• Journal of Ginseng Research
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• 제42권4호
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• pp.412-418
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• 2018

Background: Ginsenoside Rg3(S) and compound K (C-K) are pharmacologically active components of ginseng that promote human health and improve quality of life. The aim of this study was to produce Rg3(S) and C-K from ginseng extract using recombinant Lactococcus lactis. Methods: L. lactis subsp. cremoris NZ9000 (L. lactis NZ9000), which harbors ${\beta}$-glucosidase genes (BglPm and BglBX10) from Paenibacillus mucilaginosus and Flavobacterium johnsoniae, respectively, was reacted with ginseng extract (protopanaxadiol-type ginsenoside mixture). Results: Crude enzyme activity of BglBX10 values comprised 0.001 unit/mL and 0.003 unit/mL in uninduced and induced preparations, respectively. When whole cells of L. lactis harboring pNZBglBX10 were treated with ginseng extract, after permeabilization of cells by xylene, Rb1 and Rd were converted into Rg3(S) with a conversion yield of 61%. C-K was also produced by sequential reactions of the permeabilized cells harboring each pNZBgl and pNZBglBX10, resulting in a 70% maximum conversion yield. Conclusion: This study demonstrates that the lactic acid bacteria having specific ${\beta}$-glucosidase activity can be used to enhance the health benefits of Panax ginseng in either fermented foods or bioconversion processes.

• Journal of Microbiology and Biotechnology
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• 제14권3호
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• pp.576-583
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• 2004

GERI-155 is a macrolide antibiotic containing two deoxyhexose molecules and shows antimicrobial activities against Gram-positive bacteria. Deoxysugar biosynthetic gene cluster of GERI-155 from Streptomyces sp. GERI-l55 genome was cloned. Four orfs were identified and a putative orf presumed to be the dTDP g]ucose-4,6-dehydratase gene was designated as gerE. GerE was expressed in E. coli by using a recombinant expression vector pHJ1. The expressed protein was purified from E. coli cell lysate by using ammonium sulfate fractionation, and DEAE-sepharose CL-6B and hydroxylapatite column chromatography. The molecular mass of the expressed protein correlated with the predicted mass that was deduced from the cloned gene sequence data. The recombinant protein was a homodimer with a subunit relative molecular weight of 39,000 Dalton. It was found to have dTDP-glucose 4,6-dehydratase activity and also found to be highly specific for dTDP-glucose as a substrate. The values of $K_{m} and V_{max}$ for dTDP-g]ucose were $32\mu$M and 335 nmol $min^{-1}$ (mg protein)^{-1}$, respectively. dTTP and dTDP were strong inhibitors of the protein. $NAD^+$, the coenzyme for dTDP-glucose 4,6-dehydratase, was tightly bound to the expressed protein.

• 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.

• 한국미생물·생명공학회지
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• 제50권3호
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• pp.319-327
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• 2022

γ-Aminobutyric acid (GABA) is a multi-functional compound with broad applications for food industry. GABA producing bacteria were isolated from cabbage kimchi. Among them, B737 was the best GABA producer when culture supernatants were analyzed by TLC. B737 was identified as Levilactobacillus brevis by 16S rRNA gene sequencing. Its glutamate decarboxylase (GAD) gene was cloned by PCR and the nucleotide sequence determined. B737 GAD consisting of 485 amino acids is the largest in size among GADs reported from LAB so far. gadB from L. brevis B737 was overexpressed in Escherichia. coli BL21(DE3) using pET26b(+).pET26b(+). The recombinant GAD was purified and its size was 55 kDa by SDS-PAGE. Maximum GAD activity was observed at pH 5 and 40℃ and the activity was dependent on pyridoxal 5'-phosphate. K_m and V_max of recombinant GAD were 6.2 ± 0.06 mM and 0.34 ± 0.002 mM/min, respectively. L. brevis B737 can be used as a starter for fermented foods with high GABA contents.

• 대한환경공학회지
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• 제27권5호
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• pp.507-512
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• 2005

본 연구에서는 유전자 재조합 발광균주, Pseudomonas putida mt-2 KG1206을 이용하여 토양에 오염된 m-toluate 탐지 방법 및 적용 가능성에 대해 조사하였다. KG1206은 톨루엔 계열 화합물의 중요 중간 분해물질인 m-toluate 및 benzoate가 직접 생물 발광 유도제로 작용하며, 또한 톨루엔 계열 화합물들이 간접 유도제로서 발광 활성을 나타내었다. 토양에 오염된 유도제 오염원 검출을 위해 발광 균주 9.9 mL에 에탄올 추출물 0.1 mL을 첨가하여 조사하였다. 생물발광에 근거하여 작성된 m-toluate 검량선은 대략 $R^2>0.97$ 이상의 상관관계가 관찰되었다. 토양에 임의 오염된 m-toluate(직접 발광유도제)는 정립한 방법에 따라 발광활성에 근거하여 추측하였고, 기기분석치와 통계적으로 유의한 것으로 조사되었다. 본 연구 결과를 통해서 특정 화합물에 대해 발광을 생산하는 유전자 재조합 균주가 특정 오염원에 오염된 지역의 관리를 위한 수단으로 사용할 수 있는 가능성을 확인할 수 있었다.

