• 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.

• Journal of Animal Science and Technology
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• v.66 no.4
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• pp.807-833
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• 2024

Elderly people avoid eating red meat and dried meat product due to its texture and stiffness; they deprive them of rich sources of nutrients. In addition, food-related diseases are exponentially increasing due to using synthetic additives in food products. Therefore, this research aimed to develop semi-dried goat meat jerky considering geriatric preferences by using natural tenderizers and nitrate. Four treatments were formulated negative control (NC [synthetic nitrite without tenderizers]), positive control (PC [Swiss chard without tenderizers]), T1 (Swiss chard with pineapple powder), and T2 (Swiss chard with pineapple and tomato powder). T1 and T2 had higher processing yield, and rehydration capacity compared with NC and PC. The fat content of T1 and T2 was lower than the control groups. Moisture was significantly higher in T1, NC, and T2 than in PC (p < 0.05). T2 showed the lowest water activity (0.87), lowest shear force (4.82 kgf), and lowest total plate count (TPC). The lowest pH and thiobarbituric acid reactive substances (TBARS) were observed in T1 and T2. T1 showed the lowest lightness and the maximum redness (p < 0.05) while PC showed the lowest yellowness. During the storage period, moisture and pH decreased, and TPC and TBARS significantly increased whereas water activity is stable regardless of the treatment. The results of the myofibrillar fragmentation index (MFI) and sodium dodecyl sulfate-polyacrylamide gel revealed that T1 and T2 more effectively converted protein to polypeptides. In addition, tenderizers positively affected thrombogenicity, atherogenicity, and hypocholesterolemic/hypercholesterolemic indices. T2 observed the highest overall sensory acceptance by reducing goaty flavor. Overall, jerky treated with tenderizers is easily chewable and digestible for the elderly due to its tenderness and essential fatty acids that would be senior-friendly food.

• Journal of Life Science
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• v.26 no.11
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• pp.1245-1252
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• 2016

As a research of inflammation inhibitory activity using natural resource, the inflammation inhibitory activity by purified active compound from Rhododendron mucronulatum flower was experimented. Rhododendron mucronulatum flower components were purified and separated with Sephadex LH-20 and MCI gel CHP-20 column chromatography, Purified compound was confirmed as myricetin by $^1H-NMR$, $^{13}C-NMR$ and Fast atom bombardment (FAB)-Mass spectrum to have inhibition activity on inflammatory factors secreted by Raw 264.7 cells in response to lipopolysaccharide stimulation. Myricetin inhibited nitric oxide (NO) expression in a concentration dependent manner, approximately 40% inhibition was observed at a concentration of $50{\mu}M$. The inhibition effect of myricetin on inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 protein expression was 20% and 80%, respectively, at a concentration of $25{\mu}M$. Myricetin also inhibited expression of the inflammatory cytokines, tumor necrosis factor $(TNF)-{\alpha}$, interleukin $(IL)-1{\beta}$, IL-6 and prostaglandin $E_2(PGE_2)$ in a concentration dependent manner; a concentration of $50{\mu}M$, 70%, 80%, 80% and 95% inhibition was observed, respectively. Therefore myricetin isolated from Rhododendron mucronulatum flowers is expected to have an anti-inflammatory effect in Raw 264.7 cell induced by lipopolysaccharides. The results can be expected myricetin from Rhododendron mucronulatum flower to use as functional resource for anti-inflammatory activity.

• Korean Journal of Food Science and Technology
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• v.32 no.4
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• pp.755-762
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• 2000

Physicochemical properties of commercial sweet potato starches manufactured by 7 different companies were investigated in comparison with corn and potato starches. Crude ash and protein content varied from 0.36 to 1.02%, and from 0.04 to 0.14% based on dry weight, respectively. The protein contents were relatively smaller than that of corn or potato starch. But whiteness of the sweet potato starches was less than that of corn or potato starch. Mean diameter of the sweet potato starch granules varied from 14.23 to $21.08\;{\mu}m$ depending on the company and all sweet potato starches showed bimodal size distributions. Pasting viscosity measured by Rapid Viscoanalyzer(RVA) also showed variations among the starches of different companies. The starch from D company in Korea had the lowest pasting temperature$(74.00^{\circ}C)$ whereas the starch from a phillippine company(P) did the highest one$(80.35^{\circ}C)$. The peak viscosity of sweet potato starches was higher than that of corn starch but lower than that of potato starch. The D company starch also showed the highest peak viscosity(2283 cp) among the starches tested. Paste breakdown by hot shearing ranged from 524 cp (S company) to 1279 cp (HL company). Textural properties of the starch gels appeared significantly different among the starches of different manufacturers. The greatest hardness of the gel was $137.90\;g_{f}$ at 1 day storage whereas the lowest value was $31.53\;g_{f}$. Except the starches from 2 companies (P and S), the sweet potato starches formed very soft and weak gels. P or S company starches formed the gels similar to potato starch. Syneresis by freeze-thawing treatments appeared less for sweet potato starch gels than that for corn starch gels, but greater than that for potato starch gel. The overall properties of the sweet potato starches varied by the manufacturing companies, and ranged between those of corn and potato starches.

