• Journal of Microbiology and Biotechnology
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• 제24권2호
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• pp.254-263
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• 2014

Enteromorpha polysaccharides (EP) extracted from green algae have displayed a wide variety of biological activities. However, their high molecular weight leads to a high viscosity and low solubility, and therefore, greatly restrains their application. To solve this problem, bacteria from the surface of Enteromorpha were screened, and an Alteromonas macleodii strain B7 was found to be able to decrease the molecular weight of EP in culture media. Proteins harvested from the supernatant of the A. macleodii B7 culture were subjected to native gel electrophoresis, and a band corresponding to the Enteromorpha polysaccharide lyase (EPL) was detected by activity staining. The enzyme identity was subsequently confirmed by MALDI-TOF/TOF mass spectrometry as the putative ${\alpha}$-amylase reported in A. macleodii ATCC 27126. The amylase gene (amySTU) from A. macleodii B7 was cloned into Escherichia coli, resulting in high-level expression of the recombinant enzyme with EP-degrading activity. AmySTU was found to be cold-adapted; however, its optimal enzyme activity was detected at $40^{\circ}C$. The ${\alpha}$-amylase was highly stable over a broad pH range (5.5-10) with the optimal pH at 7.5-8.0. The highest enzyme activity was detected when NaCl concentration was 2%, which dropped by 50% when the NaCl concentration was increased to 16%, showing an excellent nature of halotolerance. Furthermore, the amylase activity was not significantly affected by tested surfactants or the presence of some organic solvents. Therefore, the A. macleodii strain B7 and its ${\alpha}$-amylase can be useful in lowering EP molecular weight and in starch processing.

• Journal of Microbiology
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• 제44권4호
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• pp.396-402
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• 2006

[ ${\beta}-Galactosidase$ ] is extensively employed in the manufacture of dairy products, including lactose-reduced milk. Here, we have isolated two gram-negative and rod-shaped coldadapted bacteria, BS 1 and HS 39. These strains were able to break down lactose at low temperatures. Although two isolates were found to grow well at $10^{\circ}C$, the BS 1 strain was unable to grow at $37^{\circ}C$. Another strain, HS-39, evidenced retarded growth at $37^{\circ}C$. The biochemical characteristics and the results of 16S rDNA sequencing identified the BS 1 isolate as Rahnella aquatilis, and showed that the HS 39 strain belonged to genus Buttiauxella. Whereas the R. aquatilis BS 1 strain generated maximal quantities of ${\beta}-galactosidase$ when incubated for 60h at $10^{\circ}C$, Buttiauxella sp. HS-39 generated ${\beta}-galactosidase$ earlier, and at slightly lower levels, than R. aquatilis BS 1. The optimum temperature for ${\beta}-galactosidase$ was $30^{\circ}C$ for R. aquatilis BS-1, and was $45^{\circ}C$ for Buttiauxella sp. HS-39, thereby indicating that R. aquatilis BS-1 was able to generate a cold-adaptive enzyme. These two cold-adapted strains, and most notably the ${\beta}-galactosidase$ from each isolate, might prove useful in some biotechnological applications.

• Journal of Microbiology and Biotechnology
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• 제23권9호
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• pp.1221-1228
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• 2013

Two lipase genes (bpl1 and bpl3) from Antarctic Bacillus pumilus strains were expressed in Bacillus subtilis. Both recombinant lipases BPL1 and BPL2 were secreted to the culture medium and their activities reached 3.5 U/ml and 5.0 U/ml, respectively. Their molecular masses apparent using SDS-PAGE were 23 kDa for BPL1 and 19 kDa for BPL3. Both lipases were purified to homogeneity using ammonium sulfate precipitation and HiTrap SP FF column and Superose 12 column chromatographies. The final specific activities were estimated to be 328 U/mg for BPL1 and 310 U/mg for BPL3. Both lipases displayed an optimum temperature of $35^{\circ}C$, similar to other mesophilic enzymes. However, they maintained as much as 70% and 80% of the maximum activities at $10^{\circ}C$. Accordingly, their calculated activation energy at a temperature range of $10-35^{\circ}C$ was 5.32 kcal/mol for BPL1 and 4.26 kcal/mol for BPL3, typical of cold-adapted enzymes. The optimum pH of BPL1 and BPL3 was 8.5 and 8.0, respectively, and they were quite stable at pH 7.0-11.0, showing their strong alkaline tolerance. Both lipases had a preference toward medium chain length ($C_6-C_{10}$) fatty acid substrates. These results indicate the potential for the two Antarctic B. pumilus lipases as catalysts in bioorganic synthesis, food, and detergent industries.

