• 미생물학회지
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• 제27권3호
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• pp.181-187
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• 1989

Lactobacillus casei SW-M1으로부터 lactose 이용 pPLac Plasmid를 분리하였다. 이 plasmid에 lactose이용 유전자가 존재하는지를 확이하기 위하여 plasmid curing을 실시한 결과, acriflavin 8mg/ml 과 11 mg/ml EtBr를 처리한 후 , 3차 접종 배양의 경우에 curing 빈도가 가장 높았다. Lac와 plasmid가 cured 된 $Lac^{+}$strain의 당 이용능을 조사한 결고, glucose lactosidasedldydsmd은 불변이나, lactosedldydsmd만이 $Lac^{+}$strain에서 감소하였다 pPLac plasmid의 lactose 분해능은 $\beta$-galactosidase 에 의한 것이 아니고, phospho-$\beta$-galactosidase 에 의한 것으로 확인되었다. $Lac^{+}$strain의 carbohydrate가 막투과시 PTS과 관련이 있는가를 조사한 결과ㅏ lactose-PTS가 가장 활성이 높았으며, 그 다음이 galactose-PTS, glucose-PTS 로 나타났다. 그러므로 lactose는 lactose-PTS(lactose-phosphotransferase system)에 의하여 glucose와 galactose-6-phosphate로 분해됨을 알 수 있었다. Phospho-$\beta$-galactosidase의 induction 실험에서는 galactoserk 가장 높은 induction 효과를 보여 주었으며, lactose와 glucose는 높은 수준의 induction을 나타내었으며, IPTG는 induction 효과가 없었다. Glucosedh lactose 배지에서 L. casie는 diauxic growth나 phospho-$\beta$-galactosidase합성을 조사한 결과, catabolite repression을 받지 않는 것으로 나타났다.

• Asian-Australasian Journal of Animal Sciences
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• 제17권12호
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• pp.1751-1757
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• 2004

Recombinant heterologous proteins can be produced as insoluble aggregates partially or perfectly inactive in Escherichia coli. One of the strateges to improve the solubility of recombinant proteins is fusion with a partner that is excellent in producing soluble fusion proteins. To improve the production of soluble $\beta$-galactosidase, the gene of Thermus thermophilus KNOUC112 $\beta$-galactosidase (KNOUC112 $\beta$-gal) was fused with thioredoxin gene, and optimization of its expression in E. coli TOP10 was performed. KNOUC112 $\beta$-gal in pET-5b was isolated out, fused with thioredoxin gene in pThioHis C, and transformed to E. coli TOP10. The $\beta$-galactosidase fused with thioredoxin was produced in E. coli TOP10 as dimer and trimer. The productivity of fusion $\beta$ -galactosidase expressed via pThioHis C at 37$^{\circ}C$ was about 5 times higher than that of unfused $\beta$-galactosidase expressed via pET-5b at 37$^{\circ}C$. Inclusion body of $\beta$-galactosidase was formed highly, regardless of the induction by IPTG when KNOUC112 $\beta$ -gal was expressed via pET-5b at 37$^{\circ}C$. Fusion $\beta$ -galactosidase expressed at 37$^{\circ}C$ via pThioHis C without the induction by IPTG was soluble, but the induction by IPTG promoted the formation of inclusion body. Lowering the incubation temperature for the expression of fusion gene under 25$^{\circ}C$ prevented the formation of inclusion body, optimally at 25$^{\circ}C$. 0.07 mM of IPTG was sufficient for the soluble expression of fusion gene at 25$^{\circ}C$. The soluble production of Thermus thermophilus KNOUC112 $\beta$-galactosidase could be increased about 10 times by fusion with thioredoxin, and optimization of incubation temperature and IPTG concentration for induction.

• 한국미생물·생명공학회지
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• 제40권1호
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• pp.17-22
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• 2012

가정에서 제조된 청국장으로부터 lactose를 glucose와 galactose로 가수분해하는 ${\beta}$-galactosidase의 생산균을 분리하였다. 분리균 YB-1105는 형태적 특성, 생화학적 성질 및 16S rRNA 유전자 염기서열에 근거하여 Bacillus licheniformis로 확인되었다. B. licheniformis YB-1105의 배양상등액과 균체파쇄액에서 모두 ${\beta}$-galactosidase 활성이 관찰되었으며 이들은 모두 pH 6.5와 $50^{\circ}C$의 반응조건에서 paranitrophenyl-${\beta}$-D-galactopyranoside의 가수분해 활성이 최대로 나타났다. 그러나 균체파쇄상등액에 비해 배양상등액의 ${\beta}$-galactosidase 활성은 산성 pH와 고온에서 크게 영향을 받았다. 한편 두 분획의 가수분해 활성은 낮은 농도의 galactose에 의해도 급격하게 저해되었으나, glucose와 mannose는 고농도에 의해서는 약하게 저해를 받는 것으로 확인되었다.