• 한국미생물·생명공학회지
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• 제47권3호
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• pp.343-349
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• 2019

Gul jeotgals (GJs) were prepared using solar salt aged for 3 years. One sample was fermented using starters, such as Bacillus subtilis JS2 and Tetragenococcus halophilus BS2-36 (each $10^6CFU/g$), and another sample was fermented without starters for 49 days at $10^{\circ}C$. Initial counts of bacilli and lactic acid bacteria (LAB) in non-starter GJ were found to be $3.20{\times}10^2$ and $7.67{\times}10^1CFU/g$ on day 0, and increased to $1.37{\times}10^3$ and $1.64{\times}10^6CFU/g$ on day 49. Those of starter GJ were found to be $2.10{\times}10^5$ and $3.30{\times}10^7CFU/g$ on day 49, indicating the growth of starters. The pH values of GJ were $5.93{\pm}0.01$ (non-starter) and $5.92{\pm}0.01$ (starter) on day 0 and decreased to $5.78{\pm}0.01$ (non-starter) and $5.75{\pm}0.01$ (starter) on day 49. Amino-type nitrogen (ANN) production increased continuously during fermentation, and $407.19{\pm}15.85$ (non-starter) and $398.04{\pm}13.73$ (starter) mg% on day 49. Clone libraries of 16S rRNA genes were constructed from total DNA extracted from non-starter GJ on days 7, 21, and 42. Nucleotide sequences of Escherichia coli transformants harboring recombinant pGEM-T easy plasmid containing 16S rRNA gene inserts from different bacterial species were analyzed using BLAST. Uncultured bacterium was the most dominant group and Gram - bacteria such as Acidovorax sp., Afipia sp., and Variovorax sp. were the second dominant group. Bacillus amyloliquefaciens (day 7), Bacillus velezensis (day 21 and 42), and Bacillus subtilis (day 42) were observed, but no lactic acid bacteria were detected. Acidovorax and Variovorax species might play some role in GJ fermentation. Further studies on these bacteria are necessary.

• 한국잠사곤충학회지
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• 제52권1호
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• pp.45-51
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• 2014

면역 유도된 천잠(Antheraea yamamai) 유충에서 cecropin-A 유전자를 분리하였고 이 유전자를 Ay-CecA로 명명하였다. 전체 Ay-CecA cDNA 크기는 419 bp로 64개의 아미노산 잔기를 인코딩하는 195 bp ORF로 구성되어 있다. 천잠 CecA 유전자는 22개 잔기의 signal peptide, 4개 잔기의 propeptide 및 항균활성을 갖는 37개 잔기로 구성된 성숙 단백질(mature protein) 영역으로 구성되고 예상 분자량은 4046.81 Da으로 산출되었다. 천잠 CecA의 아미노산 서열은 다른 나비목 곤충에서 분리된 cecropin와 매우 높은 상동성(62 ~ 78%)을 나타냈다. Ay-CecA 유전자의 C말단에 기존에 보고된 곤충의 cecropin에서와 동일하게 C말단 아미드화를 위한 glycine 잔기가 존재하고 있다. 이 펩타이드의 항균활성을 검정하기 위해서 대장균 발현 시스템을 이용하여 활성이 있는 재조합 Ay-CecA 발현에 성공하였다. 발현 기주인 대장균에 대한 재조합 CecA 독성 중화를 위해서 불용성 단백질인 ketosteroid isomerase(KSI) 유전자를 CecA 유전자와 융합하였다. 융합 CecA-KSI 단백질은 대부분 불용성 단백질로 발현되었다. 발현된 융합단백질은 Ni-NTA immobilized metal affinity chromatography(IMAC)에 의해서 정제하였으며 CNBr 반응을 통하여 재조합 CecA를 절단하여 용출하였다. 최종적으로 양이온 교환 chromatography 과정을 통하여 CecA를 순수 정제하였다. 정제된 재조합 Ay-CecA는 그람음성균인 E. cori ML 35, Klebsiella pneumonia 및 Pseudomonas aeruginosa에 대해 매우 높은 항균활성을 나타냈었다. 따라서 본 연구 결과, 높은 항균활성 지닌 CecA는 천잠의 면역 반응에서 중요한 역할을 담당할 것으로 사료된다.

