• 한국균학회지
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• 제25권2호통권81호
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• pp.143-151
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• 1997

Aspergillus niger와 Penicillium notatum간의 lipase 우수 생성 종간 형질전환체를 획득하고, 유전분석과 정제를 통하여 다음과 같은 결과를 얻었다. P. notatum의 영양요구물을 확인하여 본 결과 Tyrosine 요구성임을 알 수 있었다. 핵전이를 위한 원형질체 형성 및 재생 조건에서는 Novozym 234의 농도 1%, 삼투안정제는 0.6M KCl, 효소의 처리시간은 180분 그리고 최적 pH는 5.8로 나타났다. 핵전이에 의한 형질전환의 빈도는 $1.3{\times}10^{-4}{\sim}3.8{\times}10^{-4}$으로 비교적 낮은 편이었다. 유전적 안정성, conidia의 크기, DNA 함량의 측정 그리고 핵염색의 결과 형질전환체의 핵형은 aneuploid로 추정되었다. 형질전환체의 lipase 활성은 모균주와 비교하여 $1.2{\sim}1.7$배 증가하였다.

• Biomolecules & Therapeutics
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• 제23권2호
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• pp.189-194
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• 2015

P450 1A2 is responsible for the metabolism of clinically important drugs and the metabolic activation of environmental chemicals. Genetic variations of P450 1A2 can influence its ability to perform these functions, and thus, this study aimed to characterize the functional significance of three P450 1A2 allelic variants containing nonsynonymous single nucleotide polymorphisms (P450 $1A2^*8$, R456H; $^*15$, P42R; $^*16$, R377Q). Variants containing these SNPs were constructed and the recombinant enzymes were expressed and purified in Escherichia coli. Only the P42R variant displayed the typical CO-binding spectrum indicating a P450 holoenzyme with an expression level of ~ 170 nmol per liter culture, but no P450 spectra were observed for the two other variants. Western blot analysis revealed that the level of expression for the P42R variant was lower than that of the wild type, however the expression of variants R456H and R377Q was not detected. Enzyme kinetic analyses indicated that the P42R mutation in P450 1A2 resulted in significant changes in catalytic activities. The P42R variant displayed an increased catalytic turnover numbers ($k_{cat}$) in both of methoxyresorufin O-demethylation and phenacetin O-deethylation. In the case of phenacetin O-deethylation analysis, the overall catalytic efficiency ($k_{cat}/K_m$) increased up to 2.5 fold with a slight increase of its $K_m$ value. This study indicated that the substitution P42R in the N-terminal proline-rich region of P450 contributed to the improvement of catalytic activity albeit the reduction of P450 structural stability or the decrease of substrate affinity. Characterization of these polymorphisms should be carefully examined in terms of the metabolism of many clinical drugs and environmental chemicals.

• 대한의용생체공학회:의공학회지
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• 제23권2호
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• pp.139-145
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• 2002

2.2-Bis[P-(2-hydroxy-3-methacryloyloxypropoxyl)phenol]propane (Bis-GMA)는 광중합형 치과용 고분자 복합레진의 다관능성 유기물로 널리 사용되고 있다. Bis-GMA는 두 개의 히드록시기를 가지고 있어서 구강내에서 광중합되는 복합레진의 물흡수를 야기시켜 장기간 점진적으로 심미성이 감소하고 결국엔 복합레진의 기계적 특성까지 저하시키게 된다. 본 연구에서는 최종제품의 내구성과 저장안정성을 향상시키기 위하여 복합레진에 광안정제와 산화방지제를 첨가제로 사용하였다. 먼저 Tinuvin P의 광안정제를 첨가하면 색변화가 큰 것을 볼 수 있었으나 Irganox 245의 산화방지제를 첨가하였을 때 색안정성이 향상됨을 알 수 있었다. 또한 Tinuvin P와 Irganox 245가 동시에 첨가되었을 때 색안 정성이 향상되며 시료의 가속화 실험 전과 가속화 후 간접인장강도와 같은 기계적 성질도 크게 감소되지 않았다. 따라서 치과용 복합레진의 제조에 있어서 첨가제인 Tinuvin P와 Irganox 245가 각각 0.5% 및 0.1% 첨가되었을 때 내구성 및 색안정성이 향상되었으며 나아가 저장 안정성도 향상됨을 확인할 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제26권1호
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• pp.20-27
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• 2016

