• Journal of Microbiology and Biotechnology
• /
• 제31권3호
• /
• pp.483-491
• /
• 2021

Two putative genes, lip29 and est29, encoding lipolytic enzymes from the thermophilic bacterium Geobacillus thermocatenulatus KCTC 3921 were cloned and overexpressed in Escherichia coli. The recombinant Lip29 and Est29 were purified 67.3-fold to homogeneity with specific activity of 2.27 U/mg and recovery of 5.8% and 14.4-fold with specific activity of 0.92 U/mg and recovery of 1.3%, respectively. The molecular mass of each purified enzyme was estimated to be 29 kDa by SDS-PAGE. The alignment analysis of amino acid sequences revealed that both enzymes belonged to GDSL lipase/esterase family including conserved blocks with SGNH catalytic residues which was mainly identified in plants before. While Est29 showed high specificity toward short-chain fatty acids (C4-C8), Lip29 showed strong lipolytic activity to long-chain fatty acids (C12-C16). The optimal activity of Lip29 toward p-nitrophenyl palmitate as a substrate was observed at 50℃ and pH 9.5, respectively, and its activity was maintained more than 24 h at optimal temperatures, indicating that Lip29 was thermostable. Lip29 exhibited high tolerance against detergents and metal ions. The homology modeling and substrate docking revealed that the long-chain substrates showed the greatest binding affinity toward enzyme. Based on the biochemical and insilico analyses, we present for the first time a GDSL-type lipase in the thermophilic bacteria group.

• Bulletin of the Korean Chemical Society
• /
• 제16권5호
• /
• pp.401-406
• /
• 1995

The P. fragi lipase overexpressed in E. coli as a fusion protein of 57 kilodalton (kDa) has been purified through glutathione-agarose affinity chromatography by elution with free glutathione. The general properties of the purified GST-fusion protein were characterized by observing absorbance of released p-nitrophenoxide at 400 nm which was hydrolyzed from the substrate p-nitrophenyl palmitate. The optimum condition was observed at 25 $^{\circ}C$, pH 7.8 with 0.4 ${\mu}g$ of protein and 1.0 mM substrate in 0.6% (v/v) TritonX-100 solution. Also the lipase was activated by Ca+2, Mg+2, Ba+2 and Na+ but it was inhibited by Co+2 and Ni+2. pGEX-2T containing P. fragi lipase gene as expression vector was named pGL191 and used as a template for the site-directed mutagenesis by sequential PCR steps. A Ser-His-Asp catalytic triad similar to that present in serine proteases may be present in Pseudomonas lipase. Therefore, the PCR fragments replacing Asp217 to Arg and His260 to Arg were synthesized, and substituted for original fragment in pGL19. The ligated products were transformed into E. coli NM522, and pGEX-2T harboring mutant lipase genes were screened through digestion with XbaI and StuI sites created by mutagenic primers, respectively. No activity of mutant lipases was observed on the plate containing tributyrin. The purified mutant lipases were not activated on the substrate and affected at pH variation. These results demonstrate that Asp217 and His260 are involved in the catalytic site of Pseudomonas lipase.

• 한국균학회지
• /
• 제38권1호
• /
• pp.57-61
• /
• 2010

본 연구에서는 버섯으로부터 새로운 항비만 치료(예방) 효과를 갖는 물질을 추출, 정제하여 그 특성을 규명하고, 이를 기능성식품 개발의 소재로 용용하고자 lipase 저해 활성이 높은 상황버섯(Phellinus linteus)의 lipase 저해 물질을 각종 chromatography로 정제 하였다. 먼저 상황버섯(Phellinus linteus) 자실체 추출물에 대한 용매 계통분획을 실시하여 저해 활성이 가장 높은 에틸아세테이트 추출물을 얻었고 이를 3차례의 sillicas gel column chromatography와 분취용 HPLC를 실시하여 $IC_{50}$값이 175 ng의 lipase 저해활성을 가진 연한 연두색 분말의 정제된 lipase 저해 물질을 얻었다. 정제한 lipase 저해물질을 일련의 NMR spectrometry를 실시한 결과 분자량이 523.06 Da으로 추정되었고 225.1 nm에서 최대 흡수 파장을 가진 물질이었다.