• Journal of Microbiology and Biotechnology
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• 제16권10호
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• pp.1583-1590
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• 2006

T4 endonuclease V (T4 endo V) [EC 3. 1. 25. 1], found in bacteriophage T4, is responsible for excision repair of damaged DNA. The enzyme possesses two activities: a cyclobutane pyrimidine dimer DNA glycosylase (CPD glycosylase) and an apyrimidic/apurinic endonuclease (AP lyase). T4 denV (414 bp cDNA) encoding T4 en do V (138 amino acid) was synthesized and expressed using either an expression vector, pTriEx-4, in E. coli or a baculovirus AcNPV vector, pBacPAK8, in insect cells. The recombinant His-Tag/T4 endo V (rHis-Tag/T4 endo V) protein expressed from bacteria was purified using one-step affinity chromatography with a HiTrap Chelating HP column and used to make rabbit anti-His-Tag/T4 endo V polyclonal antibody for detection of recombinant T4 endo V (rT4 endo V) expressed in insect cells. In the meantime, the recombinant baculovirus was obtained by cotransfection of BacPAK6 viral DNA and pBP/T4 endo V in Spodoptera frugiperda (Sf21) insect cells, and used to infect Sf21 cells to overexpress T4 endo V protein. The level of rT4 endo V protein expressed in Sf21 cells was optimized by varying the virus titers and time course of infection. The optimal expression condition was set as follows; infection of the cells at a MOI of 10 and harvest at 96 h post-infection. Under these conditions, we estimated the amount of rT4 endo V produced in the baculovirus expression vector system to be 125 mg/l. The rT4 endo V was purified to homogeneity by a rapid procedure, consisting of ion-exchange, affinity, and reversed phase chromatographies, based on FPLC. The rT4 endo V positively reacted to an antiserum made against rHis-Tag/T4 endo V and showed a residual nicking activity against CPD-containing DNA caused by UV. This is the first report to have T4 endo V expressed in an insect system to exclude the toxic effect of a bacterial expression system, retaining enzymatic activity.

• 한국축산식품학회지
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• 제35권6호
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• pp.867-873
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• 2015

1,4-Dihydroxy-2-naphthoic acid (DHNA), a precursor of menaquinone (vitamin K2), has an effect on growth stimulation of bifidobacteria and prevention of osteoporosis, making it a promising functional food material. Therefore, we tried to clone the menB gene encoding DHNA synthase from Leuconostoc mesenteroides CJNU 0147. Based on the genome sequence of Leu. mesenteroides ATCC 8293 (GenBank accession no., CP000414), a primer set (Leu_menBfull_F and Leu_menBfull_R) was designed for the PCR amplification of menB gene of CJNU 0147. A DNA fragment (1,190 bp), including the menB gene, was amplified, cloned into pGEM-T Easy vector, and sequenced. The deduced amino acid sequence of MenB (DHNA synthase) protein of CJNU 0147 had a 98% similarity to the corresponding protein of ATCC 8293. The menB gene was subcloned into pCW4, a lactic acid bacteria - E. coli shuttle vector, and transferred to CJNU 0147. The transcription of menB gene of CJNU 0147 (pCW4::menB) was increased, when compared with those of CJNU 0147 (pCW4) and CJNU 0147 (−). The DHNA was produced from it at a detectable level, indicating that the cloned menB gene of CJNU 0147 encoded a DHNA synthase which is responsible for the production of DHNA, resulting in an increase of bifidogenic growth stimulation activity.

• Journal of Microbiology and Biotechnology
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• 제24권8호
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• pp.1096-1104
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• 2014

$\small{L}$-Asparaginase from gram-positive bacteria has been poorly explored. We conducted recombinant overexpression and purification of $\small{L}$-asparaginase from Staphylococcus sp. OJ82 (SoAsn) isolated from Korean fermented seafood to evaluate its biotechnological potential as an antileukemic agent. SoAsn was expressed in Escherichia coli BL21 (DE3) with an estimated molecular mass of 37.5 kDa, determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Consistent with asparaginases in gram-negative bacteria, size-exclusion chromatography determined SoAsn as a homodimer. Interestingly, the optimal temperature of SoAsn was $37^{\circ}C$ and over 90% of activity was retained between $37^{\circ}C$ and $50^{\circ}C$, and its thermal stability range was narrower than that of commercial E. coli $\small{L}$-asparaginase (EcAsn). Both SoAsn and EcAsn were active between pH 9 and 10, although their overall pH-dependent enzyme activities were slightly different. The $K_m$ value of SoAsn was 2.2 mM, which is higher than that of EcAsn. Among eight metals tested for enzyme activity, cobalt and magnesium greatly enhanced the SoAsn and EcAsn activity, respectively. Interestingly, SoAsn retained more than 60% of its activity under 2 M NaCl condition, but the activity of EcAsn was reduced to 48%. Overall, the biochemical characteristics of SoAsn were similar to those of EcAsn, but its kinetics, cofactor requirements, and NaCl tolerance differed from those of EcAsn.