• Biotechnology and Bioprocess Engineering:BBE
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• 제9권3호
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• pp.147-150
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• 2004

Permeabilization is known to overcome cell membrane barriers of whole cell biocatalysts. The use of organic solvents is advantageous in terms of cost, simplicity, and efficiency. In this study, Ochrobactrum anthropi SY509 was permeabilized with various organic solvents. Treatment with organic solvents resulted in lower permeability barriers due to falling out lipids of the cell membrane. Therefore, permeabilized cells showed higher enzyme activity with no cell viability. Among various organic solvents, 0.5% (v/v) chloroform was selected as the most efficient permeabilizing reagent. Changes in the cell membrane structure were observe d and the residual amounts of phospholipids of the cell membrane were measured to investigate the mechanism of the improved permeability.

• Journal of Microbiology and Biotechnology
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• 제17권8호
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• pp.1308-1315
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• 2007

Lipase B from Candida antarctica (CalB) displayed on the cell surface of H. polymorpha has been functionally improved for catalytic activity by molecular evolution. CalB was displayed on the cell surface by fusing to a cell-wall anchor motif (CwpF). A library of CalB mutants was constructed by in vivo recombination in H. polymorpha. Several mutants with increased whole-cell CalB activity were acquired from screening seven thousand transformants. The two independent mutants CalB 10 and CalB 14 showed an approximately 5 times greater whole-cell activity than the wild-type. When these mutants were made as a soluble form, CalB 10 showed 6 times greater activity and CalB 14 showed an 11 times greater activity compared with the wild-type. Sequence analyses of mutant CALB genes revealed amino acid substitutions of $Leu^{278}Pro$ in CalB10 and $Leu^{278}Pro/Leu^{219}Gln$ in CalB14. The substituted $Pro^{278}$ in both mutants was located near the proline site of the ${\alpha}$10 helix. This mutation was assumed to induce a conformational change in the ${\alpha}$10 helix and increased the $k_{cat}$ value of mutant CalB approximately 6 times. Site-directed mutagenized CalB, LQ ($Leu^{219}Gln$) was secreted into the culture supernatant at an amount of approximately 3 times more without an increase in the CalB transcript level, compared with the wild-type.

• Journal of Microbiology and Biotechnology
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• 제24권3호
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• pp.379-385
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• 2014

The purpose of this study was to compare the ability of an engineered Escherichia coli to degrade chlorpyrifos (Cp) using an organophosphorus hydrolase enzyme, encoded in both Flavobacterium sp. ATCC 27551 or Pseudomonas diminuta, by employing the Lpp-OmpA chimera and the N-terminal domain of the ice nucleation protein as anchoring motifs. Tracing of the expression location of the recombinant protein using SDS-PAGE showed the presentation of OPH by both anchors on the outer membrane. This is the first report on the presentation of OPH on the cell surface by Lpp-OmpA under the control of the T7 promoter. The results showed cell growth in the presence of Cp as the sole source of energy, without growth inhibition, and with higher whole-cell activity for both cells harboring plasmids pENVO and pELMO, at approximately 10,342.85 and 10,857.14 U/mg, respectively. Noticeably, the protein displayed by pELMO was lower than the protein displayed by pENVO. It can be concluded that Lpp-OmpA can display less protein, but more functional OPH protein. These results highlight the high potential, of both engineered bacteria, for use in the bioremediation of pesticide-contaminated sources in the environment.

• 대한수의학회지
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• 제27권2호
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• pp.185-189
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• 1987

