• Korean Journal of Microbiology
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• v.47 no.4
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• pp.302-307
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• 2011

Native chitin deacetylase of Aspergillus nidulans was purified to apparent homogeneity by a combination of phenyl-Sepharose and Q-Sepharose column chromatography. In order to analyze the amino acid residues involved in the enzyme activity, the enzyme was chemically modified with chemical agent, which selectively reacted with the specific amino acid residue on the protein. When the enzyme was chemically modified with diethylpyrocarbonate, which specifically reacted with histidine residues on the protein, the activity was eliminated. The chitin deacetylase, chemically modified with 100 ${\mu}M$ modifier at the residue of arginine or tyrosine, has shown to have decreased activities. It was shown that the modification at aspartic acid or glutamic acid did not affect the enzyme activity to a greater extent, which would not implicate that acid amino residues were directly involved in catalytic reaction and would affect on the global structures of the proteins. This results demonstrated that histidine and tyrosine residues of enzyme would participate in an important function of the chitin deacetylase activity.

• Korean Journal of Food Science and Technology
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• v.16 no.3
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• pp.279-284
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• 1984

Microstructure of milk powder and cheese made from milk powder were observed by electron microscope. Freeze dried milk powder showed apple-like appearance. The cheese made from freeze dried milk powder had relatively flat surface and homogenous deposit in compare with classical processed cheese. Imported milk powder also indicated similar surface as well as freeze dried milk powder, however, the cheese made from imported milk powder had somewhat coarse surface structures with the spaces between casein matrix and deposit. Commericial milk powder showed irregular shape in size and coagulum which were possibly denatured in the course of drying. The cheese made from commercial powder indicted irregular and small deposit and porous structure. The porousity of the cheese seemed to be influenced by the degree of heat treatment. Denatured protein would be less dispersive than native in presence of polyphosphates. Fat globule and protein micelle of cheese made from skim milk powder get very adjacent to each other and showed compactness of micelles. It is thought that melting mechanism of skim milk powder was different from the melting of typical processed cheese.

• Molecules and Cells
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• v.42 no.7
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• pp.503-511
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• 2019

As sessile organisms, plants have developed sophisticated system to defend themselves against microbial attack. Since plants do not have specialized immune cells, all plant cells appear to have the innate ability to recognize pathogens and turn on an appropriate defense response. The plant innate immune system has two major branches: PAMPs (pathogen associated molecular patterns)-triggered immunity (PTI) and effector-triggered immunity (ETI). The ability to discriminate between self and non-self is a fundamental feature of living organisms, and it is a prerequisite for the activation of plant defenses specific to microbial infection. Arabidopsis cells express receptors that detect extracellular molecules or structures of the microbes, which are called collectively PAMPs and activate PTI. However, nucleotidebinding site leucine-rich repeats (NB-LRR) proteins mediated ETI is induced by direct or indirect recognition of effector molecules encoded by avr genes. In Arabidopsis, plasmamembrane localized multifunctional protein RIN4 (RPM1-interacting protein 4) plays important role in both PTI and ETI. Previous studies have suggested that RIN4 functions as a negative regulator of PTI. In addition, many different bacterial effector proteins modify RIN4 to destabilize plant immunity and several NB-LRR proteins, including RPM1 (resistance to Pseudomonas syringae pv. maculicola 1), RPS2 (resistance to P. syringae 2) guard RIN4. This review summarizes the current studies that have described signaling mechanism of RIN4 function, modification of RIN4 by bacterial effectors and different interacting partner of RIN4 in defense related pathway. In addition, the emerging role of the RIN4 in plant physiology and intercellular signaling as it presents in exosomes will be discussed.

• Journal of Microbiology and Biotechnology
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• v.29 no.9
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• pp.1383-1390
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• 2019

