• Journal of Microbiology and Biotechnology
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• v.25 no.11
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• pp.1944-1953
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• 2015

A novel alkaline protease from Streptomyces sp. M30, SapHM, was purified by ammonium sulfate precipitation, hydrophobic interaction chromatography, and DEAE-Sepharose chromatography, with a yield of 15.5% and a specific activity of 29,070 U/mg. Tryptic fragments of the purified SapHM were obtained by electrospray ionization quadrupole time-of-flight mass spectrometry. Nucleotide sequence analysis revealed that the gene sapHM contained 1,179 bp, corresponding to 392 amino acids with conserved Asp156, His187, and Ser339 residues of alkaline protease. The first 24 amino acid residues were predicted to be a signal peptide, and the molecular mass of the mature peptide was 37.1 kDa based on amino acid sequences and mass spectrometry. Pure SapHM was optimally active at 80℃ in 50 mM glycine-NaOH buffer (pH 9.0), and was broadly stable at 0-50℃ and pH 4.0-9.0. The protease relative activity was increased in the presence of Ni²⁺, Mn²⁺, and Cu²⁺ to 112%, 113%, and 147% of control, respectively. Pure SapHM was also activated by dimethylformamide, dimethyl sulfoxide, Tween 80, and urea. The activity of the purified enzyme was completely inhibited by phenylmethylsulfonyl fluoride, indicating that it is a serine-type protease. The K_m and V_max values were estimated to be 35.7 mg/ml, and 5 × 10⁴ U/mg for casein. Substrate specificity analysis showed that SapH was active on casein, bovine serum albumin, and bovine serum fibrin.

• Journal of Microbiology and Biotechnology
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• v.24 no.6
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• pp.748-755
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• 2014

In the genome annotation of Escherichia coli MG1655, the orf382 (1,149 bp) is designated as a gene encoding an alcohol dehydrogenase that may be Fe-dependent. In this study, the gene was amplified from the genome by PCR and overexpressed in Escherichia coli BL21(DE3). The recombinant $6{\times}$His-tag protein was then purified and characterized. In an enzymatic assay using different hydroxyl-containing substrates (n-butanol, $\small{L}$-threonine, ethanol, isopropanol, glucose, glycerol, $\small{L}$-serine, lactic acid, citric acid, methanol, or $\small{D}$-threonine), the enzyme showed the highest activity on $\small{L}$-threonine. Characterization of the mutant constructed using gene knockout of the orf382 also implied the function of the enzyme in the metabolism of $\small{L}$-threonine into glycine. Considering the presence of tested substrates in living E. coli cel ls and previous literature, we believed that the suitable nomenclature for the enzyme should be an $\small{L}$-threonine dehydrogenase (LTDH). When using $\small{L}$-threonine as the substrate, the enzyme exhibited the best catalytic performance at $39^{\circ}C$ and pH 9.8 with $NAD^+$ as the cofactor. The determination of the Km values towards $\small{L}$-threonine (Km = $11.29{\mu}M$), ethanol ($222.5{\mu}M$), and n-butanol ($8.02{\mu}M$) also confirmed the enzyme as an LTDH. Furthermore, the LTDH was shown to be an ion-containing protein based on inductively coupled plasma-atomic emission spectrometry with an isoelectronic point of pH 5.4. Moreover, a circular dichroism analysis revealed that the metal ion was structurally and enzymatically essential, as its deprivation remarkably changed the ${\alpha}$-helix percentage (from 12.6% to 6.3%).

• Journal of Microbiology and Biotechnology
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• v.30 no.4
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• pp.526-532
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• 2020

