• Journal of Microbiology and Biotechnology
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• 제29권10호
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• pp.1522-1542
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• 2019

To adapt to environmental changes and to maintain cellular homeostasis, microorganisms adjust the intracellular concentrations of biochemical compounds, including metal ions; these are essential for the catalytic function of many enzymes in cells, but excessive amounts of essential metals and heavy metals cause cellular damage. Metal-responsive transcriptional regulators play pivotal roles in metal uptake, pumping out, sequestration, and oxidation or reduction to a less toxic status via regulating the expression of the detoxification-related genes. The sensory and regulatory functions of the metalloregulators have made them as attractive biological parts for synthetic biology, and the exceptional sensitivity and selectivity of metalloregulators toward metal ions have been used in heavy metal biosensors to cope with prevalent heavy metal contamination. Due to their importance, substantial efforts have been made to characterize heavy metal-responsive transcriptional regulators and to develop heavy metal-sensing biosensors. In this review, we summarize the biochemical data for the two major metalloregulator families, SmtB/ArsR and MerR, to describe their metal-binding sites, specific chelating chemistry, and conformational changes. Based on our understanding of the regulatory mechanisms, previously developed metal biosensors are examined to point out their limitations, such as high background noise and a lack of well-characterized biological parts. We discuss several strategies to improve the functionality of the metal biosensors, such as reducing the background noise and amplifying the output signal. From the perspective of making heavy metal biosensors, we suggest that the characterization of novel metalloregulators and the fabrication of exquisitely designed genetic circuits will be required.

• Journal of Microbiology and Biotechnology
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• 제17권9호
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• pp.1460-1468
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• 2007

In this study, an approx. 2.5-kb gene fragment including the catalase gene from Rhodospirillum rubrum S1 was cloned and characterized. The determination of the complete nucleotide sequence revealed that the cloned DNA fragment was organized into three open reading frames, designated as ORF1, catalase, and ORF3 in that order. The catalase gene consisted of 1,455 nucleotides and 484 amino acids, including the initiation and stop codons, and was located 326 bp upstream in the opposite direction of ORF1. The catalase was overproduced in Escherichia coli UM255, a catalase-deficient mutant, and then purified for the biochemical characterization of the enzyme. The purified catalase had an estimated molecular mass of 189 kDa, consisting of four identical subunits of 61 kDa. The enzyme exhibited activity over a broad pH range from pH 5.0 to pH 11.0 and temperature range from $20^{\circ}C$ to $60^{\circ}C$C. The catalase activity was inhibited by 3-amino-1,2,4-triazole, cyanide, azide, and hydroxylamine. The enzyme's $K_m$ value and $V_{max}$ of the catalase for $H_2O_2$ were 21.8 mM and 39,960 U/mg, respectively. Spectrophotometric analysis revealed that the ratio of $A_{406}$ to $A_{280}$ for the catalase was 0.97, indicating the presence of a ferric component. The absorption spectrum of catalase-4 exhibited a Soret band at 406 nm, which is typical of a heme-containing catalase. Treatment of the enzyme with dithionite did not alter the spectral shape and revealed no peroxidase activity. The combined results of the gene sequence and biochemical characterization proved that the catalase cloned from strain S1 in this study was a typical monofunctional catalase, which differed from the other types of catalases found in strain S1.

• 미생물학회지
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• 제51권1호
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• pp.48-60
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• 2015

순창군에서 제조된 전통 장류로부터 612종의 다양한 미생물을 분리하였고, 612종의 미생물 중에서 생리활성 분석 및 biogenic amine 생성여부를 조사하여 최종적으로 Bacillus subtilis SCJ4를 선별하였다. 최종 선별한 균주 SCJ4의 생화학적 특성과 16S rRNA 염기서열 분석에 의해 B. subtilis SCJ4로 명명하였으며, B. subtilis SCJ4의 산업적으로 사용이 적합한 최적배지조성을 확립하기 위하여 통계학적 방법인 반응표면 분석법을 사용하였다. 실험계획법은 배지조성을 예측하기 위해 Plackett-Burman design을 사용하여 모델을 설정하였다. 실험 모델을 통하여 선별된 배지 조성으로는 tryptone, peptone, $MgSO_4$로 예측되었으며, 배지조성에 대한 농도를 설정하기 위해 중심합성계획법을 사용하여 최적화를 수행하였다. 최종 예측된 배지 조성의 농도는 tryptone 15.35 g/L, peptone 12.235 g/L, $MgSO_4$ 3.5 g/L로 예측되었다. 이때 최대 균체량은 1.07497 g/L로 예측되었으며, 모델의 검증실험을 통하여 예측된 모델과 실제 결과가 동일함을 확인하였으며, 기본배양배지와 비교하였을 때 균체량이 0.8767 g/L에서 1.1222 g/L으로 약 28%증가하였다.

