• 한국해양바이오학회지
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• 제7권1호
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• pp.11-18
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• 2015

An extracellular alkaline ${\alpha}$-D-mannosidase produced by a strain named as MA-01 was produced and its preliminary enzyme activity was characterized. Upon determining the 16S rDNA sequence and its homology search, the strain was identified to be one of species of the Bacillus safensis. Localization of enzyme was elucidated that ${\alpha}$-D-mannosidase can be found in culture medium as an extracellular enzyme. In addition, partial enzyme activity of 63% compared with the extracellular enzyme activity was observed in membrane protein. The optimal pH and temperature of the ${\alpha}$-D-mannosidase were pH 7.5 and $37^{\circ}C$, respectively. The $K_m$ and $V_{max}$ values of the ${\alpha}$-D-mannosidase in crude enzyme toward p-nitrophenyl-${\alpha}$-D-mannopyranoside were determined to be $455.6{\mu}M$ and $10.8{\mu}mole/min/mg$ of protein, respectively. To the best of our knowledge, this is the first report described the alkaline ${\alpha}$-D-mannosidase from the family of B. safensis.

• Advances in nano research
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• 제6권1호
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• pp.81-92
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• 2018

Biosynthesis of nanoparticles has acquired particular attention due to its economic feasibility, low toxicity and simplicity of the process. Extracellular synthesis of nanoparticles by bacteria and fungi has been stated to be brought about by enzymes and other reducing agents that may be secreted in the culture medium. The present study was carried out to determine the underlying mechanisms of extracellular silver nanoparticle synthesis by Pseudomonas hibiscicola isolated from the effluent of an electroplating industry in Mumbai. Synthesized nanoparticles were characterized by spectroscopy and electron microscopic techniques. Protein profiling studies were done using Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (1D-SDS PAGE) and subjected to identification by Mass Spectrometry. Characterization studies revealed synthesis of 50 nm nanoparticles of well-defined morphology. Total protein content and SDS PAGE analysis revealed a reduction of total protein content in test (nanoparticles solution) samples when compared to controls (broth supernatant). 45.45% of the proteins involved in the process of nanoparticle synthesis were identified to be oxidoreductases and are thought to be involved in either reduction of metal ions or capping of synthesized nanoparticles.

• Journal of Microbiology and Biotechnology
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• 제4권2호
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• pp.134-140
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• 1994

An antagonistic bacterium Pseudomonas stutzeri YPL-1 liberated extracellular chitinase and $\beta$-1,3-glucanase which are key enzymes in the decomposition of fungal hyphal walls. The lytic enzymes caused abnormal swelling and retreating at the hyphal tips of plant pathogenic fungus Fusarium solani in a dual culture. Scanning electron microscopy revealed the hyphal degradation of F. solani in the regions interacting with P. stutzeri YPL-1. The production of chitinase and properties of a crude preparation of the enzyme from P. stutzeri YPL-1 were investigated. Peak of the chitinase activity was detected after 4 hr of cultivation. The enzyme had optimum temperature and pH of 50$^{\circ}C$ and pH 5.3, respectively. The enzyme was stable in the pH range of 3.5 to 6.0 up to 50$^{\circ}C$. The enzyme was significantly inhibited by metal compounds such as $HgCl_2$, but was stimulated by $CoCl_2$. P. stutzeri YPL-1 produced high levels of the enzyme after 84 hr of incubation. Among the tested carbon sources, chitin was the most effective for the enzyme production, at the concentration level of 3%. As a source of nitrogen, peptone was the best for the enzyme production, at the concentration level of 4%. The maximum amount of enzyme was produced by cultivating the bacterium at a medium of initial pH 6.8.