• 대한약학회:학술대회논문집
• /
• 대한약학회 2002년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2
• /
• pp.392.3-393
• /
• 2002

The increase of triglyceride in blood can be a signal of an increasing danger of arterial diseases when insulin resistance. diabetes. HDL -cholesterol decrease is accompanied. It is adjusted to triglyceride level in blood by a balance. which seems to be absorbed from VLDL metabolism in liver and by lipoprotein lipase activity. The hyper-triglyceride disease treatment proposal role should match with suppression does into liver or elimination of a triglyceride. (omitted)

• Journal of Microbiology and Biotechnology
• /
• 제17권10호
• /
• pp.1655-1660
• /
• 2007

A metagenome is a unique resource to search for novel microbial enzymes from the unculturable microorganisms in soil. A forest soil metagenomic library using a fosmid and soil microbial DNA from Gwangneung forest, Korea, was constructed in Escherichia coli and screened to select lipolytic genes. A total of seven unique lipolytic clones were selected by screening of the 31,000-member forest soil metagenome library based on tributyrin hydrolysis. The ORFs for lipolytic activity were subcloned in a high copy number plasmid by screening the secondary shortgun libraries from the seven clones. Since the lipolytic enzymes were well secreted in E. coli into the culture broth, the lipolytic activity of the subclones was confirmed by the hydrolysis of p-nitrophenyl butyrate using culture supernatant. Deduced amino acid sequence analysis of the identified ORFs for lipolytic activity revealed that 4 genes encode hormone-sensitive lipase (HSL) in lipase family IV. Phylogenetic analysis indicated that 4 proteins were clustered with HSL in the database and other metagenomic HSLs. The other 2 genes and 1 gene encode non-heme peroxidase-like enzymes of lipase family V and a GDSL family esterase/lipase in family II, respectively. The gene for the GDSL enzyme is the first description of the enzyme from metagenomic screening.

• 사상체질의학회지
• /
• 제19권1호
• /
• pp.171-185
• /
• 2007

1. Objectvies This experimental study was designed to investigate the effect of cheongpesagan-tang extract on the obstruction of the lipase activity and weight, plasma, UCP1, 2 mRNA in db/db mouse. Material and Methods: The body weight loss, food intake, feeding efficiency ratio, weight of the internal organs (liver, kidney, epididymal fat, brown adipose tissue), plasma glucose, triglyceride, total cholesterol, white adipose tissue, adipocyte size distribution, expression of UCP1, 2 mRNA were measured in db/db mouse administered Cheongpesagan-tang extract for 6 weeks. These were then compared with those of control groups administered the diet. 2. Results 1) Inhibitory effect against lipase activity was Kilgyung(81.7%), Nabokja (73.1%), Seungma(73.0%), Daewhang (68.4%), Kalgeun (55.3%), Kobon(34.5%), Hwanggeum(4.2%). 2) In the sample group, the body weight was significantly decrease than that of control group. 3) In the sample group, the weight of epididymal fat showed significantly decrease than that of control group. 4) In the sample group, triglyceride showed significantly decrease than that of control group. 5) In the sample group, distribution of adipose tissue showed significantly larger than that of control group. 6) In the sample group, UCP1, 2 mRNA in BAT showed significantly increase than that of control group. 3. Conclusions These results show that cheongpesagan-tang has an effect on the treatment of obesity.

• KSBB Journal
• /
• 제14권4호
• /
• pp.511-516
• /
• 1999

유지의 가수분해시, 반응온도, 금속이온, pH, 교반속도, 물-유지의 무게비 및 효소량 등의 환경 및 조성인자가 효소 Lipase-OF의 역가 및 가수분해특성에 미치는 영향을 규명하였다. 융점이 낮은 유지의 경우 Lipase-OF의 활성이 가장 높은 온도는 37$^{\circ}C$ 근방이었으나 융점이 4$0^{\circ}C$ 이상의 온도 범위에서는 유지의 종류에 관계없이 온도가 상승하면 Lipase-OF의 활성이 급격히 감소하여 $65^{\circ}C$ 이상에서는 효소의 활성이 정지되었다. 교반속도를 150, 250, 350, 450, 550 및 650 rmp으로 변화시켜가면서 유지의 가수분해실험을 수행하여 350 rpm 이하에서는 교반속도가 상승하면 가수분해율도 상승하였으나 교반속도 350 rpm 이상에서는 교반속도 상승에 대한 가수분해율의 변화를 찾아볼 수 없었다. 유지와 물의 무게비를 9 : 1에서 1 : 9까지 변화시켜가면서 가수분해실험을 수행하여 일정한 가수분해 시간에서 가수분해율이 가장 높은 무게비가 1 : 1 근방의 값임을 규명하였다. 금속 이온 중 $Ca^{2+}와\;Mg^{2+}$ 이온이 가수분해율 상승에 기여하였다. 금속이온이 없는 경우에 비하여 $Ca^{2+}$또는 $Mg^{2+}$ 이온 농도가 100 ppm 근방의 값일 때 2 내지 3%의 가수분해율 증가효과를 나타내었다. Lipase-OF에 대한 최적 pH는 7근방이었다. pH가 산성쪽으로 감소하면 가수분해율도 감소하였으며 알칼리쪽으로 증가하여도 가수분해율이 감소하였다. 기질의 0.00075 wt% 와 0.1 wt% 범위내에서 Lipase-OF량이 가수분해율에 미치는 영향을 규명하였다. 효소량 0.013 wt% 이하에서는 효소량이 증가하면 가수분해율도 증가하였으나 0.013 wt% 이상에서는 효소량이 증가하여도 가수분해율은 증가하지 않았다. Lipase-OF의 가수분해율에 미치는 환경 및 조성인자의 영향에 대한 실험을 통하여 최적온도는 37$^{\circ}C$, 최적 pH는 7, $Ca^{2+}\;또는\;Mg^{2+}$의 최적농도는 100 ppm, 최적교반속도는 350 rpm, 유지와 물의 최적무게비는 1 : 1 및 최적 효소량은 유지의 0.013 wt%임을 규명하였다.