A bacterial strain, designated B301^T and isolated from raw chicken meat obtained from a local market in Korea, was characterized and identified using a polyphasic taxonomic approach. Cells were gram-negative, non-motile, obligate-aerobic coccobacilli that were catalase-positive and oxidase-negative. The optimum growth conditions were 30℃, pH 7.0, and 0% NaCl in tryptic soy broth. Colonies were round, convex, smooth, and cream-colored on tryptic soy agar. Strain B301^T has a genome size of 3,102,684 bp, with 2,840 protein-coding genes and 102 RNA genes. The 16S rRNA gene analysis revealed that strain B301^T belongs to the genus Acinetobacter and shares highest sequence similarity (97.12%) with A. celticus ANC 4603^T and A. sichuanensis WCHAc060041^T. The average nucleotide identity and digital DNA-DNA hybridization values for closely related species were below the cutoff values for species delineation (95-96% and 70%, respectively). The DNA G+C content of strain B301^T was 37.0%. The major respiratory quinone was Q-9, and the cellular fatty acids were primarily summed feature 3 (C_16:1 ω6c/C_16:1 ω7c), C_16:0, and C_18:1 ω9c. The major polar lipids were phosphatidylethanolamine, diphosphatidyl-glycerol, phosphatidylglycerol, and phosphatidyl-serine. The antimicrobial resistance profile of strain B301^T revealed the absence of antibiotic-resistance genes. Susceptibility to a wide range of antimicrobials, including imipenem, minocycline, ampicillin, and tetracycline, was also observed. The results of the phenotypic, chemotaxonomic, and phylogenetic analyses indicate that strain B301^T represents a novel species of the genus Acinetobacter, for which the name Acinetobacter pullorum sp. nov. is proposed. The type strain is B301^T (=KACC 21653^T = JCM 33942^T).

• Korean Journal of Fisheries and Aquatic Sciences
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• v.42 no.3
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• pp.215-223
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• 2009

This study was conducted to investigate the effect of diets supplemented with different levels (0, 2.5, 5.0, and 7.5%) of yuza (Citrus junas Sieb ex Tanaka) on nutritional composition of olive flounder. Four groups of fish (242.2$\pm$14.2 g) were fed to apparent satiation twice daily for 4 months. There were no significant differences in proximate composition among the treatment groups (P<0.05). Vitamin C content in flounder muscle was higher in the yuza-added groups than in the control group, and the content among the treatment groups increased as amount of yuza added to diets increased (P<0.05). Of the eight organic acids in flounder muscle, lactic acid was predominant, followed by oxalic acid, succinic-acid, tartaric acid, and acetic acid. Flounders fed 2.5% yuza diet had the highest lactic acid content of all treatments. Four sugars were found in all groups and glucose was the major sugar. Glucose and ribose were detected as the highest sugars in the 2.5% treatment, while maltose and galactose were the dominant sugars in the 5.0% treatment. The abundant fatty acids in fed flounders were 22:6n-3 (DHA), 16:0, and l8:1n-9, which were composed of over 60% of total fatty acids. The control and the 7.5% treatment group had higher 22:6n-3 (DHA) content than the other groups. Major amino acids in samples were glutamic acid, aspartic acid, lysine, leucine, valine, arginine, and alanine. The 2.5% yuza treatment had the highest content of total amino acids and essential amino acids. There were little differences in the free amino acid compositions among the treatments. However, taurine was the predominant amino acid and made up over 47% of total free amino acids. The 2.5% added yuza group contained higher amount of sweet amino acids such as alanine, serine, proline, glycine than the other groups. The addition of yuza to diet of olive flounder had no or little effect on the nutritional components of olive flounder except for vitamin C. However, the 2.5% yuza added group had the highest nutritional values of the treatment groups.

• The Korean Journal of Pharmacology
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• v.26 no.1
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• pp.51-54
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• 1990

Human leukocyte cathepsin-Gs are active participant in the active phase of inflammations like rheumatoid arthritis, emphysema and glomerular injury. Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for treatment of these inflammatory diseases. Mechanism of action of NSAIDs for treatment of inflammatory diseases, especially like rheumatoid arthritis, are known as the inhibitors of prostaglandin synthesis. Inhibitions of the activities of human leukocyte cathepsin-Gs by non-steroidal anti-inflammatory drugs, however, were not same as the known pharmacological effects (inhibition of cyclooxygenase) of these drugs. Among them, especially, sulindac, salicylate, phenylbutazone, oxyphenbutazone, and salicyluric acid inhibited human leukocyte cathepsin-Gs effectively. $IC_{50}s$ of each drug were 4.3mM, 14.3mM, 6.5mM, 11mM and 15mM respectively. The drugs which have same chemical structure and same degree of inhibition effect on cyclooxygenase showed different degree or no effect on inhibition of cathepsin G. These inhibition effect might be, beside of inhibition of cyclooxygenase in the prostaglandin synthesis pathway, another benefitial antiinflammatory effect of NSAIDs by direct protection against tissue destruction in inflammatory diseases.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.6
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• pp.928-939
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• 2003

