• 생명과학회지
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• 제24권6호
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• pp.603-611
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• 2014

한국산과 중국산 녹두 종자에서 0.15 M NaCl/0.1 M sodium phosphate buffer (pH7.0)에 의한 추출, $(NH_4)_2SO_4$ 침전, 최종적으로 Sephadex G-100을 이용한 affinity chromatography에 의해 lectin을 분리한 다음, 이들의 생화학적 특성을 조사, 비교하였다. 사람의 적혈구는 trypsin의 처리 유무와 상관없이 응집반응이 일어나지 않으며, 토끼의 적혈구에서는 trypsin을 처리한 경우에만 응집반응이 일어났다. 두 녹두 종자 lectin의 분자량은 SDS-PAGE를 통해 54 kDa와 28 kDa로 확인되었다. 한국산 녹두 종자 lectin의 최적 반응 온도는 $60^{\circ}C$이며, 중국산 녹두 종자 lectin의 경우는 $50^{\circ}C$로 나타났다. 종자 lectin이 열에 가장 안정한 온도는 한국산의 경우는 $50^{\circ}C$이며, 중국산의 경우 $40-50^{\circ}C$로 밝혀졌다. 한국산 녹두 종자 lectin은 pH 3.2에서 가장 높은 활성을 보였으며, 중국산 녹두 종자 lectin은 pH 6.2에서 가장 높은 활성을 보였다. 변성제를 처리했을 때, thiourea와 guanidine-HCl과는 혈액 응집이 일어나지 않아 변성작용이 일어남을 알 수 있었고, urea와는 혈액 응집이 일어나지 않아 변성작용이 일어나지 않음을 알 수 있었다. D-glucose외 6가지의 탄수화물에 대한 특이성이 나타나지 않았다. 또한 두 녹두 종자의 lectin은 $Ca^{2+}$, $Co^{2+}$, $Cu^{2+}$, $Cu^{2+}$, $Mg^{2+}$ 및 $Mn^{2+}$ 등의 금속이온에 대한 특이성이 없었다.

• Journal of Microbiology and Biotechnology
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• 제19권4호
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• pp.362-367
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• 2009

In recombinant strains, many proteins and enzymes are expressed as inactive and insoluble inclusion bodies. For soluble expression of an active form of StyB, an NADH-flavin oxidoreductase, several recombinant Escherichia coli strains were developed and tested. Among them, strain BL21(DE3)pLysS effectively produced an active and soluble form of StyB as about 9% of the total protein content, when cultivated at $20^{\circ}C$ with 0.5 mM IPTG. The solubly expressed StyB has the highest oxidoreductase activity at pH 6.5-7.5 and $37^{\circ}C$. Substrate dependence profiles of the StyB-catalyzed reaction showed that the maximum specific activity($V_m$) and half saturation constant($K_m$) were $1,867{\pm}148\;U/mg$ protein and $51.6{\pm}11{\mu}M$ for NADH, and $1,274{\pm}34\;U/mg$ protein and $8.2{\pm}1.2{\mu}M$ for FAD, respectively. This indicates that solubly produced StyB has 6- to 9-fold higher oxidoreductase activities than the in vitro refolded StyB from inclusion bodies.

• KSBB Journal
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• 제19권5호
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• pp.363-367
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• 2004

Leuconostoc mesenteroides NRRL B-1149 produces various glucoseyltransferases for the synthesis of dextran, levan and glucose-1-phosphate using sucrose as a substrate. A sucrose phosphorylase (1149SPase) was purified from L. mesenteroides NRRL B-1149 culture by using hollow fiber filtration (30 kDa cut off), Toyopearl DEAE 650 M column chromatography and following two times of DEAE-Sepharose column chromatographies. The specific activity of the purified 1149SPase was 25.7 (U/mg) with $16\%$ yield. The 1149SPase showed a molecular size of 56 kDa on denatured $10\%$ SDS-PAGE. The N-terminal amino acid sequence of the enzyme was MEIQNKAM. The optimum pH and temperature of this enzyme were 6.2~6.5 and 37^{circ}C, respectively. It had an apparent K_{m} of 6.0 mM and K_{cat} of 1.62/s for sucrose. 1149SPase crystal was formed by hanging drop diffusion technique using 20 mM calcium chloride dihydrate, 100 mM sodium acetate trihydrate pH 4.6 and $30\%$ 2-methyl-2,4-pentanediol as vaporizing and reservation solution. The 1149SPase catalyzes transferring of glucose from isomaltose or sucrose to salicin and salicyl alcohol by disproportionation reaction or acceptor reaction and synthesized two acceptor products, respectively.