• Journal of Microbiology and Biotechnology
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• 제18권3호
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• pp.434-442
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• 2008

We observed that an inclusion body (IB) of recombinant ${\beta}$-galactosidase that was produced by the araBAD promoter system in Escherichia coli (E. coil) showed enzyme activity. In order to improve its activity, the lowering of the transcription rate of the ${\beta}$-galactosidase structural gene was attempted through competition between an inducer (L-arabinose) and an inducer analog (D-fucose). In the deep-well microtiter plate culture and lab-scale fermentor culture, it was demonstrated that the addition of D-fucose caused an improvement in specific ${\beta}$-galactosidase production, although ${\beta}$-galactosidase was produced as an IB. In particular, the addition of D-fucose after induction led to an increase in the specific activity of ${\beta}$-galactosidase IB. Finally, we confirmed that the addition of D-fucose after induction caused changes in the structure of ${\beta}$-galactosidase IB, with higher enzyme activity. Based on these results, we expect that an improved enzyme IB will be used as a biocatalyst of the enzyme bioprocess, because an enzyme IB can be purified easily and has physical durability.

• 미생물학회지
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• 제30권6호
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• pp.539-545
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• 1992

Lactobacillus casei SM-M1 의 플라스미드로부터 phospho-$\beta$-galactosidase gene 을 갖는 DNA 를 E. coli 에 클로닝한 pPLac15(13kb) 의 재조합 플라스미드를 제조하였다.(15). pPLac15 DNA 를 분리하여 제한효소로 처리하여 제한효소 지도를 작성하였다. Phospho-$\beta$-galactosidase 유전자의 발현을 높이기 위하여 lac promoter 를 가진 pUC18 의 PstI 위치에 클닝하여 pPLac18 을 제조하였으며, 이것을 다시 EcoRI 으로 절단하여 pUC 18 에 클로닝하여 얻은 pPLac23 (7.6 kb) 를 얻었다. Phospho-$\beta$-galactosidase 효소활성은 pPLac23 의 형질전환주인 E. coli SW-23 에서는 pPLac15 를 가진 형질전환주인 E. coli SW-15 보다 약 1.8 배의 효소의 활성을 나타내었으며 pPLac18 을 가진 E. coli SW-18 보다는 약간 높은 활성을 나타내었다.

• 미생물학회지
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• 제22권2호
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• pp.77-84
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• 1984

This examined some conditions for the induction of ${\beta}$-D-galactosidase synthesis in Candida kefyr CBS 834. The optimal pH, temperature, and inoculum size either for growth or${\beta}$-D-galactosidase synthesis were 5.5, $30^{\circ}C$ and above 0.2 at A610nm, respectively. Enzyme activity began to increase at 2h after the addition of inducer, and continued to increase linearly up to $2{\sim}3h$ before reaching stationary phase, and thereafter its activity was decreased. ${\beta}$-D-galactosidase was induced either by lactose or galactose but not either by glucose or ethanol. The greater activity of ${\beta}$-D-galactosidase on galactose than on lactose indicated that the former might be natural inducer for ${\beta}$-D-galactosidase synthesis. The rate of its induction as a function of lactose concentration showed that enzyme activity increased linearly above 4mM, while it was very low below that. Glucose represed the induction of ${\beta}$-D-galactosidase, and the period of adaptation to inducer from other carbon sources was relatively short.

• Journal of Microbiology and Biotechnology
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• 제16권12호
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• pp.1968-1976
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• 2006

Recombinant E. coli ACV 1003 (recA:: lacZ) was used to measure low concentrations of DNA-damaging chemicals, which produce $\beta$-galactosidase via an SOS regulon system. Very low $\beta$-galactosidase activities of less than 0.01 unit/ml, $\beta$-galactosidase produced through an SOS response corresponding to the 10 ng/ml (ppb) of DNA damaging chemicals in the environment, can be rapidly determined by using an alternative interferometric biosensor with optically flat thin films of porous silicon rather than by the conventional time-consuming Miller's enzyme assay as well as the ELISA method. fu order to enhance the sensitivity in the interferometry, it needs to obtain more uniform distribution and higher biolinking efficiency, whereas interferometric sensing is rapid, cheap, and advantageous in high throughput by using a multiple-well-type chip. In this study, pore size adjusted to 60 nm for the target enzyme $\beta$-galactosidase to be bound on both walls of a Si pore and a calyx crown derivative was apllied as a more efficient biolinker. Furthermore, anti-$\beta$-galactosidase was previously functionalized with the biolinker for the target $\beta$-galactosidase to be specifically bound. When anti-$\beta$-galactosidase was bound to the calyx-crown derivative-linked surface, the effective optical thickness was found to be three times as high as that obtained without using anti-$\beta$-galactosidase. The resolution obtained was very similar to that afforded by the time-consuming ELISA method; however, the reproducibility was still unsatisfactory, below 1 unit $\beta$-galactosidase/ml, owing to the microscopic non-uniform distribution of the pores in the etched silicon surface.