A study was carried out to determine the effect of de-hulling on apparent and true ileal amino acids digestibility of soybean meals for growing pigs. Twenty barrows (Duroc${\times}$Large white${\times}$Longer white) were fitted with a simple T-cannula at the distal ilium. Digestibility of 20 experimental diets was determined, nine of them were de-hulled soybean meal diets, and nine of them were regular soybean meal diets and two low protein casein diets for determination of endogenous amino acid correction for true digestibility determination. A TEX>$5<{\times}5<$ Latin Squares Design was adopted in this trail. The results showed that de-hulling increased apparent ileal digestibility of isoleucine, threonine, aspartic, tyrosine and indispensable and dispensable amino acid (p<0.05) in soybean meals. Furthermore, dehulling is also increased apparent digestibility of arginine, leucine, lysine, phenylalanine, alanine, glutamic acid, serine and gross amino acids (p<0.01). However, there were no significant differences found for histidine, methionine, tryptophan, cystine and glycine (p>0.05). Similar responses were found for true ileal digestibility. In three dehulled and non-dehulled pairs soybean meals from the same respective sources, de-hulling increased apparent digestibility of lysine, methionine, threonine and cystine 1.42%, 2.06%, 2.18% and 1.40% respectively. True digestibility of lysine, methionine, threonine and cystine was increased 1.65%, 1.94%, 2.30% and 1.82% respectively. A prediction equation for true ileal amino acid digestibility (including lysine and arginine) was established by multivariate linear regression. The independent variables included relevant amino acid, organic matter, crude protein, ether extract and nitrogen free extract. The coefficient R2 values of lysine and agrinine were 0.596 and 0.531 respectively. According to the crude protein content, a prediction equation for lysine and arginine content in soybean meal was also established by single linear regression. The coefficient $R^2$ values of lysine and agrinine were 0.636 and 0.636 respectively.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.2
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• pp.218-224
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• 2018

Objective: This study evaluated the effects of different levels of protein concentrate supplementation on the growth performance of yak calves, and correlated the growth rate to changes occurring in the plasma- amino acids, -insulin profile, and signaling activity of mammalian target of rapamycin (mTOR) cascade to characterize the mechanism through which the protein synthesis can be improved in early weaned yaks. Methods: For this study, 48 early (3 months old) weaned yak calves were selected, and assigned into four dietary treatments according to randomized complete block design. The four blocks were balanced for body weight and sex. The yaks were either grazed on natural pasture (control diet) in a single herd or the grazing yaks was supplemented with one of the three protein rich supplements containing low (17%; LP), medium (19%; MP), or high (21%; HP) levels of crude proteins for a period of 30 days. Results: Results showed that the average daily gain of calves increased (0.14 vs 0.23-0.26 kg; p<0.05) with protein concentrates supplementation. The concentration of plasma methionine increased (p<0.05; 8.6 vs $10.1-12.4{\mu}mol/L$), while those of serine and tyrosine did not change (p>0.05) when the grazing calves were supplemented with protein concentrates. Compared to control diet, the insulin level of calves increased (p<0.05; 1.86 vs $2.16-2.54{\mu}IU/mL$) with supplementation of protein concentrates. Addition of protein concentrates up-regulated (p<0.05) expression of mTOR-raptor, mammalian vacuolar protein sorting 34 homolog, the translational regulators eukaryotic translation initiation factor 4E binding protein 1, and S6 kinase 1 genes in both Longissimus dorsi and semitendinosus. In contrast, the expression of sequestosome 1 was down-regulated in the concentrate supplemented calves. Conclusion: Our results show that protein supplementation improves the growth performance of early weaned yak calves, and that plasma methionine and insulin concentrations were the key mediator for gene expression and protein deposition in the muscles.

