• Journal of Microbiology and Biotechnology
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• 제16권1호
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• pp.149-152
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• 2006

Leuconostoc mesenteroides SY1, a strain isolated from cabbage Kimchi, was transformed with pCW4, a shuttle vector based on a cryptic plasmid from Lactobacillus paraplantarum C7. $\alpha-Amylase$ gene, amyL, from Bacillus licheniformis was cloned into pCW4, resulting in $pCW4T{\alpha},\;and\;pCW4T{\alpha}$ was introduced into SY1 by electroporation. Transformation efficiency was $10^2cells/{\mu}g$ plasmid DNA. L. mesenteroides cells harboring $pCW4T{\alpha}$ did not show amylase activity, although amyL transcript was synthesized as determined by slot blot experiment. $pCW4T{\alpha}$ was stably maintained in SY1 in the presence of erythromycin (Em, $5\;{\mu}g/ml$) but rapidly lost when Em was omitted. Less than $1\%$ of the cells maintained $pCW4T{\alpha}$ after 5 days at $30^{\circ}C$.

• 한국식품위생안전성학회지
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• 제32권3호
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• pp.171-178
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• 2017

청국장추출물과 청국장의 주요한 플라보노이드의 하나인 genistein의 HepG2 세포에서 Trp-P-1 유도 세포독성과 DNA손상에 대한 보호효과를 평가하였다. 청국장추출물과 주요 플라보노이드성분 genistein은 Trp-P-1 유도 세포독성에 대하여 세포독성보호효과를 나타내었다. 청국장추출물은 Trp-P-1 유도 DNA single strand breaks를 억제하였다. 한편, 청국장추출물은 HepG2 세포에서 Trp-P-1 유도에 의한 CYP1A1와 CYP1A2 발현의 억제를 나타내었다. 청국장추출물과 genistein은 Trp-P-1에 의한 유도 세포독성과 DNA손상에 대하여 CYP1A1, CYP1A2 발현억제에 의하여 보호효과가 나타나는 것으로 판단된다. 한국의 전통 콩발효식품인 청국장은 게놈 불안정성(genomic instability)을 일으키는 heterocyclic amines (HCAs)과 같은 식품의 가열조리로부터 올 수 있는 발암물질에 대한 유전독성을 예방할 수 있는 유망한 기능성물질로서 활용가능성이 있을 것으로 판단된다.

• Nutrition Research and Practice
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• 제9권6호
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• pp.569-578
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• 2015

BACKGROUND/OBJECTIVES: Fermentation can increase functional compounds in fermented soybean products, thereby improving antioxidant and/or anti-inflammatory activities. We investigated the changes in the contents of phenolics and isoflavones, antioxidant activity and anti-inflammatory activity of Doenjang during fermentation and aging. MATERIALS/METHODS: Doenjang was made by inoculating Aspergillus oryzae and Bacillus licheniformis in soybeans, fermenting and aging for 1, 3, 6, 8, and 12 months (D1, D3, D6, D8, and D12). Doenjang was extracted using ethanol, and sequentially fractioned by hexane, dichloromethane (DM), ethylacetate (EA), n-butanol, and water. The contents of total phenolics, flavonoids and isoflavones, 2,2-diphenyl-1 picryl hydrazyl (DPPH) radical scavenging activity, and ferric reducing antioxidant power (FRAP) were measured. Anti-inflammatory effects in terms of nitric oxide (NO), prostaglandin (PG) E2 and pro-inflammatory cytokine production and inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 expressions were also measured using LPS-treated RAW 264.7 macrophages. RESULTS: Total phenolic and flavonoid contents showed a gradual increase during fermentation and 6 months of aging and were sustained thereafter. DPPH radical scavenging activity and FRAP were increased by fermentation. FRAP was further increased by aging, but DPPH radical scavenging activity was not. Total isoflavone and glycoside contents decreased during fermentation and the aging process, while aglycone content and its proportion increased up to 3 or 6 months of aging and then showed a slow decrease. DM and EA fractions of Doenjang showed much higher total phenolic and flavonoid contents, and DPPH radical scavenging activity than the others. At $100{\mu}g/mL$, DM and EA fractions of D12 showed strongly suppressed NO production to 55.6% and 52.5% of control, respectively, and PGE2 production to 25.0% and 28.3% of control with inhibition of iNOS or COX-2 protein expression in macrophages. CONCLUSIONS: Twelve month-aged Doenjang has potent antioxidant and anti-inflammatory activities with high levels of phenolics and isoflavone aglycones, and can be used as a beneficial food for human health.