• Journal of Microbiology and Biotechnology
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• 제28권5호
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• pp.776-783
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• 2018

The agarase gene gaa16a was identified from a draft genome sequence of Gilvimarinus agarilyticus JEA5, an agar-utilizing marine bacterium. Recently, three agarase-producing bacteria, G. chinensis, G. polysaccharolyticus, and G. agarilyticus, in the genus Gilvimarinus were reported. However, there have been no reports of the molecular characteristics and biochemical properties of these agarases. In this study, we analyzed the molecular characteristics and biochemical properties of agarases in Gilvimarinus. Gaa16A comprised a 1,323-bp open reading frame encoding 441 amino acids. The predicted molecular mass and isoelectric point were 49 kDa and 4.9, respectively. The amino acid sequence of Gaa16A showed features typical of glycosyl hydrolase family 16 (GH16) ${\beta}$-agarases, including a GH16 domain, carbohydrate-binding region (RICIN domain), and signal peptide. Recombinant Gaa16A (excluding the signal peptide and carbohydrate-binding region, rGaa16A) was expressed as a fused protein with maltose-binding protein at its N-terminus in Escherichia coli. rGaa16A had maximum activity at $55^{\circ}C$ and pH 7.0 and 103 U/mg of specific activity in the presence of 2.5 mM $CaCl_2$. The enzyme hydrolyzed agarose to yield neoagarotetraose as the main product. This enzyme may be useful for industrial production of functional neoagaro-oligosaccharides.

• Journal of Microbiology and Biotechnology
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• 제27권8호
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• pp.1472-1482
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• 2017

Bacterial cytochrome P450 (CYP) steroid hydroxylases are effectively useful in the pharmaceutical industry for introducing hydroxyl groups to a wide range of steroids. We found a putative CYP steroid hydroxylase (BaCYP106A2) from the bacterium Bacillus sp. PAMC 23377 isolated from Kara Sea of the Arctic Ocean, showing 94% sequence similarity with BmCYP106A2 (Bacillus megaterium ATCC 13368). In this study, soluble BaCYP106A2 was overexpressed to evaluate its substrate-binding activity. The substrate affinity ($K_d$ value) to 4-androstenedione was $387{\pm}37{\mu}M$. Moreover, the crystal structure of BaCYP106A2 was determined at $2.7{\AA}$ resolution. Structural analysis suggested that the ${\alpha}8-{\alpha}9$ loop region of BaCYP106A2 is intrinsically mobile and might be important for initial ligand binding. The hydroxyl activity of BaCYP106A2 was identified using in vitro enzyme assays. Its activity was confirmed with two kinds of steroid substrates, 4-androstenedione and nandrolone, using chromatography and mass spectrometry methods. The main products were mono-hydroxylated compounds with high conversion yields. This is the second study on the structure of CYP106A steroid hydroxylases, and should contribute new insight into the interactions of bacterial CYP106A with steroid substrates, providing baseline data for studying the CYP106A steroid hydroxylase from the structural and enzymatic perspectives.

• 공업화학
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• 제29권5호
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• pp.524-532
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• 2018

본 연구에서는 천연 유래의 코코넛 오일을 원료로 사용하여 2종류의 아미노산계 음이온 생체계면활성제 포타슘 코코일 글루타메이트(potassium cocoyl glutamate, CTK)와 소듐 코코일 글루타메이트(sodium cocoyl glutamate, CTN)를 합성하였으며, 합성한 계면활성제의 구조를 FT-IR, $^1H-NMR$ 및 $^{13}C-NMR$ 분석을 통하여 규명하였다. 합성한 계면활성제에 대하여 정적 및 동적 표면장력과 유화력 등의 계면 물성을 측정한 결과, CTK와 CTN 모두 계면 활성이 우수하고 계면 에너지를 낮추는데 효과적임을 알 수 있었다. 특히, CTK 계면활성제가 CTN 계면활성제와 비교하여 계면 에너지를 낮추는데 보다 효과적이었는데 이는 CTK가 소수성이 더 크고 계면활성제 단분자가 벌크 용액으로부터 공기와 수용액의 계면으로 이동하는 속도가 빨라서 공기와 수용액의 계면이 계면활성제 단분자에 의하여 더 짧은 시간에 포화되기 때문임을 알 수 있었으며, 생활용품이나 화장품 제조에 적용될 수 있을 것으로 기대된다.

• Preventive Nutrition and Food Science
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• 제9권2호
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• pp.113-120
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• 2004

Selected microbiological and biochemical characteristics of the steeping process for the production of yukwa, a traditional Korean oil-puffed snack made of waxy rice, were investigated during steeping of waxy rice in water for 15 days. The lengthy steeping process was largely predominated by lactic acid bacteria (LAB), particularly, Lactobacillus and Leuconostoc. The predominat type of bacterium isolated was the Y26 strain tentatively identified as Lactobacillus plantarum. The titratable acidity of the steeping medium increased from 0.01 to 1.13%, in parallel with the decrease in pH ranging from 6.3 to 4.2 as the steeping period increased from 0 to 15 days. A high amount of lactic acid and to a much lesser extent, butyric acid, acetic acid, propionic acid, and succinic acid were detected during the steeping process. The amount of reducing sugars in the steeping medium increased from 0.61 to 10.43 mg/mL, whereas sucrose decreased from 0.46 mg% to an undetectable level. Starch degradation products including glucose, maltose and oligosaccharides ranging G3-G7 were not initially noticed, but their content increased during the steeping process until completion. However, no oligosaccharides larger than G8 were detected in the steeping medium. The activities of $\alpha$-amylase, $\beta$-amylase and protease in the steeping medium of waxy rice tended to rise increase with time during the steeping process. From these results, the lengthy steeping process in yukwa production can be characterized as the spontaneous fermentation, dominated by lactic acid bacteria, which is a necessary process for inducing biochemical modification of waxy rice.

