• Journal of Microbiology and Biotechnology
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• 제23권3호
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• pp.397-404
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• 2013

Paenibacillus xylanilyticus KJ-03 was isolated from soil samples obtained from a field with Amorphophallus konjac plants. A gene encoding xylanase was isolated from KJ-03 and cloned using a fosmid library. The xynA gene encodes xylanase; it consists of 1,035 bp and encodes 345 amino acids. The amino acid sequence deduced from the P. xylanilyticus KJ-03 xylanase showed 81% and 69% identities with those deduced from the P. polymyxa E681 and Paenibacillus sp. HPL-001 xylanases, respectively. The xynA gene comprises a single domain, consisting of a catalytic domain of the glycosyl hydrolase (GH) 10 family. The xynA gene was expressed in Escherichia coli BL21 (trxB), and the recombinant xylanase was purified by Niaffinity chromatography. The purified xylanase showed optimum activity with birchwood xylan as a substrate at $40^{\circ}C$ and pH 7.4. Treatment with $Mg^{2+}$ and $Li^+$ showed a slight decrease in XynA activity; however, treatment with 5 mM $Cu^{2+}$ completely inhibited its activity. The results of the thin layer chromatography analysis indicated that the major hydrolysis product was xylobiose and small amounts of xylose and xylotriose. XynA showed increased activity with oat spelt xylan and birchwood xylan, but showed only slight activity with locust bean gum.

• Journal of Microbiology and Biotechnology
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• 제27권2호
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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.

• International Journal of Oral Biology
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• 제36권2호
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• pp.59-64
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• 2011

Anthocyanins are naturally occuring phytochemicals and the main components of the coloring of plants, flowers and fruits. They are known to elicit antioxidative, anti-inflammatory and cancer preventive activity. In this study, we investigated anthocyanins in black / yellow soybean seedcoats using different methods of detection - thin layer chromatography (TLC), capillary zone electrophoresis (CZE) and HPLC analysis. The anthocyanins in soybean seedcoats were extracted by five independent methods of extraction and the aglycons (anthocyanidins) of the corresponding anthocyanins were prepared by acid mediated hydrolysis. The anthocyanin / anthocyanidin in black soybean seedcoat showed characteristic TLC mobility, CZE electrophoretic retention and HPLC migration time while little of anthocyanins were detected from yellow soybean seedcoat. The extracted anthocyanins showed pH dependent retention time in CZE and spectral change in UV-Vis spectrum. HPLC analysis of the hydrolyzed extract of black soybean seedcoat identified the presence of four anthocyanidins. The major anthocyanin in black soybean seedcoat was cyanin (cyanidin-3-O-glucoside), with the relative order of anthocyanidin in cyanidin > delphinidin > petunidin > pelargonidin.

• BMB Reports
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• 제29권6호
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• pp.500-506
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• 1996

We have isolated a water-extracted novel regulator for blood coagulation from an earthworm, Lumbricus rubellus. As a folk remedy, the earthworm has been known to facilitate blood circulation. After complete heat inactivation of endogenous proteases in the earthworm, an anticoagulant(s) was purified through ammonium sulfate fractionation and three consecutive gel permeation chromatography of Sephacryl S-300, Sephadex G-75, and G-150 by measuring activated partial thromboplastin time (APTT) The anticoagulant was further purified to 2,800 fold with a C4 reversed-phase HPLC This activity was stable under heat ($100^{\circ}C$ for 30 min) and acidic conditions (0.4 N HCl). The effects of this partially purified anticoagulant on thrombin were observed with various substrates such as N${\alpha}$-benzoyl-DL-arginine-p-nitroanilide (BApNA), H-D-phenylalanyl-L-pipecoyl-L-arginine-p-nitroanilide (S-2238), N${\alpha}$-p-tosyl-L-arginine methyl ester (TAME), and fibrinogen as a natural substrate. Only TAME hydrolysis, due to an esterase activity of the enzyme, was inhibited among the chromogenic substrates. In addition, the anticoagulant not only inhibited the conversion of fibrinogen to fibrin but also prolonged the fibrin clot formation monitored with the in vitro coagulation test. Based on these observations, we suggest the significance of measuring the ability of antithrombotic drugs to inhibit the esterase activity of thrombin. In this report, it was also shown that the earthworm indeed contained a water-extractable, heat- and acid-stable anticoagulant which could be used as a novel antithrombotic agent.