• 한국미생물·생명공학회지
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• 제18권5호
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• pp.490-495
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• 1990

토양 분리균인 Bacillus spp.가 생산하는 inulinase를 ammonium sulfate 분획, 열처리, DEAE Sephadex Cl-6B ion exchange chromatography, Sephadex G-100 및 Sephadex G-150 gel 여과 등의 과정을 거쳐 단일 단백질로 분리 정제하였다. 정제 inulinase는 분자량이 약 56,000인 효소로서 pH6.0,$50^{\circ}C$에서 최대 활성을 나타내었으며 $Co^{2+}$와 $Mn^{2+}$에 의해서 현저한 활성화 효과를 보였다. 또한 본 효소는 sucrose와 raffinose에 대해서도 높은 활성을 나타내므로 $\beta$-D-fructofuranosidase(EC 3.2.1.26)로 분류되는 exo-inulinase인 것으로 확인되었다. 한편 효소활성에 필수적인 아미노산 잔기는 tryptophan과 histidine인 것으로 분석되었으며 inulin과 sucrose에 대한 $K_m$값은 각각 $2.0 \times 1.0^[-3}M, 1.0 \times 10^[-2}M$로 산출되었다.

• BMB Reports
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• 제49권11호
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• pp.585-586
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• 2016

Extracellular vesicles (EVs) are natural carriers of biomolecules that play central roles in cell-to-cell communications. Based on this, there have been various attempts to use EVs as therapeutic drug carriers. From chemical reagents to nucleic acids, various macromolecules were successfully loaded into EVs; however, loading of proteins with high molecular weight has been huddled with several problems. Purification of recombinant proteins is expensive and time consuming, and easily results in modification of proteins due to physical or chemical forces. Also, the loading efficiency of conventional methods is too low for most proteins. We have recently proposed a new method, the so-called exosomes for protein loading via optically reversible protein-protein interaction (EXPLORs), to overcome the limitations. Since EXPLORs are produced by actively loading of intracellular proteins into EVs using blue light without protein purification steps, we demonstrated that the EXPLOR technique significantly improves the loading and delivery efficiency of therapeutic proteins. In further in vitro and in vivo experiments, we demonstrate the potential of EXPLOR technology as a novel platform for biopharmaceuticals, by successful delivery of several functional proteins such as Cre recombinase, into the target cells.

• Applied Microscopy
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• 제26권2호
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• pp.157-175
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• 1996

The development of flexor digital tendon of the hand was studied by electron microscopy in human fetuses ranging from 9 mm to 260 mm crown rump length. The primordium of tendons was first identified as discrete collection of mesenchymal cells at 25 mm fetus. Synovial sheath formation had commenced by 40 mm fetus and was complete by 70 mm fetus. Cell junction or adhesion sites at all ages were noted between the tendon cells. When dilatation of the synovial cavity occurred, two types of synovial cells were observed. A-type cells had numerous vesicles and large vacuoles. In contrast, B-type cells were characterized by abundant rough endoplasmic reticulum and well-developed Golgi complex. By $150mm{\sim}260mm$ fetuses, a mojority of the synovial cells were type B. The most remarkable difference between the synovial cells of full-term fetus and adult was the larger amount of collagen fibers in the latter. The vascular buds were first observed between the individual fibril bundles in the interfascicular space at 150 mm fetus. At 25 mm fetus, collagen fibrils were first noted within narrow cytoplasmic recesses which were continued with the extracellular space. Collagen fibrils were filled in almost entire extracellular space at 150 mm fetus. Besides collagen fibrils in the extracellular space small elastic fibers were also identified and followed in their development.

