunits. The purified enzyme exhibited maximum activity at $65^{\circ}C$ and pH 5.5. It retained 89% of its initial activity up to 60 min at $60^{\circ}C$ and had a half-life of 25 min at $65^{\circ}C$. The enzyme was highly specific for pNPX as the substrate. It showed little or no activity against other p-nitrophenyl glycosides and xylans. The $K_m\;and\;V_{max}$ for pNPX was 0.53 mM and 3.18 U/mg protein, respectively. The ${\beta}-xylosidase$ was strongly inhibited by $Ag^+,\;Fe^{2+},\;Hg^{2+}\;and\;Zn^{2+}$ and slightly activated by DTT. The hydrolysis product from xylobiose, xylotriose, and xylotetraose was xylose.

• Journal of Life Science
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• v.31 no.11
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• pp.1028-1036
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• 2021

Firefly luciferase (FLuc) can function as an efficient marker in the gene and viral therapies. Nonetheless, its clinical translation has been unaccomplished with the concerns on its exogenous nature and the similarity with human fatty acyl-CoA synthetase. In this study, we aimed to show safety of FLuc by conducting a set of preclinical experiments and a human use. Initially, FLuc permeability across the plasma membrane was investigated by delivering the FLuc-carrying viral vector, OTS-412, or the FLuc recombinant protein. After in vitro infection of OTS-412 into different cancer cell lines, FLuc activity was detected only in the cell lysates, but not in culture media. In addition, recombinant FLuc protein further showed the impermeability against the plasma membrane. Similar result was also observed in the in vivo experiment. After being injected into the VX2 tumor-bearing rabbit, the FLuc exclusively resided within the tumor tissue without being detected in the blood plasma or other organs. Human cancer cell lines originated from various organs were lysed and treated to the FLuc, and none of the human substrates was reactive against the FLuc. As a final step, FLuc recombinant protein was intravenously injected into a human. The luciferase was degraded with the half-life of 20 to 30 minutes in blood, and was untraceable from 1.5 hr after the injection. In addition, the blood plasma was nonresponsive against the fatty acids. Hematological analysis was also comparable between the pre- and post-injection. Altogether, our study collectively demonstrates the safety of the firefly luciferase.

• Microbiology and Biotechnology Letters
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• v.36 no.3
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• pp.182-188
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• 2008

A hyperthermophilic bacteria (strain HJ6) was isolated from a hot springs located in the Arima-cho, Hyogo, Japan. The cells were long-rod type ($2-4{\mu}m$), about $0.4{\mu}m$ in diameter. The pH and temperature for optimal growth were 6.5 and $80^{\circ}C$, respectively. Phylogenetic analysis based on the 16S rDNA sequence and biochemical studies indicated that HJ6 belonged to the genus Thermus thermophilus (Tt). The gene encoding the Trehalose synthase (TS) was cloned and sequenced. The open reading frame (ORF) of the TtTS gene was composed of 2,898 nucleotides and encoded a protein (975 amino acids) with a predicted molecular weight of 110.56 kDa. The deduced amino acid sequence of TtTS showed 99% and 83% identities to the Thermus caldophilus TS and Meiothermus ruber TS, respectively. TtTS gene was expressed in Escherichia coli cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for Trehalose synthase activity were found to be $80^{\circ}C$ and 7.5, respectively. The half-life of heat inactivation was about 40 min at $90^{\circ}C$. The maximum trehalose conversion rate of maltose into trehalose by the enzyme increased as the substrate concentration increased, and reached 55.7% at the maltose concentration of 500 mM, implying that the enzyme conversion was dependent of the substrate concentration.

• Korean Journal of Food Science and Technology
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• v.36 no.3
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• pp.440-447
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• 2004

Characteristics of human ${\beta}-carotene$ 15,15'-dioxygenase isolated by recombinant DNA technology was elucidated. Optimal pH and temperature were 9.0 and $40^{\circ}C$, respectively. Enzyme activity was temperature-sensitive. Enzyme was stable at pH 6.0-9.0 for 24 hr and under $5^{\circ}C$. Half-life of enzyme at $35^{\circ}C$ was 40 min. Crude preparations of enzyme were inhibited by ferrous ion-chelating agent and sulfhydryl-binding agent. GSH offsets inhibitory effect of PCMB. With increasing substrate concentrations, enzyme activity gave typical Michaelis-Menten curve, Based on Hanes-Woolf plot of data, $K_{m}\;and\;V_{max$ were $3.39{\times}10^{6}\;M\;and\;1.2\;pmol/mg$ protein/min, respectively.