• Journal of Microbiology and Biotechnology
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• v.27 no.2
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• pp.277-288
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• 2017

Rhizomucor miehei NRRL 5282 and Rhizopus oryzae NRRL 1526 can produce lipases with high synthetic activities in wheat bran-based solid-state culture. In this study, the purification and biochemical characterization of the lipolytic activities of these lipases are presented. SDS-PAGE indicated a molecular mass of about 55 and 35 kDa for the purified R. miehei and Rh. oryzae enzymes, respectively. p-Nitrophenyl palmitate (pNPP) hydrolysis was maximal at $40^{\circ}C$ and pH 7.0 for the R. miehei lipase, and at $30^{\circ}C$ and pH 5.2 for the Rh. oryzae enzyme. The enzymes showed almost equal affinity to pNPP, but the $V_{max}$ of the Rh. oryzae lipase was about 1.13 times higher than that determined for R. miehei using the same substrate. For both enzymes, a dramatic loss of activity was observed in the presence of 5 mM $Hg^{2+}$, $Zn^{2+}$, or $Mn^{2+}$, 10 mM N-bromosuccinimide or sodium dodecyl sulfate, and 5-10% (v/v) of hexanol or butanol. At the same time, they proved to be extraordinarily stable in the presence of n-hexane, cyclohexane, n-heptane, and isooctane. Moreover, isopentanol up to 10% (v/v) and propionic acid in 1 mM concentrations increased the pNPP hydrolyzing activity of R. miehei lipase. Both enzymes had 1,3-regioselectivity, and efficiently hydrolyzed p-nitrophenyl (pNP) esters with C8-C16 acids, exhibiting maximum activity towards pNP-caprylate (R. miehei) and pNP-dodecanoate (Rh. oryzae). The purified lipases are promising candidates for various biotechnological applications.

• Journal of Microbiology and Biotechnology
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• v.31 no.3
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• pp.483-491
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• 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
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• v.16 no.5
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• pp.401-406
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• 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.

• The Korean Journal of Mycology
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• v.38 no.1
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• pp.57-61
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• 2010

For development of anti-obesity nutraceuticals from mushrooms, new anti-obesity lipase inhibitor from Phellinus linteus was purified by systematic solvent extraction, TLC and HPLC and characterized. Methanol extract from P. linteus most effectively inhibited(72.5%) porcine pancreatic lipase and ethyl acetate fraction showed the highest inhibitory activity in the systematic solvent extraction. A lipase inhibitor from the ethyl acetate fraction was purified through following steps including 3 times silica gel chromatography and preparative HPLC. The purified lipase inhibitor was a yellowish geen powder and its $IC_{50}$ value was 175 ng. Its molecular weight by MALDI-TOF-MS was 523.06 Da and showed maximal absorption spectrum at 225.1 nm.

• Proceedings of the PSK Conference
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• 2002.10a
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• pp.392.3-393
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• 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
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• v.17 no.10
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• pp.1655-1660
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• 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.

• Journal of Sasang Constitutional Medicine
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• v.19 no.1
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• pp.171-185
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• 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
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• v.14 no.4
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• pp.511-516
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• 1999

The effects of environmental and compositon factors, such as reaction time, metal ions, pH, agitation speed, the weight ratio of water to oil, and the weight of enzyme, on the hydrolysis of oils by Lipase-OF were investigated. In case of oils with low melting point, the optimum temperature of hydrolysis were the enzyme activity was maximum was 37$^{\circ}C$. However, when the melting temperature was higher than 4$0^{\circ}C$, the optimum temperature was around the fusion temperature. The activity of Lipase-OF decreased very rapidly with increase of temperature in the range of higher than 45$^{\circ}C$ and the activity perished above $65^{\circ}C$. The effect of agitation speed was investigated from 150 to 650 rpm. The hydrolysis of oils increased as the agitation speed increased up to 350 rpm, but it did not increase any more above 350 rpm. The weight ratio of water to oil was changed from 1 : 9 to 9 : 1 for the investigation of the effect on the hydrolusis. The weight ratio for maximum hydrolysis was 1 : 1. $Ca^{2+}\;and\;Mg^{2+}$ among various metal ions had some effect on the stimulation of hydrolysis. The optimum concentration of the ions was about 100ppm at which the hydrolysis increased, compared with that of distilled water, by 2 to 3%. The Optimum pH of Lipase-OF was 7. The hydrolysis decreased as the pH decreased as the pH decreased and also decreased as the pH increased. The content of enzyme affected the hydrolysis of oil. The hydrolysis increased with the content of Lipase-OF in the range of less than 0.013 wt% of substrate. However, the increase of hydrolysis with the content of Lipase-OF ceased above 0.013 wt%. The experiments investigating the effect of environmental and composition factors on the hydrolysis of oils showed that the optimum temperature was 37$^{\circ}C$, the pH 7, the concentration of $Ca^{2+}\;or\;Mg^{2+}$ 100 ppm, the agitation speed 350 rpm, the weight ratio of water to oil 1 : 1, and the content of Lipase-OF 0.013 wt% of substrate.

• The Korean Journal of Food And Nutrition
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• v.9 no.4
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• pp.392-397
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• 1996

To produce low salt fermented anchovy by an accelerated method with Asp. oryzae and Bacillus sp. koji, enzyme activity and variation of microflora during the 60 day fermentation were examined. Bacterial counts changed a little during the fermentation with the highest on day 40 for proteolytic and anaerobic bacteria and on day 20 for aerobic bacteria. Proteolytic, lipolytic, aerobic, and anaerobic bacteria counts were higher in the Bacillus sp. koji added anchovy paste than in others. The protease and lipase activities reached the highest point on day 20 and 30, respectively, and decreased gradually afterwards. The protease activity was higher in Asp. oryzae koji than in bacillus sp. koji, but the lipase activity was to the contrary.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2018.04a
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• pp.81-81
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• 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.