• Asian-Australasian Journal of Animal Sciences
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• 제17권10호
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• pp.1411-1416
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• 2004

The objective of this work was to isolate a microorganism, able to produce high lactate dehydrogenase (LDH) activity, for use as a microbial feed additive. The LDH is an important enzyme for lactate conversion in the rumen, thereby possibly overcoming lactic acidosis owing to sudden increases of cereal in the diets of ruminants. In the present study, various bacterial strains were screened from a variety of environments. Among the isolated microorganisms, strain FFy 111-1 isolated from a Korean traditional fermented vegetable food called Kimchi showed the highest enzyme activity, along with retaining strong enzyme activity even in rumen fluid in vitro. Based on morphological and biochemical characteristics as well as compositions of cellular fatty acids plus API analyses, this strain was identified as Lactobacillus sp. The optimum temperature and pH for growth were found to be 30$^{\circ}C$ and pH 6.5, respectively. A maximum cell growth of 2.2 at $A_{650}$ together with LDH activity of 2.08 U per mL was achieved after 24 h of incubation. Initial characterization of FFy 111-1 suggested that it could be a potential candidate for use as a direct-fed microbial in the ruminant animals.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.88.1-88
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• 2003

Antogonistic bacteria against Rhizoctonia solani and Pythium spp., causing serious damage to golf course grasses, were isolated from the top soil of several golf courses in Korea. The isolate of Limk0102 was selected as the biological agent by characterization of antifungal activity, large scale preparation, fungicides tolerance and ecological fitness to the targe environment. The isolate was identified as Bacillus subtilis by biochemical and physiological characterization, and 165 rDNA sequence analysis. The bacterial agent was formulated as a granule type by seeding it on fine sand. The formulated agent showed high recovery rate (more than 10$\^$8/ cells/g sand) even after 6 month-storage at room temperature with similar antifungal activity with that of original cells. In vitro, the biological agent successfully exhibited antagonistic performance on bentgrass inoculated with R. solani or Pythium spp. isolated from the diseased grasses on golf courses. Field evaluation on disease control activity and ecological fitness of the agent is now under going on several golf courses.

• Food Science and Biotechnology
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• 제18권4호
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• pp.878-883
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• 2009

In Brazil canned 'Biuti' peach is a very popular form of this sub-tropical fruit. This production represents an important economic agro-activity in Minas Gerais, Brazil during the summer period, in preparation for the Christmas celebrations. The aim of this work was to characterize the 'Biuti' peach polyphenoloxidase (PPO), since peach products show enzymatic oxidation of the polyphenols by oxidative enzymes, which affects the products during their shelf life. Two different hypothesis for the browning problem in processed peaches were studied: the inadequacy of the blanching treatment and the presence of a latent phenolase in the peaches. The PPO was characterized: pH optimum (5.5) and stability (5.5-6.5); optimum temperature at $20^{\circ}C$ and 80% of the activity retained after 30 min at $15-40^{\circ}C$. The test for the presence of latent PPO in the processed and canned peaches was negative. Ascorbic acid, ${\beta}$-mercaptoethanol, sodium metabisulfite, and cysteine were efficient in inhibiting the PPO.