The number of splenic lymphocyte, serum prostaglandin $E_2$ level and natural killer cell activity were assayed after single whole body irradiation of a sublethal dose of $^{60}Co-{\gamma}$ ray to C57BL/6J mice. With a view to knowing the relationships between radiation induced prostaglandin $E_2$ level and the normal natural killer cell activity after natural killer cell-target cell conjugation, The change of normal natural killer cell activity were measured by administration of prostaglandin $E_2$ containing serum from irradiated mice. The results were summarized as follows; 1. The total number of splenic lymphocyte was significantly decreased by irradiation and the number was not affected by indometacin, prostaglandin synthesis inhibitor, treatment. 2. Serum prostaglandin $E_2$ level was increased in irradiated mice, but indometacin treated mice group showed low level of prostaglandin $E_2$. 3. In the case of irradiated mice, natural killer cell activity was not shown any difference between irradiated group and indometacin combined group. But when natural killer cell-target cell conjugations were exposed to the serum of each group during cytotoxic activity assay, whereas the normal natural killer cell activity was significantly decreased by treatment of serum from irradiated mice, the activity was not changed by treatment of indometacin pretreated mice serum. This result indicated that the prostaglandin $E_2$ induced by the radiation inhibited the post-target binding cytolytic process of natural killer activity.

• 한국식품저장유통학회지
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• 제7권1호
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• pp.124-131
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• 2000

Antibacterial activities of powdered spices(garlic , ginger, cinnamon and clove) against pathogenic Escherichia coli )157:H7 and Staphyloccus auresus were investigated. Spice powder was added in was exponetial phase of each bacterial culture . Growth inhibition was determined by the absorbance at 660nm and morphological changes of the cells were observed by transmission electron microscope (TEM). Ginger powder has the highest antibacterial activity, following cinnamon , clove and garlic has the least activity.Growth of Escherichia coli O157:H7 and Staphyloccus aureus were completely inhibited within 5 hours after addition of 1 % of garlic , 0.3% of ginger or cinnamon , 0.5% of clove powder on the exponential phase of the cells. Spice untreated cells of E. coli and S. aureus, the cytoplasm was entirely surrounded by rigid cell wall and cell walls formed a smooth layer well attached to the plasma membrane. In the cells of E. coli and S. aureus treated with spice powder, cell wall and plasma membrane were lysed and severely damaged. E.coli cells growth in the presence of spice powder showed plammolysis, the loss of electron dense material, the formation of extra cellular blebs and cytoplasm burst out from the cell. S .sureus cells grown in the presence of spice powder showed swell of cell wall, the loss of electron dense material , coagulation of cell cytoplasm and formation of extra cellular blebs. Severely damaged cells of S. aureus lost whole cytoplasm and left as ghost of the cell. Spice powder stimulated autolyssi and induced cell death.

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권6호
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• pp.530-537
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• 2006

Recently isolated, Pseudomonas putida SN1 grows on styrene as its sole carbon and energy source through successive oxidation of styrene by styrene monooxygenase (SMO), styrene oxide isomerase (SOI), and phenylacetaldehyde dehydrogenase. For the production of (S)-styrene oxide, two knockout mutants of SN1 were constructed, one lacking SOI and another lacking both SMO and SOI. These mutants were developed into whole-cell biocatalysts by transformation with a multicopy plasmid vector containing SMO genes (styAB) of the SN1. Neither of these self-cloned recombinants could grow on styrene, but both converted styrene into an enantiopure (S)-styrene oxide (e.e. > 99%). Whole-cell SMO activity was higher in the recombinant constructed from the SOI-deleted mutant (130 U/g cdw) than in the other one (35 U/g cdw). However, the SMO activity of the former was about the same as that of the SOI-deleted SN1 possessing a single copy of the styAB gene that was used as host. This indicates that the copy number of styAB genes is not rate-limiting on SMO catalysis by whole-cell SN1.

• 약학회지
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• 제42권3호
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• pp.336-344
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• 1998

Effects of ochratoxin A (OTA) on embryo development were studied in cultured whole embryos from 9.5 day gestation rat for 48 h. OTA (more than $0.5{\mu}g/ml$) induced microcephaly in the cultured rat whole embryos. Protein and DNA content, and DNA synthesis were significantly inhibited by OTA. We next examined whether the microcephaly seen in cultured whole embryo partially results from inhibition of differentiation of embryonic midbrain cells. Embryonic midbrain cells were extracted from 12 day gestation rat embryos, and cultured for 96 hr. OTA ibhibited cell differentiation about 50% over control. We also tested whether OTA-induced embryotoxicity would be associated with oxidative damages. We measured the ${\gamma}$-glutamyltranspeptidase (${\gamma}$-GT) and glutathione peroxidase (GPX) activities, and glutathione (GSH) content in both cultured whole embryos and embryonic midbrain cells. OTA decreased GSH content, whereas slightly increased ${\gamma}$-GT activity, but GPX activity was not significantly changed. These results show that OTA caused the microcephaly and its effect may be partially due to the inhibition of cell differentiation of embryonic midbrain cells, but the role of oxidative damages is not clear in embryotoxicity.