• Korean Journal of Agricultural Science
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• v.16 no.2
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• pp.225-238
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• 1989

These studies were conducted to investigate the extraction, purification and characterization of oriental pear (Niitaka. Pyrus pyrifolia Nak.) protease, and the results obtained were as follows: 1. Oriental pear protease was effectively extracted by the method of homogenizing pear pulp with 0.7 volume of 0.1M-sodium phosphate buffer, pH 6.5 containing 5mM-cysteine, 40mM-2-mercaptoethanol and 2mM-EDTA at 10,000 rpm for 5 min. 2. The protease was purified by ammonium sulfate fractionation, Sephadex G-100 filtration and DEAE-Sephadex A-50 column chromatography, and the purified enzyme gave a single protein band on polyacrylamide gel electrophoresis. 3. The specific activity of purified enzyme was 29.65 unit/mg protein and the yield was 7.22%. 4. The moecular weight of the protease was estimated to be about 51,000 by SDS-polyacrylamide gel electrophoresis, and the enzyme had Km value of 54.5 mg/ml for casein. 5. The purified enzyme had a maximum activity at pH 6.0 and $50^{\circ}C$, and was stable from pH 5.5-6.5 and at temperatures below $50^{\circ}C$ 6. Casein was a better substrate for this protease compared to hemoglobin. 7. The enzyme activity was markedly inhibited by p-chloromercuribenzoic acid and heavy metal salts such as $HgCl_2$ and $MnSO_4$ also considerably inhibited the enzyme activity.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.19 no.1
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• pp.27-35
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• 1986

In succession to the previous paper, the present study was directed to investigate the effect of keeping freshness of conger eel (Astroconger myriaster) and yellowtail (Seriola quinqueradiata) by partial freezing, and the changes in the physical properties of fish meat paste product prepared with the muscle of conger eel during storage were also examined. The results obtained are summarized as follows: The period of keeping freshness (days in which k value reaches $20\%$) of conger eel and yellowtail by partial freezing was 10 days and 6 days, respectively. VBN content in the conger eel muscle showed 39.5 mg/100g by icing for 15 days, and did not show a great change by partial freezing and freezing, while that of yellowtail muscle reached at 32 mg/100g by icing, 20 mg/100g by partial freezing and 18 mg/100g by freezing for 15 days. The lipids extracted from the muscles of both fishes by icing were remarkably oxidized than those by partial freezing. The myofibrillar protein in the conger eel muscle during storage for 9 days decreased $3\%,\;10%\;and\;11\%$ by icing, partial freezing and freezing, respectively, and that of yellowtail muscle did $16\%,\;10%\;and\;4\%$ by icing, partial freezing and freezing, respectively. On the other hand, the alkali-soluble protein in both fishes increased with storage time. Gel strength of fish meat paste product prepared with the muscle of conger eel decreased to $35\%$ by icing, $74\%$ by partial freezing and $76\%$ by freezing for 10 days compared to control, and the expressible water increased 1.6 times, 1.2 times and 1.1 times by icing, partial freezing and freezing, respectively, as much as that of control product.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.38 no.3
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• pp.264-274
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• 1993

This study was carried out to assess the agronomic characters and physicochemical properties of floury and chalky-endosperm mutant lines induced by chemical mutagen treatment to rice varieties, Hwacheongbyeo and IR24. Linkage analysis of a floury-endosperm gene was carried out using linkage testers. The grain size of brown rice of the mutants was smaller than that of the original varieties. The l, 000-grain and 1$\ell$ weight were lighter in the mutants compared with those in the original varieties. The compound starch granules in the endosperm cell of the mutants showed a loosely-packed crystalline structure. Amylose contents in mutants ranged from 16.9 to 28.5%. Crude protein contents of the mutants were not significantly different from the original rice variety, Hwacheongbyeo, but white core mutant(line 47106) derived from IR24 showed higher protein(l1.32%) compared with IR24(8.30%). The mutants showed slightly harder gel characteristics, and much lower viscosity in Amylograph than original varieties. Steamed rice-cakes from mutant lines showed greater volume than those from original varieties. During the process of alcohol fermentation, Brix in the mutants(especially floury mutants) decreased faster and the alcohol production after 10-day fermentation was much greater in the mutants than in the original varieties. Three different gene loci for floury endosperm characteristics were identified from the allelism test among mutant lines, and the genes were tentatively symbolized as flo-a, flo-b and flo-c, respectively. A floury gene, flo-a, was linked with lg(liguleless) gene in the linkage group N, with R.V. 5.76$\pm$1.72%.

