• Natural Product Sciences
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• 제16권1호
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• pp.1-5
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• 2010

$\beta$-Glucuronidases (E.C. 3.2.1.31) from Escherichia coli, Helix pomatia, and bovine liver activity have been investigated on 7-O-glucuronides (baicalin, wogonoside, and luteolin-7-O-glucuronide) and 3-O-glucuronides (quercetin-3-O-glucuronide and kaempferol-3-O-glucuronide). Bovine liver enzyme was not active on any of these substrates. E. coli and H. pomatia enzymes were active on 7-O-glucuronides, however, 3-O-glucuronides were resistant to $\beta$-glucuronidase hydrolysis. These results suggest that glucuronic acid at 7-position is more susceptible to E. coli and H. pomatia $\beta$-glucuronidases than that at 3-position. In addition, the subtle difference of aglycone structure on 7-O-glucuronides affected the preference of enzyme. E. coli enzyme was favorable for the hydrolysis of baicalin, however, H. pomatia enzyme was found to be efficient for the hydrolysis of wogonoside. Both enzymes showed the similar hydrolytic activity towards luteolin-7-O-glucuronide. When the Scutellaria baicalensis crude extract was subjected to enzymatic hydrolysis, baicalin and wogonoside were successfully converted to their aglycone counterparts with H. pomatia at 50 mM sodium bicarbonate buffer pH 4.0. Accordingly, the enzymatic transformation of glycosides may be quite useful in preparing aglycones under mild conditions.

• Journal of Microbiology and Biotechnology
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• 제31권3호
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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.

• Journal of Animal Science and Technology
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• 제49권5호
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• pp.667-676
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• 2007

본 연구는 톱밥주원료의 병재배 버섯폐배지의 퇴적발효 간 섬유소분해성 미생물 첨가가 발효물의 발효 성상, 균수 및 효소 활력에 미치는 영향을 평가하고, 미생물 처리한 버섯폐배지 급여시 면양의 영양소 이용성에 미치는 효과를 구명하고자 실시하였다. 면양 대사시험의 경우, 면양 6두를 공시하여 배합사료, 볏짚 그리고 버섯폐배지를 각각 70:15:15(건물기준)의 비율로 급여하였다. 처리구의 버섯폐배지는 퇴적발효 시 4종의 섬유소 분해균(201-3, 201-7, 206-3, 3)을 접종하였다. 1.2 M/T 규모의 버섯폐배지의 퇴적발효 온도는 발효 7일 이내에 50 ℃ 내외에 도달하였다. 고온의 발효열에 의해 발효 전과 비교해서 발효 후에는 대조구와 균처리구 모두 총균수가 현저히 감소하였고(P<0.05), 처리에 따른 차이는 없었다(P>0.05). 섬유소 분해효소의 활력에 있어서, CMCase와 xylanase의 활력은 퇴적발효 처리에 의해 공히 현저히 감소하였다(P<0.05). 발효 후에는 대조구와 비교해서 미생물 처리구에서 CMCase 활력은 2.5배 정도, xylanase 활력은 4배 정도 더 높았다(P<0.05). 리그닌 분해 효소인 laccase와 MnP의 활력은 미생물 처리에 따른 차이는 없었다. 면양대사시험 결과, 버섯폐배지를 볏짚 급여량의 50%(총사료의 15%)와 대체하였을 때(건물기준) 미생물 처리한 폐배지 발효물 급여 시에는 무처리 폐배지 발효물 급여시와 비교하여 면양체내에서의 ash(P=0.051), NFE(P=0.071), hemicellulose(P=0.087) 및 NDF(P=0.096)의 전장 소화율은 증가하는 경향이었으며, 체내 질소 균형은 차이가 없었다(P>0.05). 따라서 이러한 연구 결과는 톱밥주원료 버섯폐배지의 발효사료화 시 섬유소분해성 미생물 처리는 반추동물에 의한 폐배지 영양소의 체내 이용성을 향상시킬 수 있음을 시사해 주고 있다.