• 한국식품영양학회지
• /
• 제9권4호
• /
• pp.392-397
• /
• 1996

저식염 멸치젓을 속성으로 제조하기 위하여 Asp. oryzae와 Bacillus sp.로 만든 zdh지를 첨가하고 숙성중 미생물상 변화와 효소활성을 비교 검토하였다. 젓갈 숙성 중 미생물상은 단백질분해균과 혐기성 균수는 숙성 40일경, 호기성 균수는 20일경에 많았다. 단백질분해균과 지방질분해균, 호기성균, 혐기성 균수는 Bacillus sp. 코오지 첨가구에서 높았다. 젓갈 숙성중 단백질 가수분해효소 활성은 숙성 20일 경에, 지방질 가수분해효소는 숙성 30일 경에 높았다가 점진적으로 감소하였으며, 단백질 가수분해효소는 Asp. oryzae 코오지 첨가구가, 지방질 가수분해효소는 Bacillus sp. 코오지 첨가구에서 높았다.

• 한국자원식물학회:학술대회논문집
• /
• 한국자원식물학회 2018년도 춘계학술발표회
• /
• pp.81-81
• /
• 2018

The present study was investigated on in vitro anti-obesity effect of 4-hydroxybenzyl alcohol from Cudrania tricuspidata. We isolated various compounds from Cudrania tricuspidata. Among these compounds, anti-obesity effects of 4-hydroxybenzyl alcohol was examined by lipase activity assay, cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase type IV (PDE4) activity assay, and citrate synthase activity assay. 4-hydroxybenzyl alcohol and Cudrania tricuspidata extracts inhibited the enzymatic activities of lipase, PDE4, and citrate synthase. Lipase is known to mediate the hydrolysis of triacylglycerol in adipose tissue and cholesterol esters in other tissue or cells. Also, PDE4 hydrolyses cAMP, a crucial secondary messenger for in metabolic pathways including glucose and lipid metabolism, lipolysis, and thermogenic function. 4-hydroxybenzyl alcohol and Cudrania tricuspidata extracts induced the inhibitory effect against each enzymatic activity on several specific substrates as observed by detection at 405 or 412 nm. These findings might be attributable to the inhibition of adipogenesis, and partial prevention of obesity. In conclusion, these results show that 4-hydroxybenzyl alcohol and Cudrania tricuspidata may be a critical candidate as a natural anti-obesity source.

• Journal of Ginseng Research
• /
• 제5권1호
• /
• pp.49-55
• /
• 1981

The effects of total, protopanaxadiol-and protopanaxatriol-saponins on the in vitro activities of several enzymes in rat serum were observed Alkaline phosphatase activity was increased 61 % by total saponin and 46% by protopanaxatriol-saponin, compared to control group. While SCOT activity was slightly decreased by total saponin and protopanaxatriol- saponin, it was slightly increased by Protopanaxadiol-saponin And while SCPT activity was slightly decreased by total saponin, it was increased by protopanaxadiol-saponin and protopanaxatriol-saponin. Creatine phosphokinase activity had a tendency to be increased by protopanaxatriol-saponin. Lactate dehydrogenase activities were increased in three saponin treated groups, but those were nonignificant. Compared to the control group, lipase activity was increased by all saponin samples. It was increased 157% by total saponin The increase in lipase activity by total saponin corresponded with the decrease in serum t total lipid by total saponin .