• Journal of Ginseng Research
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• 제39권4호
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• pp.392-397
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• 2015

Background: Red ginseng (RG) is processed from Panax ginseng via several methods including heat treatment, mild acid hydrolysis, and microbial conversion to transform the major ginsenosides into minor ginsenosides, which have greater pharmaceutical activities. During the fermentation process using microbial strains in a machine for making red ginseng, a change of composition occurs after heating. Therefore, we confirmed that fermentation had occurred using only microbial strains and evaluated the changes in the ginsenosides and their chemical composition. Methods: To confirm the fermentation by microbial strains, the fermented red ginseng was made with microbial strains (w-FRG) or without microbial strains (n-FRG), and the fermentation process was performed to tertiary fermentation. The changes in the ginsenoside composition of the self-manufactured FRG using the machine were evaluated using HPLC, and the 20 ginsenosides were analyzed. Additionally, we investigated changes of the reducing sugar and polyphenol contents during fermentation process. Results: In the fermentation process, ginsenosides Re, Rg1, and Rb1 decreased but ginsenosides Rh1, F2, Rg3, and Compound Y (C.Y) increased in primary FRG more than in the raw ginseng and RG. The content of phenolic compounds was high in FRG and the highest in the tertiary w-FRG. Moreover, the reducing sugar content was approximately three times higher in the tertiary w-FRG than in the other n-FRG. Conclusion: As the results indicate, we confirmed the changes in the ginsenoside content and the role of microbial strains in the fermentation process.

• 한국결정성장학회지
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• 제22권6호
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• pp.291-298
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• 2012

자기 조립 분자 집합체 물질인 CMPO로 표면개질 된 메조 다공성 실리케이트를 가수분해와 축합반응을 이용하여 합성하였다. 손님 물질인 CMPO는 2-(diphenylphosphoryl) acetic acid와 3-(triethoxysilyl) propan-1-amine의 아마이드 결합반응을 이용하여 합성하였으며, MCM-41, SBA-15 그리고 실리카 나노입자와 같은 다양한 메조 다공성 실리케이트는 주인물질로 채택하였다. 메조 다공성 실리케이트의 비표면적은 680 $m^2/g$~1310 $m^2/g$의 넓이로 측정되었으며 BJH 방법을 이용해서 동공의 크기를 확인한 결과 2.3~9.1 nm 범위의 다양한 크기를 가지고 있었다. 메조 다공성 실리카 중에서는 SBA-15(II)가 가장 높은 약 35 wt%의 CMPO 함유량을 나타내었다. 메조 다공성 실리케이트의 표면에 개질된 CMPO 실란 작용기와 란탄족 이온과의 접근성에 관한 연구 결과, CMPO로 개질 된 모든 흡착제의 경우 상대적으로 이온 반경이 큰 La(III)보다는 크기가 작은 Nd(III)와 Eu(III) 이온을 더 선호하였다.

• Bulletin of the Korean Chemical Society
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• 제27권1호
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• pp.65-70
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• 2006

An amperometric glucose biosensor has been developed based on the use of the nanoporous composite film of sol-gel-derived zirconia and perfluorosulfonated ionomer, Nafion, for the encapsulation of glucose oxidase (GOx) on a platinized glassy carbon electrode. Zirconium isopropoxide (ZrOPr) was used as a sol-gel precursor for the preparation of zirconia/Nafion composite film and the performance of the resulting glucose biosensor was tuned by controlling the water content in the acid-catalyzed hydrolysis of sol-gel stock solution. The presence of Nafion polymer in the sol-gel-derived zirconia in the biosensor resulted in faster response time and higher sensitivity compared to those obtained at the pure zirconia- and pure Nafion-based biosensors. Because of the nanoporous nature of the composite film, the glucose biosensor based on the zirconia/Nafion composite film can reach 95% of steady-state current less than 5 s. In addition, the biosensor responds to glucose linearly in the range of 0.03-15.08 mM with a sensitivity of 3.40 $\mu$A/mM and the detection limit of 0.037 mM (S/N = 3). Moreover, the biosensor exhibited good sensor-to-sensor reproducibility (~5%) and long-term stability (90% of its original activity retained after 4 weeks) when stored in 50 mM phosphate buffer at pH 7 at 4 ${^{\circ}C}$.