Lactobacillus kefiranofaciens ZW3 was obtained from kefir grains, which have high lactose hydrolytic activity. In this study, a heterodimeric LacLM-type β-galactosidase gene (lacLM) from ZW3 was isolated, which was composed of two overlapping genes, lacL (1,884 bp) and lacM (960 bp) encoding large and small subunits with calculated molecular masses of 73,620 and 35,682 Da, respectively. LacLM, LacL, and LacM were expressed in Escherichia coli BL21(DE3) and these recombinant proteins were purified and characterized. The results showed that, compared with the recombinant holoenzyme, the recombinant large subunit exhibits obviously lower thermostability and hydrolytic activity. Moreover, the optimal temperature and pH of the holoenzyme and large subunit are 60℃ and 7.0, and 50℃ and 8.0, respectively. However, the recombinant small subunit alone has no activity. Interestingly, the activity and thermostability of the large subunit were greatly improved after mixing it with the recombinant small subunit. Therefore, the results suggest that the small subunit might play an important role in maintaining the stability of the structure of the catalytic center located in the large subunit.

• 한국생물정보학회:학술대회논문집
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• 한국생물정보시스템생물학회 2005년도 BIOINFO 2005
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• pp.421-426
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• 2005

In this paper, we investigate the nonlinear dynamic behavior of TPP (thiamine pyrophosphate) riboswitches in E. coli (Escherichia coli). TPP riboswitches are highly conserved RNA regulatory elements, embedded within the 5’'untranslated region of three TPP biosynthesis operons. The three operons thiCEFSGH, thiMD, and thiBPQ are involved in the biosynthesis, salvage, and transport of TPP, respectively. TPP riboswitches modulate their expressions in response to changing TPP concentration, without involving protein cofactors. Interestingly, the expression of thiMD is regulated at the translational level, while that of thiCEFSGH at both levels of transcription and translation. We develop a mathematical model of the TPP riboswitch’s regulatory system possessed by thiCEFSGH and thiMD, so as to simulate the time-course experiments of TPP biosynthesis in E. coli. The simulation results are validated against three sets of reported experimental data in order to gain insight into the nature of steady states and the stability of TPP riboswitches, and to explain the biological significance of regulating at level of transcription or translation, or even both. Our findings suggest that in the TPP biosynthesis pathway of E. coli, the biological effect of down-regulating thiCEFSGH operon at the translational level by TPP riboswitch is less prominent than that at the transcriptional level.

• 한국환경보건학회:학술대회논문집
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• 한국환경보건학회 2005년도 국제학술대회
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• pp.337-340
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• 2005

Ethylene oxide is a genotoxic carcinogen with widespread uses as industrial chemical intermediate and gaseous sterilant. 2-hydroxyethylated N-terminal valine in Hb is a good biomarker for biological monitoring of ethylene oxide exposure, because of its stability. We studied the determination method of (N-2-hydroxy-ethyl)valine in hemoglobin adduct by using GC/MS. PFPITC and TBMS were used as appropriate derivatives. Ethylene oxide formed Hb adducts as (N-2-hydroxy-ethyl)valine(HEV) in mouse with ethylene oxide inhalation exposure. Standard HEV can be synthesized with 2-amino-ethanol and 2-bromo-3-methylbutyric acid. GC/MS can measured them after derivatization with pentafluorophenylisothiocianate(PFPITC) and N-(tertiary butyl dimethylsiiyl)-N-methyl-trifluoroacetamide(TBDMS-TFA) by using Edman procedure. Concentrations of Hb adduct were proportionally increased with exposure levels. They were 230${\pm}$35(nmol g$^{-1}$ globin) and 410${\pm}$72(nmol $g^{-1}$ globin) at 200ppm and 400ppm ethylene oxide inhalation exposure, respectively.