• BMB Reports
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• 제35권2호
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• pp.213-219
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• 2002

Malonyl-CoA decarboxylase (E.C.4.1.1.9) catalyzes the conversion of malonyl-CoA to acetyl-CoA. Although the metabolic role of this enzyme has not been fully defined, it has been reported that its deficiency is associated with mild mental retardation, seizures, hypotonia, cadiomyopathy, developmental delay, vomiting, hypoglycemia, metabolic acidosis, and malonic aciduria. Here, we isolated a cDNA clone for malonyl CoA decarboxylase from a rat brain cDNA library, expressed it in E. coli, and characterized its biochemical properties. The full-length cDNA contained a single open-reading frame that encoded 491 amino acid residues with a calculated molecular weight of 54, 762 Da. Its deduced amino acid sequence revealed a 65.6% identity to that from the goose uropigial gland. The sequence of the first 38 amino acids represents a putative mitochondrial targeting sequence, and the last 3 amino acid sequences (SKL) represent peroxisomal targeting ones. The expression of malonyl CoA decarboxylase was observed over a wide range of tissues as a single transcript of 2.0 kb in size. The recombinant protein that was expressed in E. coli was used to characterize the biochemical properties, which showed a typical Michaelis-Menten substrate saturation pattern. The $K_m$ and $V_{max}$ were calculated to be $68\;{\mu}M$ and $42.6\;{\mu}mol/min/mg$, respectively.

• 한국미생물·생명공학회지
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• 제47권3호
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• pp.401-411
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• 2019

All Idiomarina species are isolated from saline environments; microorganisms in such extreme habitats develop metabolic adaptations and can produce compounds such as proteases with an industrial potential. ARDRA and 16S rRNA gene sequencing are established methods for performing phylogenetic analysis and taxonomic identification. However, 16S-23S ITS is more variable than the 16S rRNA gene within a genus, and is therefore, used as a marker to achieve a more precise identification. In this study, ten protease producing Idiomarina strains isolated from the Peruvian salterns were characterized using biochemical and molecular methods to determine their bacterial diversity and industrial potential. In addition, comparison between the length and nucleotide sequences of a 16S-23S ITS region allowed us to assess the inter and intraspecies variability. Based on the 16S rRNA gene, two species of Idiomarina were identified (I. zobellii and I. fontislapidosi). However, biochemical tests revealed that there were differences between the strains of the same species. Moreover, it was found that the ITS contains two tRNA genes, $tRNA^{Ile(GAT)}$ and $tRNA^{Ala(TGC)}$, which are separated by an ISR of a variable size between strains of I. zobellii. In one strain of I. zobellii (PM21), we found nonconserved nucleotides that were previously not reported in the $tRNA^{Ala}$ gene sequences of Idiomarina spp. Thus, based on the biochemical and molecular characteristics, we can conclude that protease producing Idiomarina strains have industrial potential; only two I. zobellii strains (PM48 and PM72) exhibited the same properties. The differences between the other strains could be explained by the presence of subspecies.

• Journal of Microbiology and Biotechnology
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• 제29권1호
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• pp.44-54
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• 2019

Geldanamycin and its derivatives, inhibitors of heat shock protein 90, are considered potent anticancer drugs, although their biosynthetic pathways have not yet been fully elucidated. The key step of conversion of 4,5-dihydrogeldanamycin to geldanamycin was expected to catalyze by a P450 monooxygenase, Gel16. The adequate bioconversions by cytochrome P450 mostly rely upon its interaction with redox partners. Several ferredoxin and ferredoxin reductases are available in the genome of certain organisms, but only a few suitable partners can operate in full efficiency. In this study, we have expressed cytochrome P450 gel16 in Escherichia coli and performed an in vitro assay using 4,5-dihydrogeldanamycin as a substrate. We demonstrated that the in silico method can be applicable for the efficient mining of convenient endogenous redox partners (9 ferredoxins and 6 ferredoxin reductases) against CYP Gel16 from Streptomyces hygroscopicus. The distances for ligand FDX4-FDR6 were found to be $9.384{\AA}$. Similarly, the binding energy between Gel16-FDX4 and FDX4-FDR6 were -611.88 kcal/mol and -834.48 kcal/mol, respectively, suggesting the lowest distance and binding energy rather than other redox partners. These findings suggest that the best redox partners of Gel16 could be NADPH ${\rightarrow}$ FDR6 ${\rightarrow}$ FDX4 ${\rightarrow}$ Gel16.