• 한국식품과학회지
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• 제20권5호
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• pp.659-665
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• 1988

식품에서의 쓴 맛 펩타이드의 형성 기작 및 분해에 의한 제거 방법을 알아보기 위하여 카제인 가수 분해물에서 쓴 맛 펩타이드를 분리하여 그 특성을 조사하였다. Bacillus subtilis가 생산하는 염기성 단백부내 효소를 순수 분리된 ${\alpha}_{s1}$-카세인과 ${\beta}$-카제인에 처리하여 카제인 가수 분해물을 만들고, 이 가수 분해물에서 용매 추출, Sephadex G-25 겔 크로마토그래피, 고성능 액체 크로마토그래피를 이용해 쓴 맛 펩타이드를 순수 분리했다. 고성능 액체 크로마토그래피는 역상 칼럼인 octadecylsilica 칼럼을 사용했고, 0.1% TFA와 80% $CH_3CN$의 linear gradient 방법을 이용했다. ${\alpha}_{s1}$-카제인 가수분해물에서 3가지 쓴 맛 펩타이드를 분리하였는데, BP-I은 $CH_3CN$ 34%에서, BP-II는 35%, BP-III는 26%에서 각각 용출되었다. ${\beta}$-카제인 가수 분해물에서 2가지 쓴 맛 펩타이드를 분리했는데, BP-IV는 $CH_3CN$ 40%에서, BP-V는 42%에서 용출되었다. 분리된 다섯 가지 펩타이드 중 BP-V가 가장 소수도가 높았으며, 쓴 맛은 BP-I과 BP-II가 가장 강했다.

• Asian Pacific Journal of Cancer Prevention
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• 제15권20호
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• pp.8947-8950
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• 2014

Immunological functions of heat shock proteins (HSPs) have long been recognized. In this study we aimed to efficiently purify HSP70 from renal cell carcinoma and test it as a tumor antigen for pulsing dendritic cells in vitro. HSP70 was purified from renal cell carcinoma specimens by serial column chromatography on Con A-sepharose, PD-10, ADP-agarose and DEAE-cellulose, and finally subjected to fast protein liquid chromatography (FPLC). Dendritic cells derived from the adherent fraction of peripheral blood mononuclear cells were cultured in the presence of IL-4 and GM-CSF and exposed to tumor HSP70. After 24 hours, dendritic cells were phenotypically characterized by flow cytometry. T cells obtained from the non-adherent fraction of peripheral blood mononuclear cells were then co-cultured with HSP70-pulsed dendritic cells and after 3 days T cell cytotoxicity towards primary cultured renal cell carcinoma cells was examined by Cell Counting Kit-8 assay. Dendritic cells pulsed in vitro with tumor-derived HSP70 expressed higher levels of CD83, CD80, CD86 and HLA-DR maturation markers than those pulsed with tumor cell lysate and comparable to that of dendritic cells pulsed with tumor cell lysate plus TNF-${\alpha}$. Concomitantly, cytotoxic T-lymphocytes induced by HSP70-pulsed dendritic cells presented the highest cytotoxic activity. There were no significant differences when using homologous or autologous HSP70 as the tumor antigen. HSP70 can be efficiently purified by chromatography and induces in vitro dendritic cell maturation in the absence of TNF-${\alpha}$. Conspecific HSP70 may effectively be used as a tumor antigen to pulse dendritic cells in vitro.

• 한국수산과학회지
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• 제34권6호
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• pp.632-637
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• 2001