• Journal of Microbiology and Biotechnology
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• 제8권4호
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• pp.378-387
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• 1998

The DNA sequence immediately following the xynA gene of Bacillus stearothermophilus 236 ［about l-kb region downstream from the translational termination codon (TAA) of the xynA gene］was found to have an ability to enhance the xylanase activity of the upstream xynA gene. An 849-bp ORF was identified in the downstream region, and the ORF was confirmed to encode a novel protein of 283 amino acids designated as XaiF (xylanase-activity-increasing factor). From the nucleotide sequence of the xaiF gene, the molecular mass and pI of XaiF were deduced to be 32,006 Da and 4.46, respectively. XaiF was overproduced in the E. coli cells from the cloned xaiF gene by using the T7 expression system. The transcriptional initiation site was determined by primer extension analysis and the putative promoter and ribosome binding regions were also identified. Blast search showed that the xaiF and its protein product had no homology with any gene nor any protein reported so far. Also, in B. subtilis, the xaiF trans-activated the xylanase activity at the same rate as in E. coli. In contrast, xaiF had no activating effect on the co-expressed ${\beta}-xylosidase$ of the xylA gene derived from the same strain of B. stearothermophilus. In addition, the intracellular and extracellular fractions from the E. coli cells carrying the plasmid-borne xaiF gene did not increase the isolated xylanase activity, indicating that the protein-protein interaction between XynA and XaiF was not a causative event for the xylanase activating effect of the xaiF gene.

• ALGAE
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• 제30권2호
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• pp.163-170
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• 2015

Tyrosinase inhibitors are an important component of cosmetic products. Our previous studies have proposed that eckol isolated from the brown alga Ecklonia cava, can be explored as a tyrosinase inhibitor. However, cellular activities and mechanism of action of eckol remain unknown. Therefore, the current study analyzed the eckol binding modes using the crystal structure of Bacillus megaterium tyrosinase. The effects of eckol on melanin synthesis induced by ${\alpha}$-melanocyte stimulating hormone in B16F10 melanoma cells were also investigated. We predicted the 3D structure of tyrosinase and used a docking algorithm to simulate binding between tyrosinase and eckol. These molecular modeling studies were successful (calculated binding energy value, $-115.84kcal\;mol^{-1}$) and indicated that eckol interacts with Asn205, His208, and Arg209. Furthermore, eckol markedly inhibited tyrosinase activity and melanin synthesis in B16F10 melanoma cells. We also found that eckol decreased the expression of tyrosinase, tyrosinase-related protein (TRP) 1, and TRP2. These results indicate that eckol is a potent inhibitor of melanogenesis, and this finding may be useful for the development of novel pharmaceutical and cosmetic agents.

• Archives of Pharmacal Research
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• 제29권12호
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• pp.1154-1157
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• 2006

The ermK gene from Bacillus lichenformis encodes an inducible rRNA methylase that confers resistance to the macrolide-lincosamide-streptogramin B antibiotics. The ermK mRNA leader sequence has a total length of 357 nucleotides and encodes a 14-amino acid leader peptide together with its ribosome binding site. The secondary structure of ermK leader mRNA and a leader peptide sequence have been reported as the elements that control expression. In this study, the contribution of specific leader peptide amino acid residues to induction of ermK was studied using the PCR-based megaprimer mutation method. ermK methylases with altered leader peptide codons were translationally fused to E. coli ${\beta}-galactosidase$ reporter gene. The deletion of the codons for Thr-2 through Ser-4 reduced inducibility by erythromycin, whereas that for Thr-2 and His-3 was not. The replacement of the individual codons for Ser-4, Met-5 and Arg-6 with termination codon led to loss of inducibility, but stop mutation of codon Phe-9 restored inducibility by erythromycin. Collectively, these findings suggest that the codons for residue 4, 5 and 6 comprise the critical region for induction. The stop mutation at Leu-7 expressed constitutively ermK gene. Thus, ribosome stalling at codon 7 appears to be important for ermK induction.

• BMB Reports
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• 제34권6호
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• pp.509-516
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• 2001

A genomic DNA fragment that contains the gene, which codes for a novel extracellular serine protease in Burkholderia pseudomallei, was cloned by using pQE40 as a vector. It was maintained in Escherichia coli JM109. The expression of the gene(s) resulted in the production of a 52 kDa protease. The recombinant protease was purified from the culture filtrate via ammonium sulfate fractionation, gel filtration, and anion-exchange chromatography. The purified protease had an optimum pH and temperature of pH 8.9 and $38^{\circ}C$, respectively. The protease activity was inhibited by EGTA, EDTA, and PMSF, but not 1,10-phenanthroline. The first 11 amino acid residues from the N-terminus of the purified protease were identified as LAPNDPYYYGY. PNDPYY was found to show homology to the Bacillus cereus microbial serine protease and B. subtilis PD498 serine protease. These results indicate that the protease that was purified in this study is an extracellular calcium-dependent serine protease. The purified protease was able to digest the human serum 19A, IgG, albumin, and transferrin, as well as bovine muscle actin and myosin. Furthermore, it was able to promote or cause dermonecrosis in experimental rabbits. These results propose the possible role of a novel B. pseudomallei extracellular calcium-dependent serine protease in the virulence of the pathogen.