• 생명과학회지
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• 제27권1호
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• pp.57-66
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• 2017

약용식물의 발효는 새로운 식품의 소재 개발이 가능하나, 발효 균주는 대부분 이스트, 유산균, 박테리아 등이 이용되고 있다. 본 연구에서는 감초와 번데기 단독 또는 감초에 발효원인 번데기를 20%와 50%로 첨가한 혼합물의 배지에 눈꽃 동충하초(Paecilomyces tenuipes)와 밀리타리스 동충하초(Cordyceps militaris)를 이용하여 고체배양방법을 확립하였다. 동충하초 발효물을 식품소재로 개발하기 위하여 식용 가능한 용매인 에탄올 95%, 70%, 50%, 25% 및 물로서 추출한 다음 동충하초로부터 생성된 cordycepin과 감초의 지표성분인 liquiritin, liquiritigenin과 glycirrhizin의 함량 및 NO생성 억제효과를 조사하였다. Cordycepin함량은 감초에 번데기를 50%로 혼합한 배지에 밀리타리스 동충하초 균주을 접종하여 발효한 발효물을 70% EtOH추출하였을 경우에 가장 많았으며, 번데기를 첨가하지 않은 밀리타리스 동충하초 발효물 추출물보다 함량이 33배 정도 증가하였다. 또한 추출용매의 극성이 70% EtOH보다 높거나 낮아지면 감소하는 경향이었으며, 특히 발효원으로서 번데기의 첨가는 cordycepin의 함량을 현저하게 증가시켰다. Liquiritin의 함량은 발효하지 않은 감초보다 눈꽃 동충하초와 밀리타리스 동충하초로 발효한 모든 추출물에서 감소하였다. Liquiritigenin의 함량은 눈꽃 동충하초로 발효한 추출물이 밀리타리스 동충하초 발효 추출물보다 현저히 증가하였으나, 밀리타리스 동충하초 균쥬의 발효 추출물은 발효하지 않은 감초 추출물과 거의 차이가 없었으며, 두 균주 모두 번데기의 첨가량이 증가할수록 liquiritigenin의 함량이 감소하는 경향이었다. 감초에 번데기의 첨가량 또는 추출 용매의 극성이 증가하면 liquiritin과 glycyrrhizin의 함량은 현저히 감소하였다. 이상의 결과로부터, cordycepin 함량은 C. militaris 균주로 liquiritigenin은 P. tenuipes로 발효시에 현저하게 증가하였으나, liquiritin과 glycyrrhizin은 감소하였다. 감초를 동충하초로 발효시에 번데기의 첨가는 주요 성분의 변화를 현저하게 유도하였다. 동충하초 발효 추출물은 NO생성 억제효과가 증가하였으며, 고극성 용매 추출물에서 그 효과가 현저하였다. 감초의 발효시에 생성된 cordycepin과 liquiritin, liquiritigenin 및 glycyrrhizin의 함량은 발효원으로서 첨가되는 번데기, 추출용매의 극성, 발효 균주의 종류 등에 따라서 현저한 차이가 있었다. 이러한 결과는 동충하초 균주를 이용한 기능성 식품 소재를 개발하기 위한 기초 자료로서 활용이 가능할 것으로 기대된다.

• Journal of Animal Science and Technology
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• 제50권6호
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• pp.831-838
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• 2008

본 연구에서는 병재배 방식에서 발생되는 톱밥주원료 버섯부산물의 저장성 향상을 목적으로 복합생균제를 버섯부산물(1톤 규모)에 접종하여 현장 혐기발효하였을 때 저장기간에 따른 물리화학적, 발효 및 미생물 성상에 미치는 변화를 추적하고자 하였다. 복합생균제(Enter- obacter ludwigii KU201-3, Bacillus cereus KU206-3, Bacillus subtilis KU3, Bacillus subtilis KU201-7, Saccharomyces cerevisiae, Lactobacillus plantarum)를 버섯 부산물의 1% 수준(원물기준)에서 첨가하여 1일간 퇴적 저장 후 3일, 1주, 2주, 4주 그리고 8주간 혐기발효 시켰다. 버섯부산물은 혐기저장과정에서 CP, NDF, ADF 함량은 증가하였으며(P<0.05), DM과 NFC의 함량은 감소하였으나(P<0.05), 그 변화폭은 적은 편이었다. In situ 건물 및 NDF 반추위 소실율은 버섯부산물의 발효저장 기간이 길어짐에 따라 감소하였다. 발효성상에 있어서 발효 전과 비교해서 발효 후에는 pH가 감소하고, 유산 생성량은 2배 이상 증가하였다. 그러나, 4주경과 시와 비교해서 8주경과 시에는 pH가 다시 증가하였으나, 여전히 양호한 발효상태를 보여주었다. 유산균수는 발효 8주경과 시까지 유의적 차이가 없었고, 총세균수와 효모수는 4주째부터 감소하였다. 8주경과 시의 유산균과 효모수는 모두 108cfu/g 수준이었다. 이러한 결과는 혐기발효와 복합생균제 처리는 버섯부산물의 장기간(8주) 저장에 도움이 되었음을 시사해주고 있다.