• 한국미생물·생명공학회지
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• 제15권2호
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• pp.84-88
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• 1987

Poly(ethylene glycol)/salt가 형성하는 액상 2상계에서 균체내 효소인 $\beta$-galactosidase 분리를 최적화하는 실험을 수행하였다. L. sporogenes에 의해 생산된 $\beta$-galactosidase의 분획은 2상계를 형성하는 PEG 분자량이 작을수록 상층부로 이동하였으며, 상층부 PEG 농도가 증가할 때 salting-out 현상이 나타나서 단백질 뿐아니라 $\beta$-galactosidase는 상층부로 크게 이동하였다. 또한 균체내 효소 정제에서 문제가 되는 세포벽잔사(cell debris)들은 조성이 binodial line에 접근할수록 효소 fraction과 반대방향으로 이동되었다. PEG-salt를 이용한 $\beta$-galactosidase 분리는 조성을 binodial line레 접근시키면서 동시에 상층부의 부피를 줄이는 것이 효과적이었다. PEG1000, 300을 각각 단계적으로 이용한 2단계 추출(two-step extraction)로써 세포벽 잔사, bulk protein, 색소물질 및 핵산 등을 제거할 수 있었으며, 이때 효소의 회수율은 74%, 단백질 회수율 35%로서 정제도는 2 배 이상의 효과를 보였다.

• 한국미생물·생명공학회지
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• 제20권6호
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• pp.647-654
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• 1992

장내 세균의 생리적 연구를 목적으로 한국인의 장내 상재균을 분리하여 효소 pattern을 관찰하였다. 분리된 Bifidobacterium sp. Int-57은 다른 장내 균종에 비하여 $\alpha$-glucosidase, $\beta$-glucosidase, $\alpha$-galactosidase, $\beta$-galactosidase, $\beta$-xylosidase, $\alpha$-arabinofuranosidase역가가 높았다. Bifidobacterium sp. Int-57의 각 효소 생산에 미치는 탄소원의 영향을 조사하였다. $\alpha$-glucosidase는 maltose, $\beta$-glucosidase는 cellobiose, $\alpha$-galactosidase는 raffinose, 는 lactose, $\beta$-xylosidase와 $\alpha$-arabinofuranosidase는 xyloserk 각각 최적의 탄소원이었다. 또한 각 효소들의 최적 조건과 pH 안정성을 조사 하였다. $\alpha$-glucosidase는 pH 6.0 $40^{\circ}C$에서 $\beta$-glucosidasessm pH 7.0 50에서, $\beta$-galactosidase는 pH 7.0 50에서, $\beta$-xylosidase는 pH 6.0 $40^{\circ}C$에서, $\alpha$-arabinofurnaosidase는 pH 5.0 $50^{\circ}C$에서 각각 최적이었다. $\alpha$-glucosidase는 pH 4.0~9.0 $\beta$-glucosidase는 pH 4.0~7.0 $\beta$-galactosidase는 pH 4.0~9.0, $\beta$-xylosidase는 pH 4.0~6.0, $\alpha$-arabinofuranosidase는 pH 7.0~9.0에서 각각 안정하였다.

• Applied Biological Chemistry
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• 제46권4호
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• pp.299-304
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• 2003

토양으로부터 lactose의 가수분해를 촉매하는 균체외 ${\beta}-galactosidase$를 생산하는 YB-9가 분리되었다. 분리균 YB-9는 분리균의 배양, 형태, 생리적 특성을 조사한 결과 Streptomyces속 균주로 동정되었다. 분리균의 배양상등액을 ammonium $sulfate(15{\sim}70%)$로 처리하고 투석하여 부분정제된 ${\beta}-galactosidase$를 $para-nitrophenyl-{\beta}-D-galactopyranoside(pNP-{\beta}Gal)$와 lactose를 기질로 하여 반응특성을 분석하기 위해 조효소액으로 사용하였다. ${\beta}-Galactosidase$는 pH $6.0{\sim}6.5$와 $60^{\circ}C$에서 최대활성을 보였다. $pNP-{\beta}Gal$과 lactose에 대한 ${\beta}$-galactosidase의 가수분해 활성은 galactose에 의해 감소되었다. Lactose에 대한 가수분해 활성은 glucose에 의해 미미하게 감소하였으나, glucose에 의해 $pNP-{\beta}Gal$에 대한 활성은 1.3배 증가하였다. 특히, xylose에 의한 lactose의 가수분해 활성에는 영향이 없었고, $pNP-{\beta}Gal$에 대한 활성은 1.6배 증가시켰다.