• Journal of Applied Biological Chemistry
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• 제57권2호
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• pp.137-140
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• 2014

A gene encoding thermostable pectinase (TmPec) was isolated from hyperthermophilic microorganism, Thermotoga maritima. The open reading frame (ORF) of TmPec gene is 1,104 bp long and encodes 367 amino acid residues with a molecular weight of 40,605 Da. To analyze the enzymatic activity and biochemical properties, the ORF of TmPec gene excluding putative signal sequence of 27 amino acids was introduced into the E. coli expression vector, pRSET-B, and overexpressed in E. coli BL21. Protein concentration of purified recombinant TmPec was 1.1 mg/mL with specific activity of 56 U/mg protein on pectin. The recombinant TmPec showed the highest activity at around $85-95^{\circ}C$, and at around pH 6.5. It was stable at temperature below $85^{\circ}C$. In the presence of $Ca^{2+}$, the activity of recombinant TmPec was increased to 146.3% of normal level. In contrast, $Ba^{2+}$ and Mn2+ showed strong inhibition to the recombinant TmPec.

• Journal of Microbiology and Biotechnology
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• 제16권1호
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• pp.77-83
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• 2006

A gene encoding a $\gamma-butyrolactone$ autoregulator receptor was cloned from Saccharopolyspora erythraea, and the biochemical characteristics, including the autoregulator specificity, were determined with the purified recombinant protein. Using primers designed for the conserved amino acid sequence of Streptomyces $\gamma-butyrolactone$ autoregulator receptors, a 120 bp S. erythraea DNA fragment was obtained by PCR. Southern and colony hybridization with the 120 bp fragment as a probe allowed to select a genomic clone of S. erythraea, pESG, harboring a 3.2 kb SacI fragment. Nucleotide sequencing analysis revealed a 615 bp open reading frame (ORF), showing moderate homology (identity, $31-34\%$; similarity, $45-47\%$) with the $\gamma-butyrolactone$ autoregulator receptors from Streptomyces sp., and this ORF was named seaR (Saccharopolyspora erythraea autoregulator receptor). The seaR/pET-3d plasmid was constructed to overexpress the recombinant SeaR protein (rSeaR) in Escherichia coli, and the rSeaR protein was purified to homogeneity by DEAE-Sephacel column chromatography, followed by DEAE-ion-exchange HPLC. The molecular mass of the purified rSeaR protein was 52 kDa by HPLC gel-filtration chromatography and 27 kDa by SDS-polyacrylamide gel electrophoresis, indicating that the rSeaR protein is present as a dimer. A binding assay with tritium-labeled autoregulators revealed that rSeaR has clear binding activity with a VB-C-type autoregulator as the most effective ligand, demonstrating for the first time that the erythromycin producer S. erythraea possesses a gene for the $\gamma-butyrolactone$autoregulator receptor.

• Journal of Microbiology and Biotechnology
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• 제18권9호
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• pp.1544-1549
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• 2008

MhMTS and MhMTH are trehalose ($\alpha$-D-glucopyranosyl-[1,1]-$\alpha$-D-glucopyranose) biosynthesis genes of the thermophilic microorganism Metallosphaera hakonensis, and encode a maltooligosyltrehalose synthase (MhMTS) and a maltooligosyltrehalose trehalohydrolase (MhMTH), respectively. In this study, the two genes were fused in-frame in a recombinant DNA, and expressed in Escherichia coli to produce a bifunctional fusion enzyme, MhMTSH. Similar to the two-step reactions with MhMTS and MhMTH, the fusion enzyme catalyzed the sequential reactions on maltopentaose, maltotriosyltrehalose formation, and following hydrolysis, producing trehalose and maltotriose. Optimum conditions for the fusion enzyme-catalyzed trehalose synthesis were around $70^{\circ}C$ and pH 5.0-6.0. The MhMTSH fusion enzyme exhibited a high degree of thermostability, retaining 80% of the activity when pre-incubated at $70^{\circ}C$ for 48 h. The stability was gradually abolished by incubating the fusion enzyme at above $80^{\circ}C$. The MhMTSH fusion enzyme was active on various sizes of maltooligosaccharides, extending its substrate specificity to soluble starch, the most abundant natural source of trehalose production.