• Molecules and Cells
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• 제23권1호
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• pp.1-10
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• 2007

Invading pathogens are recognized by diverse germline-encoded pattern-recognition receptors (PRRs) which are distributed in three different cellular compartments: extracellular, membrane, and cytoplasmic. In mammals, the major extracellular PRRs such as complements may first encounter the invading pathogens and opsonize them for clearance by phagocytosis which is mediated by membrane-associated phagocytic receptors including complement receptors. The major membrane-associated PRRs, Toll-like receptors, recognize diverse pathogens and generate inflammatory signals to coordinate innate immune responses and shape adaptive immune responses. Furthemore, certain membrane-associated PRRs such as Dectin-1 can mediate phagocytosis and also induce inflammatory response. When these more forefront detection systems are avoided by the pathogens, cytoplasmic PRRs may play major roles. Cytoplasmic caspase-recruiting domain (CARD) helicases such as retinoic acid-inducible protein I (RIG-I)/melanoma differentiation-associated gene 5 (MDA5), mediate antiviral immunity by inducing the production of type I interferons. Certain members of nucleotide-binding oligomerization domain (NOD)-like receptors such as NALP3 present in the cytosol form inflammasomes to induce inflammatory responses upon ligand recognition. Thus, diverse families of PRRs coordinately mediate immune responses against diverse types of pathogens.

• 한국수정란이식학회지
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• 제8권2호
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• pp.83-90
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• 1993

To investigate the effect of extracellular matrix proteins on the in vitro development of ethanol-induced parthenogenetic eggs of ICR strain mice, those were cultured in vitro in fibronectin, gelatin, or collagen precoated culture dishes containing 1.5 ml of NaH-C03$_3$-BMOC-3 medium at 37$^{\circ}C$ for 96 hrs. under the atmosphere of 5% $CO_2$ and 95% air. Fibronectin, gelatin, or collagen significantly(P$\pm$1.4, 45.4i1.4, and 44.8$\pm$O.9, respectively. And the diameter of those eggs ranged 104.6$\pm$1.9, 102.8$\pm$2.3, and 103.4$\pm$O.8 $\mu$m, respectively.

• Journal of Microbiology and Biotechnology
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• 제6권6호
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• pp.439-444
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• 1996

A bacterial strain, designated as WY22, producing extracellular chitinase was isolated from the soil around the Youngduck area, after enrichment culture in a medium containing $1{\%}$ (w/v) wet colloidal chitin as a sole carbon source. The isolate was identified as a strain of Bacillus sp. based on its morphological and physiological characteristics. It was observed that Bacillus sp. WY22 could inhibit the growth of Fusarium oxysporum with hyphal extention-inhibition assay on potato dextrose agar plate supplemented with $1{\%}$ collidal chitin. Optimum culture conditions of Bacillus sp. WY22 were examined for chitinase production in a chitin medium. High level production of chitinase was observed not only in the chitin medium but in a medium supplemented with $1{\%}$ N-glucosamine or lactose instead of chitin. The optimum concentrations of colloidal chitin and yeast extract were 3.0 and $0.5{\%}$, and the optimum culture conditions for initial pH of medium and temperature were 7.0 and $30^{\circ}C$, respectively, for the production of chitinase.

• 한국미생물·생명공학회지
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• 제24권4호
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• pp.408-414
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• 1996

The SecY is a central component of the protein export machinery that mediate the translocation of secretory proteins across the plasma membrane, and has been known to be rate-limiting factor of secretion in Escherichia coli. In order to study the extracellular protein secretion in Gram-positive microorganism, we have, constructed strains harboring more than one copy of the gene for SecY. Firstly, the gene, for B. subtilis SecY and its promoter region was subcloned into pDH32 and the chimeric vector was inserted into amyE locus by homologous recombination. Secondly, low copy number vector, pCED6, was also used for subcloning the secY gene and for constructing a strain which harbors several copies of secY. The KH1 cell which harbor two copies of secY on the chromosome excreted more extracellular proteins than the wild type PB2. Moreover, the KH2 cells which harbor several copies of secY in pCED6 vector excreted more extracellular proteins than the KH1 cells. Here, we found that the capacity of protein secretion is partly controlled by the number of secY and it is suggested that SecY has also an important role in protein secretion in B. subtilis, a gram positive microorganism, as like in E. coli. This will promote the use of B. subtilis as a host for the expression of useful foreign gene and excretion of precious proteins.