• Journal of Microbiology and Biotechnology
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• 제19권2호
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• pp.128-135
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• 2009

Acinetohacter baumannii BD5 was isolated from waters of Baek-du mountain, and the lipase gene was cloned using a PCR technique. The deduced amino acid sequence of the lipase and lipase chaperone were found to encode proteins of 325 aa and 344 aa with a molecular mass of 35 kDa and 37 kDa, respectively. The lipase gene was cloned and expressed in Escherichia coli BL21(trxB) as an inclusion body, which was subsequently solubilized by urea, and then purified using Ni-affinity chromatography. After being purified, the lipase was refolded by incubation at $4^{\circ}C$ in the presence of a 1:10 molar ratio of lipase:chaperone. The maximal activity of the refolded lipase was observed at a temperature of $35^{\circ}C$ and pH 8.3 when p-NP caprate(C10) was used as a substrate; however, 28% of the activity observed at $35^{\circ}C$ was still remaining at $0^{\circ}C$. The stability of the purified enzyme at low temperatures indicates that it is a cold-adapted enzyme. The refolded lipase was activated by $Ca^{2+},\;Mg^{2+},\;and\;Mn^{2+}$, whereas $Zn^{2+}\;and\;Cu^{2+}$ inhibited it. Additionally, 0.1% Tween 20 increased the lipase activity by 33%, but SDS and Triton X-100 inhibited the lipase activity by 40% and 70%, respectively.

• 미생물학회지
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• 제46권1호
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• pp.73-79
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• 2010

극지연구소(KOPRI)는 국내외적으로 유일하게 남북극 지역에서 분리한 저온적응성 박테리아 균주를 대상으로 culture collection(약 6,300균주)을 구축하여 운영하고 있다. 보유 중인 프로테아제(protease) 생산 균주들(총 874균주) 중에서 활성이 높은 프로테아제를 생산하는 78개의 균주들을 1차 선발한 후, 1% skim milk가 포함된 0.1${\times}$ ZoBell 고체배지에 접종하고 다양한 온도($5-30^{\circ}C$)에서 배양하면서 세포외분비성 프로테아제의 활성을 비교하였다. 위의 신속하고 직접적인 균주 스크리닝 방법을 통해서, 최종적으로 저온활성 프로테아제를 생산하는 15개의 저온적응성 균주들을 선발하였다. 최종 선발된 균주들은 16S rRNA 유전자의 분석결과 Pseudoalteromonas (13균주)와 Flavobacterium (2균주) 속(genus)으로 분류되었고, $5-15^{\circ}C$ 저온에서도 활성을 나타내는 저온성 프로테아제를 생산하였다. 15개 균주들이 생산하는 각각의 프로테아제는 특이적 화합물에 의한 효소활성 억제 정도에 따라 5개의 그룹(serine protease, aspartic protease, cysteine protease, metalloprotease, 그리고 미분류 프로테아제)으로 분류되었다. 본 실험을 통해서 선발한 남북극 유래 박테리아 균주들은 새로운 저온활성 프로테아제를 발굴하기 위한 유용한 생물자원으로서의 가치를 가지고 있다.