• Journal of Microbiology and Biotechnology
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• 제24권1호
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• pp.127-131
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• 2014

While searching for lactic acid bacteria that can restore aging-impaired immune responses, we isolated the Toll-like receptor (TLR) 2/NF-${\kappa}B$-activating strain Lactobacillus plantarum HY7712 from kimchi and investigated its immunomodulating effect in whole-body ${\gamma}$-irradiated mice. Exposure to HY7712 strongly activated NF-${\kappa}B$ signaling in RAW264.7 cells, but inhibited lipopolysaccharide-stimulated NF-${\kappa}B$ activation. Moreover, HY7712 protected against the downregulation of interferon (IFN)-${\gamma}$ and upregulation of interleukin (IL)-13 caused by ${\gamma}$-irradiation in mice. In mice, ${\gamma}$-irradiation impaired NK-cell activity against YAC-1 tumor cells, but following HY7712 exposure, the activity of NK cells was restored to 91.5% of the level measured in control mice (p < 0.05). These findings suggest that HY7712 activates the TLR2/NF-${\kappa}B$ signaling pathway and protects against the impairment of NK-cell activity caused by ${\gamma}$-irradiation or aging.

• 미생물학회지
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• 제27권1호
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• pp.70-76
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• 1989

Progestrone bioconversion by immobilized Aspergillus phoenicis was studied. Progesterone was converted into 11$\alpha$-hydroxyprogesterone and 3-minor byproducts. Whole cells of A. phoenicis were immobillized by enreappment with calcium-alginate, K-carrageenan, or polyacrylamide. Of these materials tested, cell immobilized in $Ca^{2+}$ -alginate gels showed the highest activity for 11$\alpha$-hydroxylation of progesterone. In the case of mycelia immobilized in $Ca^{2+}$-alginate, futher progressing hydroxylation of 11$\alpha$-hydroxyprogesterone was greatly reduced. Spores of A. phoenicis which were immobillized with $Ca^{2+}$-alginate and germinatedin situ for 25 hours showed higher 11$\alpha$-hydroxylase activity than those of entrapped whole mycelia and maintained initial enzyme activity for all 8 times of repeated use. After 16 times of reuse, the activity was declined 30% or more. When culture media and $Zn^{2+}$ were introduced into the reaction media, the activity of the immobilized mycelia which had been lowered due to many times of reuse was effectively reactivated.

• 유기물자원화
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• 제14권3호
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• pp.110-116
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• 2006

본 연구에서는 재조합 대장균으로 부터 Organophosphorus Hydrolase (OPH)를 이용하여 유기인 살충제 화합물인 paraoxon의 생분해 속도를 향상시켰다. OPH의 비 활성도 (Specific whole cell OPH Activity)를 증가시키기 위한 배지의 최적 조건은 초기 pH 8.5의 조절과 5.0 % acetone 첨가가 필요하다는 것을 알 수 있었다. 또한, 이 최적의 조건에서 498 Unit/L의 OPH가 생산될 때, 275 mg/L paraoxon은 반응 10분 동안 98% 생분해 효율을 나타내었고, 그 결과 생분해 속도를 $29.2mg/g{\cdot}min$까지 향상시킬 수 있었다. 이러한 실험 결과들은 지하수 또는 토양에 잔류하는 유기인 살충제를 빠른 시간 안에 효과적으로 생분해시키는 실질적인 생물 복원 기술로 사용될 수 있을 것이다.