• Journal of Microbiology and Biotechnology
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• 제15권6호
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• pp.1317-1322
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• 2005

This work describes neo-fructooligosaccharides (neo-FOSs) production using the immobilized mycelia of Penicillium citrinum. Some critical factors were evaluated to optimize maximal production of neo-FOS. Optimal alginate and cell concentrations were determined to be $1.96\%$ alginate and $7.17\%$ cell, respectively, by statistical analysis. The optimal concentration of $CaCl_{2}$, which is related to bead stability, was determined to be 2 M. It was possible to increase the neo-FOS production by adding 15 units of glucose oxidase to the batch reaction. By co-immobilizing cells and glucose oxidase, neoFOS productivity increased $123\%$ compared with the whole-cell immobilization process. Based on the results above, a co-immobilization technique was developed and it can be utilized for large-scale production.

• Journal of Microbiology and Biotechnology
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• 제19권8호
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• pp.836-844
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• 2009

A denitrification bacterium was isolated from riverbed soil and identified as Ochrobactrum sp., whose specific enzymes for denitrification metabolism were biochemically assayed or confirmed with specific coding genes. The denitrification activity of strain G3-1 was proportional to glucose/nitrate balance, which was consistent with the theoretical balance (0.5). The modified graphite felt cathode with neutral red, which functions as a solid electron mediator, enhanced the electron transfer from electrode to bacterial cell. The porous carbon anode was coated with a ceramic membrane and cellulose acetate film in order to permit the penetration of water molecules from the catholyte to the outside through anode, which functions as an air anode. A non-compartmented electrochemical bioreactor (NCEB) comprised of a solid electron mediator and an air anode was employed for cultivation of G3-1 cells. The intact G3-1 cells were immobilized in the solid electron mediator, by which denitrification activity was greatly increased at the lower glucose/nitrate balance than the theoretical balance (0.5). Metabolic stability of the intact G3-1 cells immobilized in the solid electron mediator was extended to 20 days, even at a glucose/nitrate balance of 0.1.

• Applied Biological Chemistry
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• 제41권4호
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• pp.235-240
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• 1998

The myristoyl-acyl carrier protein (ACP) specific thioesterase from Iris tectorum was purified to a considerable homogeneity and characterized. The enzyme was eluted with a considerable stability by double-gradients using Triton X-100 and low ionic KCl or Na-phosphate through DEAE-52, Octyl-Sepharose, Q-Sepharose, and hydroxyapatite chromatoraphy. SDS-PAGE analysis showed a single band of 39 kDa. The native molecular weight was estimated to be 82 kDa by Sephacryl S-200 chromatography, indicating that the enzyme was a dimer. The thioesterase showed a chain-length specificity to myristoyl-ACP in preference to other-ACPs. The enzyme activity decreased by 1.0 mM myristate to about 27% of the original activity, whereas the remaining activity with decanoate was about 90%. The purified thioesterase was inhibited by myristoyl-CoA more than by myristate, suggesting that the myristoyl-AGP thiolesterase might be controlled by myristic acid and/or a subsequent product myristoyl-CoA. In addition, some biochemical characteristics of the enzyme were described.

• Journal of Microbiology and Biotechnology
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• 제12권1호
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• pp.96-105
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• 2002

A bacteriocin-producing strain identified as Pediococcus acidilactici was isolated from kimchi. The bacteriocin was identified to belong to the pediocin family and exhibited bactericidal activity against most Gram-positive bacteria as well as some Gram-negative bacteria. The bacteriocin was stable up to $80^{\circ}C with wide pH ranges (5.0-10.0). The bactericidal activity remained unchanged after treatment with nonproteolytic enzymes such as nuclease and ${\alpha}$-amylase, however, it was destroyed after treatment with protease. The bacteriocin was effectively extracted by the pH-mediated adsorption-desorption method and purified effectively by semi-preparative RP-HPLC. The molecular weight of the bacteriocin was 4,622, as determined by electrospray mass spectrometry. The amino acid sequence consisted of 44 amino acid residues with four cysteines. The high solubility and pH stability of the isolated pediocin provide definite advantages over nisin and other bacteriocins in regards to its potential applications.