In this study, we expressed cotA laccase from Bacillus subtilis on the surface of B. subtilis spores for efficient decolorization of synthetic dyes. The cotE, cotG, and cotY genes were used as anchoring motifs for efficient spore surface display of cotA laccase. Moreover, a $His_6$ tag was inserted at the C-terminal end of cotA for the immunological detection of the expressed fusion protein. Appropriate expression of the CotE-CotA (74 kDa), CotG-CotA (76 kDa), and CotY-CotA (73 kDa) fusion proteins was confirmed by western blot. We verified the surface expression of each fusion protein on B. subtilis spore by flow cytometry. The decoloration rates of Acid Green 25 (anthraquinone dye) for the recombinant DB104 (pSDJH-EA), DB104 (pSDJH-GA), DB104 (pSDJH-YA), and the control DB104 spores were 48.75%, 16.12%, 21.10%, and 9.96%, respectively. DB104 (pSDJH-EA) showed the highest decolorization of Acid Green 25 and was subsequently tested on other synthetic dyes with different structures. The decolorization rates of the DB104 (pSDJH-EA) spore for Acid Red 18 (azo dye) and indigo carmine (indigo dye) were 18.58% and 43.20%, respectively. The optimum temperature for the decolorization of Acid Green 25 by the DB104 (pSDJH-EA) spore was found to be $50^{\circ}C$. Upon treatment with known laccase inhibitors, including EDTA, SDS, and $NaN_3$, the decolorization rate of Acid Green 25 by the DB104 (pSDJH-EA) spore decreased by 23%, 80%, and 36%, respectively.

• Mass Spectrometry Letters
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• v.12 no.3
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• pp.66-75
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• 2021

Interleukin-4 (IL-4) and IL-13 are cytokines secreted by immune cells. Cytokines induce the proliferation of macrophages or promote the differentiation of secretory cells. The initiation and progression of allergic inflammatory diseases, such as asthma, are dependent on cytokines acting through related receptor complexes. IL-4 and IL-13 are N-glycoproteins. Glycan structures in glycoproteins play important roles in protein folding, protein stability, enzymatic function, inflammation, and cancer development. Therefore, the glycan structure of IL-4 and IL-13 needs to be elucidated in detail for the development of effective therapies. We report the first attempt to characterize the site-specific N-glycosylation of recombinant IL-4 and IL-13 via liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The tandem mass spectra of intact N-glycopeptides were identified using the Integrated GlycoProteome Analyzer (I-GPA) platform, which can automatically and rapidly analyze multiple N-glycopeptides, including their glycan composition and amino acid sequences. The recombinant IL-4 and IL-13 were identified with amino acid sequence coverages of 84% and 96%, respectively. For IL-4, 52 glycoforms on one N-glycosylation site were identified and quantified. In IL-13, 232 N-glycopeptides from three N-glycosylation sites were characterized, with the site Asn52 being the most extensively glycosylated (~80%). The complex glycans were the most abundant glycan on IL-4 and IL-13 (~96% and 91%, respectively), and the biantennary glycans were the most abundant in both recombinant IL-4 and IL-13 proteins.

• Journal of Microbiology and Biotechnology
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• v.31 no.6
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• pp.882-889
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• 2021

In order to use an enzyme industrially, it is necessary to increase the activity of the enzyme and optimize the reaction characteristics through molecular evolution techniques. We used the error-prone PCR method to improve the reaction characteristics of LipCA lipase discovered in Antarctic Croceibacter atlanticus. Recombinant Escherichia coli colonies showing large halo zones were selected in tributyrin-containing medium. The lipase activity of one mutant strain (M3-1) was significantly increased, compared to the wild-type (WT) strain. M3-1 strain produced about three times more lipase enzyme than did WT strain. After confirming the nucleotide sequence of the M3-1 gene to be different from that of the WT gene by four bases (73, 381, 756, and 822), the secondary structures of WT and M3-1 mRNA were predicted and compared by RNAfold web program. Compared to the mean free energy (MFE) of WT mRNA, that of M3-1 mRNA was lowered by 4.4 kcal/mol, and the MFE value was significantly lowered by mutations of bases 73 and 756. Site-directed mutagenesis was performed to find out which of the four base mutations actually affected the enzyme expression level. Among them, one mutant enzyme production decreased as WT enzyme production when the base 73 was changed (T→ C). These results show that one base change at position 73 can significantly affect protein expression level, and demonstrate that changing the mRNA sequence can increase the stability of mRNA, and can increase the production of foreign protein in E. coli.

• Journal of Microbiology and Biotechnology
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• v.31 no.11
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• pp.1545-1551
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• 2021

Exopolysaccharides (EPSs) such as capsular polysaccharide (CPS) are important bioactive carbohydrate compounds and are often used as bioenrichment agents and bioabsorbers to remove environmental pollutants like di-n-butyl phthalate (DBP). Among the EPS-producing bacteria, lactic acid bacteria (LAB) have gained the most attention. As generally recognized as safe (GRAS) microorganisms, LAB can produce EPSs having many different structures and no health risks. However, EPS production by LAB does not meet the needs of large-scale application on an industrial scale. Here, the capA gene (encoding CPS biosynthesis protein) was overexpressed in Lactobacillus plantarum P1 to improve the production of EPSs and further enhance the DBP adsorption capability. Compared with P1, the CPS production in capA overexpressed strain was increased by 11.3 mg/l, and the EPS thickness was increased from 0.0786 ± 0.0224 ㎛ in P1 to 0.1160 ± 0.0480 ㎛ in P1-capA. These increases caused the DBP adsorption ratio of P1-capA to be doubled. Overall, the findings in this study provide a safe method for the adsorption and removal of DBP.