• Journal of Microbiology and Biotechnology
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• 제23권6호
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• pp.785-793
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• 2013

We studied the effects of 2 different dosages of high-molecular-weight poly-${\gamma}$-glutamic acid (hm ${\gamma}$-PGA) derived from Bacillus subtilis chungkookjang on lipid metabolism in a high-fructose diet-induced hypertriglyceridemic animal model. For 4 weeks, rats were fed either AIN-93 diet (normal control, NC; n = 10) or modified AIN-93 diet in which cornstarch was substituted with 63% fructose (n = 30) to induce hypertriglyceridemia. After 4 weeks, the hypertriglyceridemic rats were treated with daily oral doses of 0 mg (hypertriglyceridemic control, HC), 2.5 mg (hypertriglyceridemic, low hm ${\gamma}$-PGA, HL), or 5 $mg{\cdot}kg{\cdot}bw^{-1}{\cdot}d^{-1}$ (hypertriglyceridemic, high hm ${\gamma}$-PGA, HH) hm ${\gamma}$-PGA for 4 weeks. The HL and HH groups exhibited significantly lower levels of serum triglyceride, total cholesterol, LDL cholesterol, and free fatty acids than the HC group. The administration of hm ${\gamma}$-PGA reduced serum ALT and AST levels. The activities of lipogenic enzymes such as hepatic malic enzyme and glucose-6-phosphate dehydrogenase as well as glucose-6-phosphate dehydrogenase mRNA expression were significantly decreased by hm ${\gamma}$-PGA administration (p < 0.05). These results indicate that hm ${\gamma}$-PGA has an anti-hypertriglyceridemic effect in high-fructose diet-induced hypertriglyceridemic rats.

• Journal of Microbiology and Biotechnology
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• 제18권7호
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• pp.1235-1244
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• 2008

Indole-3-acetic acid (IAA) is produced commonly by plants and many bacteria, however, little is known about the genetic basis involving the key enzymes of IAA biosynthetic pathways from Bacillus spp. IAA intermediates from the Gram-positive spore-forming bacterium Paenibacillus polymyxa E681 were investigated, which showed the existence of only an indole-3-pyruvic acid (IPA) pathway for IAA biosynthesis from the bacterium. Four open reading frames (ORFs) encoding indole-3-pyruvate decarboxylase-like proteins and putative indole-3-pyruvate decarboxylase (IPDC), a key enzyme in the IPA synthetic pathway, were found on the genome sequence database of P. polymyxa and cloned in Escherichia coli DH5$\alpha$. One of the ORFs, PP2_01257, was assigned as probable indole-3-pyruvate decarboxylase. The ORF consisted of 1,743 nucleotides encoding 581 amino acids with a deduced molecular mass of 63,380 Da. Alignment studies of the deduced amino acid sequence of the ORF with known IPDC sequences revealed conservation of several amino acids in PP2_01257, essential for substrate and cofactor binding. Recombinant protein, gene product of the ORF PP2_01257 from P. polymyxa E681, was expressed in E. coli BL21 (DE3) as a glutathione S-transferase (GST)-fusion protein and purified to homogeneity using affinity chromatography. The molecular mass of the purified enzyme showed about 63 kDa, corresponding closely to the expected molecular mass of IPDC. The indole-3-pyruvate decarboxylase activity of the recombinant protein, detected by HPLC, using IPA substrate in the enzyme reaction confirmed the identity and functionality of the enzyme IPDC from the E681 strain.

• 농업과학연구
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• 제46권1호
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• pp.113-124
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• 2019

Insect-resistant transgenic (Bt-9) rice was generated by inserting mCry1Ac1, a modified gene from the soil bacterium Bacillus thuringiensis, into the genome of a conventional variety of rice (Ilmi). With regard to potential problems such as safety, an evaluation of non-target organisms is necessary as an essential element of an environmental risk assessment of genetically modified (GM) crops. We studied the effects of the Bt-9 rice on the survival of cantor Daphnia magna, a commonly used model organism in ecotoxicological studies. D. magna fed on the Bt-transgenic rice (Bt-9) and its near non-GM counterparts (Ilmi) grown in the same environment (a 100% ground rice suspension). The Bt-9 rice was confirmed to have the inserted T-DNA and protein expression evident by the PCR and ELISA analyses. The feeding study showed a similar cumulative immobility and abnormal response of the Daphnia magna between the Bt-9 rice and Ilmi. Additionally, the 48 h-EC50 values of the Bt-9 and Ilmi rice were 4,400 mg/L (95% confidence limits: 3861.01 - 5015.01 mg/L) and 5,564 mg/L (95% confidence limits: 4780.03 - 6476.93 mg/L), respectively. The rice NOEC (No observed effect concentration) value for D. magna was suggested to be 1,620 mg/L. We conclude that the tested Bt-9 and Ilmi have a similar cumulative immobility for D. magna, a widely used model organism, and the growth of Bt-9 did not affect non-target insects.