• Applied Biological Chemistry
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• v.29 no.2
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• pp.122-129
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• 1986

The lipid compositions of corns produced in Korea were analyzed. Free and bound lipids of the corn kernels were fractionated, quantitated and compared by silicic acid column, thin layer and gas liquid chromatography. Corn kernels contained 5.02% total lipids, which is consisted of 4.09% free lipids and 0.93% bound lipids. Free lipids comprised of 89.61% neutral lipids, 3.75% glycolipids and 6.40% phospholipids, while bound lipids contained 14.26% neutral lipids, 46.06% glycolipids and 37.18% phospholipids. In the neutral lipids of free lipids, triglycerides were predominant (67.68%) and minor components such as esterified sterols, free sterols, free fatty acids, 1,3-diglycerides, 1,2-diglycerides were present. But in the neutral lipids of bound lipids, esterified sterols were not present and the contents of triglycerides were lower (47.68%) and free fatty acids were higher than those of free lipids. Among the phospholipids in free and bound lipids, phosphatidyl choline and phosphatidyl serines and phosphatidyl inositols were also present as minor components. The major fatty acids in the three lipid classes were linoleic, oleic and palmitic acids.

• Microbiology and Biotechnology Letters
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• v.30 no.3
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• pp.235-240
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• 2002

Streptomycetes is a group of Gram-positive soil bacteria that growas a branching vegetative mycelium leading to the formation of spores, and display a physiological differenti-ation related to the synthesis of many secondary metabolites including antibiotics. Their complex life cycle and multicellular differentiation require various levels of regulation and types of signal transduction systems including eukaryotic-type serine/threonine protein kinases and prokaryotic-type histidine/aspartic acid protein kinases. Akt kinase that was found in cells is a sorine/threonine kinase controlling signal pathway for multi-tude of important cellular events. The activation or inactivation of Akt kinase in the cell is one of the critical regulatory points to deliver cell proliferation, differentiation, survival or apoptosis signal. To find the regula-tory protein homologous to Akt in Streptomyces, the fluorescien-labeled synthetic peptide (FITC-TRRSR-TESIT) was designed from the consensus sequence of target proteins for Akt kinase. From the difference of the mobility between the nonphosphorylated and phosphorylated synthetic peptides on Agarose gel electro-phoresis, the Akt-phosphorylating activity was monitored. The cell-free extract prepared from Streptomyces griseus IFO 13350 and the Akt homologous protein was purified by ammonium sulfate fractionation and many steps of column chromatographies such as, DEAE-Sepharose, Mono Q, Resource Phenyl-Soporose and Gel permeation column chromatographies. As a result, the protein phosphorylating the fluorescien-labeled Akt substrate was identified and it's molecular weight was estimated as 39 kDa on SDS-PAGE.

• Journal of Microbiology and Biotechnology
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• v.25 no.12
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• pp.2125-2134
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• 2015

IrrE is a highly conserved global regulator in the Deinococcus genus and contributes to survival from high doses of UV radiation, ionizing radiation, and desiccation. Drad-IrrE and Dgob-IrrE from Deinococcus radiodurans and Deinococcus gobiensis I-0 each share 66% sequence identity. However, Dgob-IrrE showed a stronger protection phenotype against UV radiation than Drad-IrrE in the D. radiodurans irrE-deletion mutant (ΔirrE), which may be due to amino acid residues differences around the DNA-binding HTH domain. Site-directed mutagenesis was used to generate a Drad-IrrE A184S single mutant, which has been characterized and compared with the ΔirrE mutant complemented strain with Drad-irrE, designated ΔirrE-E. The effects of the A184S mutation following UV radiation and mitomycin C (MMC) shock were determined. The A184S mutant displayed significantly increased resistance to UV radiation and MMC shock. The corresponding A184 site in Dgob-IrrE was inversely mutated, generating the S131A mutant, which exhibited a loss of resistance against UV radiation, MMC shock, and desiccation. qPCR analysis revealed that critical genes in the DNA repair system, such as recA, pprA, uvrA, and ddrB, were remarkably induced after UV radiation and MMC shock in the ΔirrE-IE and A184S mutants. These data suggested that A184S improves the ability against UV radiation and MMC shock, providing new insights into the modification of IrrE. We speculated that the serine residue may determine the efficiency of DNA binding, leading to the increased expression of IrrE-dependent genes important for protection against DNA damage.