• Journal of Microbiology and Biotechnology
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• 제24권11호
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• pp.1484-1489
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• 2014

A novel esterase gene, est01, was successfully unearthed from a biogas digester microbiota metagenomic library. The 1,194 bp est01 gene encodes a protein of 44,804 Da (designated Est01). The amino acid sequence of Est01 shows only moderate (33%) identity to a lipase/esterase. Phylogenetic analysis and biochemical characterization confirmed that Est01 is a new member of family VIII esterases. The purified Est01 from recombinant Escherichia coli BL21 (DE3) showed high hydrolytic activity against short-chain fatty acid esters, suggesting that it is a typical carboxylesterase rather than a lipase. Furthermore, the Est01 was even active at $10^{\circ}C$ (43% activity remained), with the optimal temperature at $20^{\circ}C$, and had a broad pH range from 5.0 to 10.0, with the optimal pH of 8.0. These properties suggest that Est01 is a cold-adaptive esterase and could have good potential for low-temperature hydrolysis application.

• 미생물학회지
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• 제55권1호
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• pp.83-85
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• 2019

남극 연안 툰드라 토양에서 리그닌 분해능이 있는 Pseudomonas sp. PAMC 29040를 분리하였으며, 이후 토양 유기물의 주요 구성성분인 복합유기화합물 부식질 분해능을 확인하였다. 부식질 초기 저분자화 효소(예, dye-decolorizing peroxidase)와 부식질 유래의 다양한 저분자 분해산물들을 분해하는 효소들(예, vanillate O-demethylase)를 탐색하기 위해 PAMC 29040 게놈 염기서열을 분석하였다. 분석을 통해서 최종 확보한 효소유전자 정보는 저온환경에 서식하는 토양 세균의 부식질 분해경로 제안에 활용될 것이다.

• 대한간호학회지
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• 제13권2호
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• pp.87-104
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• 1983

It has been well documented that animals exposed to cold show increased activity of thyroid gland. The calorigenic action of thyroid hormone has been demonstrated by a variety of in vivo and in vitro studies. According to Edelman et al., the thyroid thermogenesis is due to activation of energy consuming processes, especially the active sodium transport by the hormone in target tissues. If so, the increase in thyroid activity during cold exposure should induce increased capacity of sodium transport in target tissue and the change in tissue metabolism should be precisely correlated with the change in Na+_K+_ATPase activity of the tissue. This possibility was tested in the present study: in one series, changes in oxygen consumption and Na+_K+_-ATPase activity of liver preparations were measured in rats as a function of thyroid status, in order to establish the effect of thyroid hormone on the tissue respiration and enzyme system in another series, the effect of cold stimulus on the serum thyroid hormone level, hepatic tissue oxygen consumption and Na+_K+_ATPase activity in rats. The results obtained are as follows: 1. The Na+_dependent oxygen consumption of liver slices, the oxygen consumption of liver mitochondria and the Na+_K+_ATPase activity of liver preparations were significantly inhibited in hypothyroidism and activated in hyperthyroidism. Kinetic analysis indicated that the Vmax. of Na+_K+_ATPase was decreased in hypothyroidism and increased in hyperth)'roidism. 2. In cold exposed rats, the serum triiodothyronine (T₃) level increased rapidly during the initial one day of cold exposure, then declined slowly to the control level after two weeks. The serum thyroxine (T₄) level decreased gradually throughout the cold exposure. Accordingly the T₃/T₄ratio increased. The mitochondrial oxygen consumption and the Na+_dependent oxygen consumption of liver slices increased during the first two days and then remained unchanged thereafter The activity of the Na+_K+_ATPase in liver preparations increased during cold exposure with a time course similar to that of oxygen consumption. Kinetic analysis indicated that the Vmax. of Na+_K+_ATPase increased. 3. Once the animal was adapted to cold, induction of hypothyroidism did not significantly alter the hepatic oxygen consumption and Na+_K+_ATPase activity. These results indicate that: 1) thyroid hormone increases capacities of mitochondrial respiration and active sodium transport in target tissues such as liver; 2) the increased T₃level during the initial period of cold exposure facilitates biosynthesis of Na+_K+_ATPase and mitochondrial enzymes for oxidative phosphorylation, leading to enhanced production and utilization of ATP, hence heat production.