• Bulletin of the Korean Chemical Society
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• 제24권4호
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• pp.431-436
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• 2003

Solvolytic rate constants at 25℃ are reported for solvolyses of cinnamyl bromide (1) in binary mixtures of water with acetone, ethanol, methanol, methanol-d, and 2,2,2-trifluoroethanol. Product selectivities are reported for solvolyses of 1 in aqueous ethanol and methanol. Rate ratios in solvents of the same $Y_{Br}$ value and different nucleophilicity provide measures of the minimum extent of nucleophilic solvent assistance (e.g. $[k_{40EW}/k_{97TFE}]$Y = 2.88, EW = ethanol-water). With use of the extended Grunwald-Winstein equation, the l and m values are similar to the values of 0.43 and 0.88 obtained for the solvolyses of 1 using the equation (see below) which includes a parameter (I) for solvation of aromatic rings. The magnitude of l and m values associated with a change of solvent composition predicts the $S_{N1}$ reaction mechanism rather than an $S_{N2}$ channel. Product selectivities (S), defined by S = [ether product]/[alcohol product]×[water]/[alcohol solvent] are related to four rate constants for reactions involving one molecule of solvent as nucleophile and another molecule of solvent as general base catalyst. A linear relationship between 1/S and molar ratio of solvent is derived theoretically and validated experimentally for solvolyses of the above substrates from water up 75% 1/S = $(k_{wa}/k_{aw})$([alcohol solvent]/[water]) + $k_{ww}/k_{aw}$ alcohol-water. The results are best explained by product formation from a “free” carbocation intermediate rather than from a solvent-separated ion pair.

• Toxicological Research
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• 제17권
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• pp.97-104
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• 2001

Three types of proteases (MEF-1, MEF-2 and MEF-3) were purified from the egg cases of Ten-odera sinensis using ammonium sulfate fractionation, gel filtration on Bio-Gel P-60 and affinity chromatography on DEAE Affi-Gel blue gel. The proteases were assessed homogeneous by SDS-polyacrylamide gel electrophoresis and have molecular weight of 31,500, 32,900 and 35,600 Da, respectively. The N-terminal regions of the primary structure were compared and they were found to be different each other. MEFs readily digested the $A\alpha$ - and B$\beta$-chains of fibrinogen and more slowly the ${\gamma}$-chain. The action of the enzymes resulted in extensive hydrolysis of fibrinogen and fibrin, releasing a variety of fibrinopeptides. MEF-1 was inactivated by Cu$^{2+}$ and Zn$^{2+}$ and inhibited by PMSF and chymostatin. MEF-2 was inhibited by PMSF, TLCK. soybean trypsin inhibitor. MEF-3 was only inhibited by PMSF and chymostatin. Antiplasmin was not sensitive to MEF-1 but antithrombin III inhibited the enzymatic activity qf MEF-1. MEF-2 specifically bound to anti plasmin Among the chromogenic protease substrates, the most sensitive one to the hydrolysis of MEFs was benzoyl-Phe-Val-Arg-p-nitroanilide with maximal activity at pH 7.0 and 3$0^{\circ}C$. MEF-1 preferentially cleaved the oxidized B-chain of insulin between Leu15 and Tyr16. In contrast, MEF-2 specifically cleaved the peptide bond between Arg23 and Gly24. D-dimer concentrations increased on incubation of cross-linked fibrin with MEF-1, indicating the enzyme has a strong fibrinolytic activity.ity.