• Molecules and Cells
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• v.45 no.8
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• pp.537-549
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• 2022

Preproenkephalin (PPE) is a precursor molecule for multiple endogenous opioid peptides Leu-enkephalin (ENK) and Met-ENK, which are involved in a wide variety of modulatory functions in the nervous system. Despite the functional importance of ENK in the brain, the effect of brain-derived factor(s) on PPE expression is unknown. We report the dual effect of neural epidermal growth factor (EGF)-like-like 2 (NELL2) on PPE gene expression. In cultured NIH3T3 cells, transfection of NELL2 expression vectors induced an inhibition of PPE transcription intracellularly, in parallel with downregulation of protein kinase C signaling pathways and extracellular signal-regulated kinase. Interestingly, these phenomena were reversed when synthetic NELL2 was administered extracellularly. The in vivo disruption of NELL2 synthesis resulted in an increase in PPE mRNA level in the rat brain, suggesting that the inhibitory action of intracellular NELL2 predominates the activation effect of extracellular NELL2 on PPE gene expression in the brain. Biochemical and molecular studies with mutant NELL2 structures further demonstrated the critical role of EGF-like repeat domains in NELL2 for regulation of PPE transcription. These are the first results to reveal the spatio-specific role of NELL2 in the homeostatic regulation of PPE gene expression.

• Biomolecules & Therapeutics
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• v.32 no.5
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• pp.627-634
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• 2024

Sesquiterpene lactones, a class of natural compounds abundant in the Asteraceae family, have gained attention owing to their diverse biological activities, and particularly their anti-proliferative effects on human cancer cells. In this study, we systematically investigated the structure-activity relationship of ten sesquiterpene lactones with the aim of elucidating the structural determinants for the STAT3 inhibition governing their anti-proliferative effects. Our findings revealed a significant correlation between the STAT3 inhibitory activity and the anti-proliferative effects of sesquiterpene lactones in MDA-MB-231 breast cancer cell lines. Among the compounds tested, alantolactone and isoalantolactone emerged as the most potent STAT3 inhibitors, highlighting their potential as candidates for anticancer drug development. Through protein-ligand docking studies, we revealed the structural basis of STAT3 inhibition by sesquiterpene lactones, emphasizing the critical role of hydrogen-bonding interactions with key residues, including Arg609, Ser611, Glu612, and Ser613, in the SH2 domain of STAT3. Furthermore, our conformational analysis revealed the decisive role of the torsion angle within the geometry-optimized structures of sesquiterpene lactones in their STAT3 inhibitory activity (R=0.80, p<0.01). These findings not only provide preclinical evidence for sesquiterpene lactones as promising phytomedicines against diseases associated with abnormal STAT3 activation, but also highlight the importance of stereochemical aspects in their activity.

• Parasites, Hosts and Diseases
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• v.62 no.3
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• pp.330-341
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• 2024

The Trichinella spiralis novel cystatin (TsCstN) inhibits cathepsin L (CatL) activity and inflammation of macrophages during lipopolysaccharide (LPS) induction. To identify the protease inhibitory region, this study applied an in silico modeling approach to simulate truncation sites of TsCstN (Ts01), which created four truncated forms, including TsCstN^∆1-39 (Ts02), TsCstN^∆1-71 (Ts03), TsCstN^{∆1-20, ∆73-117} (Ts04), and TsCstN^{∆1-20, ∆42-117} (Ts05). The superimposition of these truncates modeled with AlphaFold Colab indicated that their structures were more akin to Ts01 than those modeled with I-TASSER. Moreover, Ts04 exhibited the closest resemblance to the structure of Ts01. The recombinant Ts01 (rTs01) and truncated proteins (rTs02, rTs03, and rTs04) were successfully expressed in a prokaryotic expression system while Ts05 was synthesized, with sizes of approximately 14, 12, 8, 10, and 2.5 kDa, respectively. When determining the inhibition of CatL activity, both rTs01 and rTs04 effectively reduced CatL activity in vitro. Thus, the combination of the α1 and L1 regions may be sufficient to inhibit CatL. This study provides comprehensive insights into TsCstN, particularly regarding its protein function and inhibitory domains against CatL.