• 한국미생물·생명공학회지
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• 제33권1호
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• pp.29-34
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• 2005

새만금 갯벌로부터 리파제를 생산하는 균주(S3)를 분리하였다. 생리적, 발효적 특성 및 계통분류학적 특성을 통해서 이 분리균이 Psychrobacter속에 속하는 것으로 판명되어서 Psychrobacter sp. S3로 명명하였다 이 균의 온도에 따른 배양특성을 구한 결과, $30^{\circ}C$에서 생장속도가 가장 빨랐으나, 리파제 효소의 활성은 $20^{\circ}C$에서 가장 높았다. S3리파제의 온도에 따른 p-nitrophenyl caproate 분해활성을 측정한 결과, 최적 온도가 $30^{\circ}C$로 판명되었으며 $10^{\circ}C$에서도 최고활성의 $80\%$ 이상의 활성을 유지하였다. 또한, $10-30^{\circ}C$범위에서의 효소활성에너지가 1.5 kcal/mol로 매우 낮게 계산되었다. 이것을 통해 S3 리파제가 전형적인 저온성 효소임이 확인되었다. 이 효소는 최적 pH가 $9.0\~9.5$인 알칼리성 효소로 확인되었다. 여러 길이의 트리글리세리드 기질을 분해할 수 있으며 그 중에서 $C_4,\;C_{14},\; C_{16}$기질을 가장 빠르게 분해하였다. S3리파제를 트리뷰티린-아가로스 젤에 가하여 온도별로 반응시킨 결과, $30^{\circ}C$와 $40^{\circ}C$에서 반응이 빠르게 진행되었으나, $4^{\circ}C$에서도 분해가 진행되었다.

• 한국미생물·생명공학회지
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• 제37권2호
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• pp.118-124
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• 2009

에스터라제 EM2L8 유전자를 E. coli 균에서 발현하고 에스터라제 활성을 분석한 결과, $40-45^{\circ}C$에서 최적의 효소활성을 보였다. $15^{\circ}C$에서 최대활성의 45% 활성을 보였고 $15-45^{\circ}C$ 사이의 활성화에너지는 4.9 kcal/mol로 계산됨으로써 전형적인 저온 적응효소인 것으로 밝혀졌다. 또한, $4^{\circ}C$에서 장기보관해도 효소활성이 전혀 줄어들지 않음을 통해서 저온에서 안정한 효소임을 알게 되었다. 반응액에 에탄올, 메탄올, 아세톤을 15% 농도까지 첨가해도 효소활성이 줄어들지 않았으며 DMSO의 경우, 40% 농도까지 첨가해도 효소활성이 유지되는 것으로 나타났다. 이 효소 40 U을 Tris-HCl 용액(1.2 mL, pH 9.0)에 넣고 $30^{\circ}C$에서 (R,S)-ECHB(0.5%, 38 mM)의 분해반응을 수행한 결과, 기질이 가수분해되어 CHBacid가 생성되며 기질의 분해속도는 $6.8\;{\mu}mole/h$로 계산되었다. (R)-ECHB 보다 (S)-ECHB 기질을 빠르게 분해하였으며 전환수율이 80%일 때, e.e.s 값이 40%로 측정되었다. 반응액에 DMSO를 10% (v/v) 농도로 각각 첨가한 결과, 기질의 분해 속도는 $10.4\;{\mu}mole/h$로 증가되었다. 하지만 DMSO의 유무와 상관없이 전환수율에 따른 e.e.s 값은 유사하게 나타났다. 결론적으로 이 효소는 저온과 각종 유기용매 하에서도 높은 안정성과 활성을 갖고 있기 때문에 각종 의약품의 유기합성공정에서 효소촉매로 활용될 수 있을 것으로 기대된다.