• Journal of Microbiology and Biotechnology
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• 제26권1호
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• pp.20-27
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• 2016

Lactobacillus kefiranofaciens ZW3 was obtained from kefir grains, which have high lactose hydrolytic activity. In this study, a heterodimeric LacLM-type β-galactosidase gene (lacLM) from ZW3 was isolated, which was composed of two overlapping genes, lacL (1,884 bp) and lacM (960 bp) encoding large and small subunits with calculated molecular masses of 73,620 and 35,682 Da, respectively. LacLM, LacL, and LacM were expressed in Escherichia coli BL21(DE3) and these recombinant proteins were purified and characterized. The results showed that, compared with the recombinant holoenzyme, the recombinant large subunit exhibits obviously lower thermostability and hydrolytic activity. Moreover, the optimal temperature and pH of the holoenzyme and large subunit are 60℃ and 7.0, and 50℃ and 8.0, respectively. However, the recombinant small subunit alone has no activity. Interestingly, the activity and thermostability of the large subunit were greatly improved after mixing it with the recombinant small subunit. Therefore, the results suggest that the small subunit might play an important role in maintaining the stability of the structure of the catalytic center located in the large subunit.

• 한국미생물·생명공학회지
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• 제36권4호
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• pp.314-319
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• 2008

Penicillin G amidase(PGA, benzylpenicillinamidohydrolase, EC 3.5.1.11)는 penicillin G를 phenylacetic acid(PAA)와 6-aminopenicillanic acid(6-APA)로 분해하는 효소이다. Escherichia coli(E. coli) ATCC 11105의 PGA는 24 kDa의 small subunit과 65 kDa의 large subunit으로 구성되어 있고, precursor polypeptide에서 signal peptide와 spacer peptide가 절단되어 활성을 가진 heterodimer가 형성된다. 본 연구에서는 E. coli ATCC 11105에서 PCR(polymerase chain reaction)을 통해 증폭한 pga gene을 expression vector에 넣어 pET-pga plasmid를 제작하였고, 이것을 E. coli BL21 (DE3) 균주에 형질 전환하여 PGA를 발현하고 그 활성을 분석하였다. E. coli BL21(DE3)/pET-pga 균주의 고밀도 배양액을 SDS-PAGE로 분석 했을 때, PGA의 precursor, large subunit, 그리고 small subunit으로 보이는 protein band가 나타났으며, PGA가 soluble form의 precursor로 발현되어 processing을 거쳐서 large subunit과 small subunit으로 절단되기도 하고, 일부는 insoluble form의 precursor로 발현되기도 하는 것으로 생각된다. 유가배양시 온도변화 전략을 사용하여 고농도 배양에서 발현을 유도하였다. 온도변화 전략은 $37^{\circ}C$에서 $28^{\circ}C$를 거쳐 $22^{\circ}C$로 3단계로 변화시켰다. 이러한 전략으로 PGA활성은 19.6 U/mL이며 균체량은 600 nm에서 흡광도가 62까지 도달하였다.

• 한국신재생에너지학회:학술대회논문집
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• 한국신재생에너지학회 2005년도 제17회 워크샵 및 추계학술대회
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• pp.631-633
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• 2005

For biohydrogen production, hydrogenase is a key enzyme. In the present work we performed search of [NiFe]-hydrogenases from hydrogen producing microorganisms using degenerate polymerase chain reaction (PCR) strategy. Degenerate primers were designed from the conserved region of [NiFe]-hydrogenase group I especially on structural genes encoding for catalytic subunit of [NiFe]-hydrogenase from bacteria producing hydrogen. Most of [NiFe]-hydrogenase (group I) are expressed via complex mechanism with aid of auxiliary protein and localized through twin-arginine translocation pathway. [NiFe]-hydrogenase is composed of large and small subunits for catalytic activity. It is known that only small subunit has signal peptide for periplasmic localization and large & small subunitscome together before localization. During this process, large subunit is treated by endopeptidase for maturation. Based on these information we used signal peptide sequence and C-terminal of large subunit by recognized by endopeptidase as templates for degenerate primers. About 2,900 bp of PCR products were successfully amplified using the designed degenerate primers from genomic DNAs of several microorganisms. The amplified PCR products were inserted into T-vector and then sequenced to confirm.

• Journal of Ginseng Research
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• 제19권1호
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• pp.51-55
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• 1995

The DNA fragment containing ginseng ribulose-1,5-bisphosphate carboxytase/oxygenase large subunit(rbcL) gene was cloned from the ginseng chloroplast EcoRl library by colony lift hybridization with tobacco rbcL gene probe. From the screened clone, the DNA fragment containing ginseng rbcL gene was digested with several restriction enzyme and analyzed by Southern blot hybridization for the construction of restriction map. The ginseng rbcL gene fragment was subcloned in pBluescript II SK + vector and sequence analysis was performed. The nucleotide sequence of ginseng rbcL gene was compared with those of petunia, tobacco, alfalfa, rice and barley, which showed a homology of 93.1%, 95.2%, 90.5%, 85.5% and 84.3%, respectively.