• Journal of Microbiology and Biotechnology
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• 제13권4호
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• pp.628-631
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• 2003

The recombinant alanine dehydrogenase (ADH) from E. coli containing Thermus caldophilus ADH was purified to homogeneity from a cell-free extract. The enzyme was purified 38-fold with a yield of 68% from the starting cell-free extract. The purified enzyme gave a single band in polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 45 kDa. The pH optimum was 8.0 for reductive amination of pyruvate and 12.0 for oxidative deamination of L-alanine. The enzyme was stable up to $70^{\circ}C$. The activity of the enzyme was inhibited by 1 mM $Zn^{2+}$, 20% hexane, and 20% $CHCl_3$. However, 10 mM $Mg^{2+}$ and 40% propanol had no effect on the enzyme activity. The Michaelis constants ($K_m$) for the substrates were $50\;\mu\textrm{M}$ for NADH, 0.2 mM for pyruvate, 39.4 mM for $NH_4+$, 2.6 mM for L-alanine, and 1.8 mM for $NAD^+$.

• Journal of Microbiology and Biotechnology
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• 제23권9호
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• pp.1229-1243
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• 2013

In this paper, an air isolate (NITT6L) has been screened based on hemolytic activity, emulsification activity, drop collapsing test, and oil displacement test, as well as lipase activity. It was found that strain NITT6L was able to reduce the surface tension of the medium from 61.5 to 39.83 mN/m and could form stable emulsions with tested vegetable oils. Morphological, biochemical, 16S rRNA sequencing analyses, and fatty acid methyl ester analysis using gas chromatography confirmed that the air isolate under study was Pseudomonas aeruginosa. Characterization of the biosurfactant using agar double diffusion assay revealed that the biosurfactant was anionic in nature, and CTAB-methylene blue assay and Molisch test revealed its glycolipid nature. The FT-IR spectrum confirmed that the crude biosurfactant was a rhamnolipid. Using unoptimized medium containing sucrose as the carbon source, the isolate was found to produce 0.3 mg/ml of rhamnolipid in batch cultivation (shake flask) at $37^{\circ}C$ and pH 7. Optimization of the medium components was carried out using design of experiments and the yield of rhamnolipid has been enhanced to 4.6 mg/ml in 72 h of fermentation.

• Journal of Microbiology and Biotechnology
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• 제13권5호
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• pp.738-744
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• 2003

The gene ADH encoding NAD-dependent alcohol dehydrogenase from Bacillus stearothennophilus was cloned and overexpressed as a GST fusion protein at a high level in Escherichia coli. The expressed fusion protein was purified simply by glutathione affinity chromatography. GST fusion protein was then cleaved by thrombin, while soluble enzyme was further purified by glutathione affinity chromatography. The recombinant enzyme had the same elctrophoretic mobility as the native enzyme from Bacillus stearothennophilus. The recombinant enzyme catalyzed the oxidation of a number of alcohols and exhibited high activities towards secondary alcohols. The $K_m\;and\;V_{max}$ values of the recombinant enzyme for ethanol were 5.11 mM and 61.35 U/mg, respectively. Pyridine and imidazole notably inhibited the enzymatic activity. The activity of the recombinant enzyme optimally proceeded at pH 9.0 and $70^{\circ}C$. The midpoint of the temperature-stability curve for the recombinant enzyme was approximately $68^{\circ}C$, and the enzyme was not completely inactivated even at $85^{\circ}C$. The recombinant enzyme showed a high resistance towards denaturing agents (0.05% SDS, 0.1 M urea). Therefore, due to its stability and relatively broad substrate specificity, the recombinant enzyme could be utilized in bio-industrial processes and biosensors.

• BMB Reports
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• 제35권3호
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• pp.343-347
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• 2002

The human protein tyrosine kinase-6 (PTK6) polypeptide that is deduced from the cDNA sequence contains a Src homology (SH) 3 domain, SH2 domain, and catalytic domain of tyrosine kinase. We initiated biochemical and NMR characterization of PTK6 SH3 domain in order to correlate the structural role of the PTK6 using circular dichroism and heteronuclear NMR techniques. The circular dichroism data suggested that the secondary structural elements of the SH3 domain are mainly composed of $\beta$-sheet conformations. It is most stable when the pH is neutral based on the pH titration data. In addition, a number of cross peaks at the low-field area of the proton chemical shift of the NMR spectra indicated that the PTK6 SH3 domain retains a unique and folded conformation at the neutral pH condition. For other pH conditions, the SH3 domain became unstable and aggregated during NMR measurements, indicating that the structural stability is very sensitive to pH environments. Both the NMR and circular dichroism data indicate that the PTK6 SH3 domain experiences a conformational instability, even in an aqueous solution.