• Journal of Life Science
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• v.34 no.2
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• pp.86-93
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• 2024

Since replication of plasmids must be strictly controlled, plasmids that generally perform rolling circle replication generally maintain a constant copy number by strictly controlling the replication initiator Rep at the transcriptional and translational levels. Plasmid pJB01 contains three orfs (copA, repB, repC or repABC) consisting of a single operon. From analysis of amino acid sequence, pJB01 CopA was homologous to the Cops, as a copy number control protein, of other plasmids. When compared with a CopG of pMV158, CopA seems to form the RHH (ribbon-helix-helix) known as a motif of generalized repressor of plasmids. The result of gel mobility shift assay (EMSA) revealed that the purified fusion CopA protein binds to the operator region of the repABC operon. To examine the functional role of CopA on transcriptional level, 3 point mutants were constructed in coding frame of copA such as CopA R16M, K26R and E50V. The repABC mRNA levels of CopA R16M, K26R and E50V mutants increased 1.84, 1.78 and 2.86 folds more than that of CopA wt, respectively. Furthermore, copy numbers owing to mutations in three copA genes also increased 1.86, 1.68 and 2.89 folds more than that of copA wt, respectively. These results suggest that CopA is the transcriptional repressor, and lowers the copy number of pJB01 by reducing repABC mRNA and then RepB, as a replication initiator.

• Microbiology and Biotechnology Letters
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• v.20 no.5
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• pp.519-526
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• 1992

The gene coding for urease of alkalophilic Bacillus pasteurii had been cloned in Escherichia coli previously. The urease protein was purified 63.1-fold by TEAE-cellulose, DEAE-Sephadex A-50, Sephadex G-150 and Sephadex G-200 chromatographies with a 7.3% yield from the sonicated fluid of the E. coli HB1Ol(pBUll) encoding B. pasteurii urease gene. The ureases of E. coli (pBUll) and B. pasteurii possessed as a $K_m$ for urea, 42.1 mM and 40.4 mM, respectively. They hydrolyzed urea with $V_{max}$ of 86.9$\mu$mol/min and 160$\mu$mol/min, respectively. Both ureases were composed with four subunits (Mrs 67,000) and a subunit (Mr 20,000). The molecular weight of both native enzymes was Mr 280,OOO$pm$10,000 determined by gel filtration chromatography and Coomassie blue staining of the subunits. The optimal reaction pH of both ureases were pH 7.5. The ureases were stabled in pH 5.5-10.5. The optimal reaction temperature of both ureases were $60^{\circ}C$, and the ureases were stable for an hour at $50^{\circ}C$, 40min at $60^{\circ}C$ and 10 min at $70^{\circ}C$ The activity of both enzymes were inhibited completely by $Ag^{2+}$, $Hg^{2+}$, $Zn^{2+}$, $Cu^{2+}$, and were inhibited 60% by CoH, 30% by $Fe^{2+}$ and 10% by $Pb^{2+}$. However it was increased by the addition of $Sn^{2+}$, $Mn^{2+}$, $Mg^{2+}$ at concentration of $1{\times}10^{-3}$M. Both ureases were inhibited completely by p-CMB and acetohydroxamic acid. The urease expressed in E. coli (pBU11) was inhibited 70% by SDS. The urease of B. pasteurii was inhibited 40% by hydroxyurea, whereas the recombinant urease of E. coli strain was inhibited 17%. Both enzymes were not inhibited by cyclohexanediaminetetraacetic acid (CDTA) and ethylendiaminetetraacetic acid (EDTA).

• Journal of the Korean Wood Science and Technology
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• v.32 no.5
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• pp.51-58
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• 2004

This study was performed to investigate compositions of inorganic elements, amino acids and glycoprotein fractions as biological substances in fruit body of Sarcodon aspratus. The fruit body of Sarcodon aspratus contained Ca, Mg, Zn, Mn, Fe, Cu, and Pb, in particular high Ca and Na. Hot water extracts consisted of 54% of polysaccharide fraction and 32.6% of protein. In amino acids composition, fourteen free amino acids were detected, mainly glutamic acid, alanine and arginine. Fifteen kinds of total amino acids were contained with major components of glutamic acid, aspartic acid, serine and threonine. Concerned to glycoprotein extraction, 95% ethyl alcohol concentration gave the highest yields with 70.6% sugar fraction, 332% glycoprotein. Different ethyl alcohol concentration resulted in different protein precipitations, and lower concentration ethyl alcohol in the range of 30 to 70% gave more than 92% of higher sugar fraction. Crude glycoprotein (GP) was fractionated by P fraction of more than MW 300,000, P-1 fraction unadsorbed by DEAE-Sephadex, P-2 fractionated from P-1 by Sepharose 2B gel chromatography and P-3 fraction adsorbed by DEAE-Sephadex. Total sugars were increased and protein contents decreased during fractionation. GP and P-3 contained glucose, galactose, mannose and fucose. GP had high glucose with high contents of glutamic acid, serine, alanine and glycine. P-3 fraction contained high mannose with aspartic acid, glutamic acid, and glycine. P-2 fraction was 700,000 MW with high glucose and fucose, and low protein of 1.1%, high amounts of aspartic acid, glutamic acid and alanine, but no mannose and no cysteine.