• Journal of Plant Biotechnology
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• 제49권1호
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• pp.39-45
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• 2022

Conserved domains are defined as recurring units in molecular evolution and are commonly used to interpret the molecular function and biochemical structure of proteins. Herein, the ADP-glucose pyrophosphorylase (AGPase) amino acid sequences of three species of the Ipomoea genus [Ipomoea trifida, I. triloba, and I. batatas (L.) Lam. (sweetpotato)] were identified to investigate their physicochemical and biochemical characteristics. The molecular weight, isoelectric point, instability index, and grand average of hyropathy markedly differed among the three species. The aliphatic index values of sweetpotato AGPase proteins were higher in the small subunit than in the large subunit. The AGPase proteins from sweetpotato were found to contain an LbH_G1P_AT_C domain in the C-terminal region and various domains (NTP_transferase, ADP_Glucose_PP, or Glyco_tranf_GTA) in the N-terminal region. Conversely, most of its two relatives (I. trifida and I. triloba) were found to only contain the NTP_transferase domain in the N-terminal region. These findings suggested that these conserved domains were species-specific and related to the subunit types of AGPase proteins. The study may enable research on the AGPase-related specific characteristics of sweetpotatoes that do not exist in the other two species, such as starch metabolism and tuberization mechanism.

• 한국약용작물학회:학술대회논문집
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• 한국약용작물학회 2007년도 춘계학술발표회
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• pp.274-276
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• 2007

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2002년도 제9회 학술 발표회 프로그램과 논문초록
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• pp.32-32
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• 2002

To understand the functional roles of the neuron-specific $\beta$-subunit of large-conductance calcium-activated potassium ($BK_{Ca}$) channel, we isolate the full-length complementary DNA of $\beta$4-subunit from rat brain library and investigated its effects on the function of $\alpha$-subunit (Slo). The deduced amino acid sequence of rat $\beta$4 (r$\beta$4), 210 amino acids in length, was closely related to those of $\beta$4 subunits in other mammalian species but showed only a limited sequence homology to the other $\beta$-subunits, $\beta$1 to $\beta$3.(omitted)d)

• ALGAE
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• 제33권1호
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• pp.49-54
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• 2018

Red algal mitochondrial genomes (mtDNAs) can provide useful information on species identification. mtDNAs of Pyropia / Porphyra (Bangiales, Rhodophyta) have shown diverse variation in their size and gene structure. In particular, the introns and intronic open reading frames found in the ribosomal RNA large subunit gene (rnl) and cytochrome c oxidase subunit 1 gene (cox1) significantly vary the mitochondrial genome size in Pyropia / Porphyra species. In this study, we examined the exon / intron structure of rnl and cox1 genes of Pyropia yezoensis at the intraspecific level. The combined data of rnl and cox1 genes exhibited 12 genotypes for 40 P. yezoensis strains, based on the existence of introns. These genotypes were more effective to identify P. yezoensis strains in comparison to the traditional DNA barcode cox1 marker (5 haplotypes). Therefore, the variation in gene structure of rnl and cox1 can be a novel molecular marker to discriminate the strains of Pyropia species.

• 한국균학회지
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• 제48권1호
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• pp.55-61
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• 2020

꼬마초롱이끼의 헛뿌리와 가는털깃털이끼의 잎에서 내생균을 분리하였다. 분리된 균주는 형태적 특성과 internal transcribed spacer, large subunit rDNA, Beta-tublin 영역 및 RNA polymerase II second largest subunit gene의 분자적 분석을 토대로 동정하였다. 연구 과정에서 2종의 국내 미기록종 내생균인 Cercophora thailandica와 Biscogniauxia petrensis를 확인하였다. 분리된 미기록종 내생균 균주의 형태적 특성 확인 및 계통 분석 결과에 대해 기술하였다.

• 한국균학회지
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• 제48권3호
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• pp.217-228
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• 2020

본 연구에서는 경남 통영시의 한산도에서 서식하는 식물의 잎을 채취하여 내생균을 분리하였다. 분리한 균주는 배양특성과 포자등의 형태성특성 및 rDNA의 internal transcribed spacer 영역, small subunit 및 large subunit 영역, 그리고 translation elongation factor 1-α 영역의 DNA 염기서열을 분석하여 동정하였다. 연구결과 5종의 국내 미기록 내생균 균주를 확인하였으며, 확인된 종은 Arthrinium camelliae-sinensis, Beltraniella humicola, B. portoricensis, Microxiphium theae, Piceomphale pinicola이다. 확인된 미기록 균주의 형태적 특성 및 분자생물학적 계통분석의 결과에 대해 서술하였다.