• 한국지하수토양환경학회지:지하수토양환경
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• 제18권4호
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• pp.32-39
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• 2013

This research focused on the effects of heavy metals on the biomonitoring activity of genetically engineered bioluminescent bacteria, Pseudomonas putida mt-2 KG1206. KG1206 was exposed to single or binary mixtures of different heavy metals as well as soils contaminated with heavy metals. In case of single exposure with different inducer pollutant, the toxicity order was as followings : As(III) > Cd, As(V) >> Cu, Cr(VI). The toxic effects of the binary mixtures was compared to the expected effect based on a simple theory of probabilities. The interactive effects were mostly additive, while in few cases antgonistic and synergistic mode of action was observed for some concentration combinations. No considerable correlation was found between the total metal contents in soils and the bioluminescence activity of each sample. However, statistically significant differences (p = 0.0102) were observed between two groups, classified based on arsenite contamination. These results demonstrate the usage of recombinant bioluminescent microorganism in biomonitoring and the complex interactive effects of metals.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 1986년도 추계학술대회
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• pp.509.1-509
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• 1986

Recent advances in recombinant DNA techniques have provided a tool for breeding of microorganisms of hyper production. Enzyme production by cloned microorganism has some advantages. They are ⅰ) Enzymes can be produced by a microorganism easily cultured ⅱ) Hyper production. ⅲ) In some cases, such as thermophilic enzyme gene is cloned in a mesophilic bacteria, the enzyme purification procedure can be simplified. One example, production of thermophilic ${\beta}$-galactosidase in B. subtilis will be presented. Bacillus stearothermophilus IAM 11001 produced three ${\beta}$-galactosidases, ${\beta}$-galactosidase I, II and III (${\beta}$-gal-I, II and III). By connecting restriction fragments of the chromosomal DNA to plasmid vector, followed by transformation of Escherichia coli, two ${\beta}$-galactosidase genes (bgaA and bgaB) located close to each other on the chromosome were cloned.

• Journal of Microbiology and Biotechnology
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• 제2권1호
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• pp.26-34
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• 1992

The effect of induction temperature on fermentation parameters has been investigated extensively using Escherichia coli M5248[pNKM21], a producer of recombinant human interleukin-2 (rhIL-2). In this recombinant microorganism, the gene expression of rhIL-2 is regulated by the cI857 repressor and $P_L$ promoter system. The recombinant fermentation parameters studied in this work include the cell growth, protein synthesis, cell viability, plasmid stability, $\beta$-lactamase activity, and rhIL-2 productivity. Interrelationships of such fermentation parameters have been analyzed through a quantitative assessment of the experimental data set obtained at eight different culture conditions. While the expression of rhIL-2 gene was repressed at culture temperatures below $34^\circ{C}$ with little effect on other fermentation parameters, under the conditions of rhIL-2 production $>(36~44^\circ{C})$ the cell growth, plasmid stability, and $\beta$-lactamase activity were, as induction temperature was increased, more profoundly reduced. Although the rhIL-2 content in the insoluble protein fraction was maximum at $40^\circ{C}$, total rhIL-2 production in the culture volume was found to be highest at the induction temperature of $36^\circ{C}$. This was in contrast to the previously known optimum induction temperature of the P$_{L}$ promoter system $>(40~42^\circ{C})$.Explanations for such a discrepancy have been proposed based on a product formation kinetics, and their implications have been discussed in detail.l.

• Journal of Microbiology and Biotechnology
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• 제28권5호
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• pp.739-747
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• 2018

The low solubility and high-cost recovery of Propionibacterium acnes polyunsaturated fatty acid isomerase (PAI) are key problems in the bioproduction of high value-added conjugated linoleic acid (CLA). To improve the solubility of recombinant PAI, six chaperone proteins were coexpressed with PAI. Introduction of GroELS proteins dramatically improved the PAI solubility from 29% to 97%, with increased activity by 57.8%. Combined expression of DnaKJ-GrpE and GroELS proteins increased the activity by 11.9%. In contrast, coexpression of DnaKJ-GrpE proteins significantly reduced the activity by 57.4%. Plasmids pTf16 harboring the tig gene and pG-Tf2 containing the tig and groEL-groES genes had no visible impact on PAI expression. The lytic protein E was then introduced into the recombinant Escherichia coli to develop a cell autolysis system. A 35% activity of total intracellular PAI was released from the cytoplasm by suspending the lysed cells in distilled water. The PAI recovery was further improved to 81% by optimizing the release conditions. The lipase from Rhizopus oryzae was also expressed in E. coli, with an extracellular activity of 110.9 U/ml. By using the free PAI and lipase as catalysts, a joint system was established for producing CLA from sunflower oil. Under the optimized conditions, the maximum titer of t-10,c-12-CLA reached 9.4 g/l. This work provides an effective and low-cost strategy to improve the solubility and recovery of the recombinant intracellular PAI for further large-scale production of CLA.