• Journal of Microbiology and Biotechnology
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• 제30권2호
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• pp.271-278
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• 2020

Glycerol dehydrogenase (GlyDH) catalyzes the oxidation of glycerol to dihydroxyacetone (DHA), which is the first step in the glycerol metabolism pathway. GlyDH has attracted great interest for its potential industrial applications, since DHA is a precursor for the synthesis of many commercially valuable chemicals and various drugs. In this study, GlyDH from Klebsiella pneumoniae (KpGlyDH) was overexpressed in E. coli and purified to homogeneity for biochemical and molecular characterization. KpGlyDH exhibits an exclusive preference for NAD⁺ over NADP⁺. The enzymatic activity of KpGlyDH is maximal at pH 8.6 and pH 10.0. Of the three common polyol substrates, KpGlyDH showed the highest k_cat/K_m value for glycerol, which is three times higher than for racemic 2,3-butanediol and 32 times higher than for ethylene glycol. The k_cat value for glycerol oxidation is notably high at 87.1 ± 11.3 sec^-1. KpGlyDH was shown to exist in an equilibrium between two different oligomeric states, octamer and hexadecamer, by size-exclusion chromatography analysis. KpGlyDH is structurally thermostable, with a T_m of 83.4℃, in thermal denaturation experiment using circular dichroism spectroscopy. The biochemical and biophysical characteristics of KpGlyDH revealed in this study should provide the basis for future research on its glycerol metabolism and possible use in industrial applications.

• Asian Pacific Journal of Cancer Prevention
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• 제16권10호
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• pp.4383-4386
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• 2015

Background: The C1561T variant of the glutamate carboxypeptidase II (GCPII) gene is critical for natural methylfolylpolyglutamte (methylfolate) absorption, and has been associated with perturbations in folate metabolism and disease susceptibility. However, little is known on C1561T-GCPII as a risk factor for colorectal cancer. Therefore, this study examined whether C1561T-GCPII influences folate metabolism and adenomatous polyp occurrence. Materials and Methods: 164 controls and 38 adenomatous polyp cases were analysed to determine blood folate and plasma homocysteine (Hcy) level, dietary intake of natural methylfolate, synthetic pteroylglutamic acid (PteGlu), vitamin C and C1561T-GCPII genotype. Results: In controls and cases, 7.3 and 18.4 percent of subjects respectively, were found to have the CT genotype, increasing the risk for adenomatous polyp occurrence 2.86 times (95% CI:1.37-8.0, p=0.035). Total dietary folate, methylfolate and PteGlu intake and the level of erythrocyte folate and plasma Hcy did not predict the occurrence of an adenomatous polyp. However, dietary natural vitamin C intake was associated with adenomatous polyp risk within C1561T-GCPII CT genotype subjects (p=0.037). Conclusions: The findings suggest that C1561T-GCPII variation may be associated with risk for adenomatous polyp, and vitamin C may modify risk by interacting with the variant gene, its expression product and/or folate substrates.

• Journal of Microbiology and Biotechnology
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• 제20권10호
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• pp.1378-1385
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• 2010

A novel agarolytic bacterium, KY-YJ-3, producing extracellular agarase, was isolated from the freshwater sediment of the Sincheon River in Daegu, Korea. On the basis of Gram-staining data, morphology, and phylogenetic analysis of the 16S rDNA sequence, the isolate was identified as Cellvibrio sp. By ammonium sulfate precipitation followed by Toyopearl QAE-550C, Toyopearl HW-55F, and MonoQ column chromatographies, the extracellular agarase in the culture fluid could be purified 120.2-fold with a yield of 8.1%. The specific activity of the purified agarase was 84.2 U/mg. The molecular mass of the purified agarase was 70 kDa as determined by dodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal temperature and pH of the purified agarase were $35^{\circ}C$ and pH 7.0, respectively. The purified agarase failed to hydrolyze the other polysaccharide substrates, including carboxymethyl-cellulose, dextran, soluble starch, pectin, and polygalacturonic acid. Kinetic analysis of the agarose hydrolysis catalyzed by the purified agarase using thin-layer chromatography showed that the main products were neoagarobiose, neoagarotetraose, and neoagarohexaose. These results demonstrated that the newly isolated freshwater agarolytic bacterium KY-YJ-3 was a Cellvibrio sp., and could produce an extracellular ${\beta}$-agarase, which hydrolyzed agarose to yield neoagarobiose, neoagarotetraose, and neoagarohexaose as the main products.