• Archives of Pharmacal Research
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• 제28권7호
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• pp.816-822
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• 2005

Catharsius protease-2 (CPM-2) was isolated from the body of dung beetles, Catharsius molossus, using a three step purification process (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and affinity chromatography on DEAE Affi-Gel blue). The purified CPM-2, having a molecular weight of 24 kDa, was assessed homogeneously by SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of CPM-2 was composed of X Val Gin Asp Phe Val Glu Glu lie Leu. CPM-2 was inactivated by $Cu^{2+}\;and\;Zn^{2+}$ and strongly inhibited by typical serine proteinase inhibitors such as TLCK, soybean trypsin inhibitor, aprotinin, benzamidine, and ${\alpha}_1$-antitrypsin. However, EDTA, EGTA, cysteine, $\beta$-mercaptoethanol, E64, and elastatinal had little effect on enzyme activity. In addition, antiplasmin and antithrombin III were not sensitive to CPM-2. Based on the results of a fibrinolytic activity test, CPM-2 readily cleaved $A{\alpha}-$ and $B{\beta}$-chains of fibrinogen and fibrin, and y-chain of fibrinogen more slowly. The nonspecific action of the enzyme resulted in extensive hydrolysis, releasing a variety of fibrinopeptides of fibrinogen and fibrin. Polyclonal antibodies of CPM-2 were reactive to the native form of antigen. The ELISA was applied to detect quantities, in nanograms, of the antigen in CPM-2 protein.

• 한국식품과학회지
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• 제21권5호
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• pp.691-696
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• 1989

감자를 물에 침지시켜 썩히고 전분을 분리 이용하는 것은 우리나라 특유의 식문화이다. 본 실험에서는 감자를 $30^{\circ}C$의 물에 7일간 침지시키면서 전분의 성질 변화를 조사하였다. 침지 중 큰 입자$(61-70\;{\mu}m)$는 침지 1일에 크게 감소하였고 침지 2일 이후에는 거의 존재하지 않았으며, 전분입자는 침지 2일부터 구멍을 보이기 시작하였다. 침지 중 수침액의 pH는 감소하였고, 총 당과 환원당은 증가하였다. 전분입자의 밀도, 아밀로오스 함량, 인 함량 및 지방질 함량은 감소하였다. 전분의 상대결정도는 증가하여 침지 4일에서 가장 높았고 다시 감소하였다. 호화엔탈피의 변화도 비슷한 결과이었다. 침지전분은 산 또는 효소에 의한 가수분해 정도가 낮았고, 팽윤력, 아밀로그라프의 점도 등도 낮았다.

• Food Science and Biotechnology
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• 제18권3호
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• pp.776-781
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• 2009

A putative cyclomaltodextrinase (BHCD) gene was found from the genome of Bacillus halodurans C-125, which encodes 578 amino acids with a predicted molecular mass of 67,279 Da. It shares 42-59% of amino acid sequence identity with common cyclomaltodextrinase (CDase)-family enzymes. The corresponding gene was cloned by polymerase chain reaction (PCR) and the dimeric enzyme with C-terminal 6-histidines was successfully overproduced and purified from recombinant Escherichia coli. BHCD showed the highest activity against ${\beta}-CD$ at pH 7.0 and $50^{\circ}C$. Due to its versatile hydrolysis and transglycosylation activities, BHCD has been confirmed as a member of CDases. However, BHCD can be distinguished from other typical CDases on the basis of its novel multisubstrate specificity. While typical CDases have over 10 times higher activity on ${\beta}-CD$ than starch or pullulan, the CD-hydrolyzing activity of BHCD is only 2.3 times higher than pullulan. In particular, it showed significantly higher activity ratio of maltotriose to acarbose than other common CDase-family enzymes.