• 한국환경농학회지
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• 제38권3호
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• pp.197-204
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• 2019

BACKGROUND: Living modified microorganisms (LMMs) have been focused in two very different aspects of positive and negative effects on ecology and human health. As a model experiment, wild type and a foreign origin gene-harboring modified E. coli strains were subjected to comparison of their metabolomes and potential effects on soil microbiota in the laboratory sets. This study assumes the unintentional release of LMMs and tries to suggest potential effects on the soil microbiota even at minimal settings. METHODS AND RESULTS: Metabolomes from the wild type and LM E. coli were analyzed by NMR and the profiles were compared. In the laboratory soil experiments, the two types of E. coli were added to the soils and monitored for the bacterial community compositions. Those metabolomic profiles did not show significant differences. The microbial community structures from the time series soil DNAs for both the sets using wild type and LMO also did not indicate significant changes, but minor by the addition of foreign organisms regardless of wild or LMO. CONCLUSION: Even if the recombinant microorganism (LMO) is released into the soil environment, the survival of microorganisms in the environment would be one of the major factors for the transfers of foreign genes to other organisms and diffusion into the soil environment.

• 한국미생물·생명공학회지
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• 제49권1호
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• pp.9-17
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• 2021

A γ-aminobutyric acid (GABA)-producing microorganism was isolated from galchi (hairtail fish, Trichiurus lepturus) jeotgal, a Korean salted and fermented seafood. The G144 isolate produced GABA excessively when incubated in MRS broth containing monosodium glutamate (MSG, 3%, w/v). G144 was identified as Lactobacillus brevis through 16S rRNA and recA gene sequencing. gadB and gadC encoding glutamate decarboxylase (GAD) and glutamate/GABA antiporter, respectively, were cloned and gadB was located downstream of gadC. The operon structure of gadCB was confirmed by reverse transcription (RT)-polymerase chain reaction. gadB was overexpressed in Escherichia coli and recombinant GAD was purified and its size was 54.4 kDa as evidenced by SDS-PAGE results. Maximum GAD activity was observed at pH 5.0 and 40℃ and the activity was dependent on pyridoxal 5'-phophate. The K_m and V_max of GAD were 8.6 mM and 0.01 mM/min, respectively.

• 한국지하수토양환경학회지:지하수토양환경
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• 제11권5호
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• pp.43-50
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• 2006

Bioreporter 균주는 복잡한 환경매체의 특정 오염원 탐지를 위해 유용하게 사용되고 있다. 특히 발광 유전자 재조합 균주는 민감하고 배경에 의한 영향을 받지 않는 장점이 있다. 사용한 유전자 재조합 균주(Pseudomonas putida mt-2 KG1206)는 TOL 플라즈미드와 pUCD615 벡터에 $P_{m}\;promoter$가 삽입된 재조합 플라즈미드를 함유하고 있으며, 톨루엔 계열 및 중요 분해산물에 대해 분해와 함께 발광을 생산하는 특성을 갖고 있다. 본 연구에서는 균주 동결 및 동결건조 준비 및 적용과정에 필요한 다양한 조건들을 조사하여, 향후 환경매체에 적용하기 위한 최적 방법에 대한 프로토콜을 작성하였다. 조사한 최적 조건들은 다음과 같다. 동결보호시약(24% sucrose), 동결건조 시간(12시간), 균주 농도($OD_{600}=0.6$), 동결균주 활성회복($35^{\circ}C$에서 빠르게 해동), 동결건조 균주 활성회복(LB배지에 $3{\sim}6$시간 노출), 현장 운반 조건(활성 회복 후 $20^{\circ}C$ 정도의 실온). 본 연구 결과는 재조합 균주 환경 적용을 위해 필요한 균주 동결 및 동결 건조에 대한 중요한 자료들을 제시하고 있다.