우렁쉥이 껍질에서 분리한 황산다당을 부분정제하여 몇 가지 화학조성과 전기영동을 하였다. 추출된 황산다당의 화학적 조성은 sulfate, uronic acid, protein, chondroitin sulfate, amino sugar, hexosamine 등으로 이루어져 있었다. 전기영동결과 autoclave 처리에 의해 추출된 조다당류는 폭넓은 단일 band를 나타내었으나, neutrase 처리구는 세 개의 band로 구분되었다. Autocalve 처리구의 분자량은 약 40,000 정도로 추정되며, neutrase 처리구의 경우 고분자 band는 100,000 이상, 두 개의 저분자 band 중 하나는 약 22,000 정도, 또 하나의 band는 약 5,000 정도인 것으로 추정되었다. 이온교환수지 및 겔 여과하여 얻은 전당획분의 화학적 조성은 부분정제 전과 거의 유사하였다. Sephadex G-25로 gel 여과한 시료의 조성당은 arabinose, xylose 및 glucose는 흔적량 정도이었으나 galactose는 $71.4\%$로 대부분을 차지하였고, N-acetylgalactosamine 함량이 $14.5\%$ 정도이었고, D-glucuronic acid가 $6.3\%$ 정도 검출되었다. Sephadex G-100과 G-100-25로 정제한 당의 전기영 동 패턴은 거의 유사하게 단일 band의 양상이나 G-25 처리구가 다소 좁은 band를 나타내었다. 부분정제한 황산다당과 표준품인 chondroitin sulfates의 FT-IR spectrometer 분석 결과, 거의 유사한 결과를 나타내었다. 황산기나 에스테르 등 몇몇 중요한 원자단의 신축 진동수가 IR spectrum의 지문영역 ($500\sim700cm^{-1}$$)에서 나타났다. 황산기 중의 S=O 신축진동이 $1,240cm^{-1}$에서, C-O-S신축진동의 경우 수평향배는 $820cm^{-1}$에서 나타났다.

• Journal of Microbiology and Biotechnology
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• 제6권3호
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• pp.213-218
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• 1996

Hot-water extract, Fr. 1, of Phellinus linteus mycelium was fractionated into Fr. 2, 3, 4, and 5 by the difference of solubility in ethanol. The polysaccharide fractions were studied for their immunostimulating activity on in vitro T-independent polyc1onal antibody response to trinitrophenyl-haptened SRBC (sheep red blood cell). The Fr. 4 with the highest immunostimulating activity was subjected to DEAE-cellulose ion exchange chromatography and gave five fractions, 4-I, II, III, IV, and V. The in vitro immunostimulating assay of the five fractions showed that 4-I and 4-III had a similar activity to that of LPS but the other fractions had low activity. By analyses of chemical composition and HPLC, all fractions obtained were found to be heteropolysaccharide-protein complex. The molecular weights ranged from 9, 000 to 15, 000. Sugar analyses showed that glucose, galactose, mannose, arabinose, and xylose were main component. Uronic acid and amino sugar were also detected in the fractions. It should be noted that the molecular weight (15, 000) of 4-III was very small and the structure of 4-III may be different from the known immunostimulating branched $\beta$-(1longrightarrow3)-glucan.

• Journal of Applied Biological Chemistry
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• 제44권3호
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• pp.113-118
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• 2001

Xylose (glucose) isomerase was purified to homogeneity from cell-extracts of Streptomyces chibaensis J-59 via ammonium sulfate precipitation followed by chromatography on DEAE-cellulose, and gel filtration on Sephacryl S-300. The purified enzyme is a homotetramer with a native molecular mass of 180 kDa and a subunit molecular mass of 44 kDa. The amino acid N-terminal sequence of glucose isomerase from S. chibaensis J-59 was determined to be Ser-Tyr-Gln-Pro-Thr-Pro-Glu-Asp-Arg-Phe-Thr-Phe-Gly-Leu. The first 14 amino acids of the N-terminal sequence of the enzyme showed strong analogies with N-terminal sequences of glucose isomerase produced by other Streptomyces spp. The optimum pH and temperature for activity were 7.5 and 85, respectively. The purified enzyme required $Mg^{2+}$, $Co^{2+}$, and $Mn^{2+}$ for the activity, $Mg^{2+}$ being the most effective. The enzyme was not inhibited by $Ca^{2+}$, but was inhibited by $Hg^{2+}$, $Ag^+$, and $Cu^{2+}$. The $K_m$, $V_{max}$, and $k_{cat}$ values of S. chibaensis J-59 isomerase for glucose were 83 mM, 40.9 U/mg, and $1,843min^{-1}$, respectively. In the presence of $Co^{2+}$, cell-free enzymes retained 100% without loss of activities by the heat-treatment at $70^{\circ}C$ for 7 days. The enzyme retained 50% residual activity after heating at $85^{\circ}C$ for 13.5 h, at $90^{\circ}C$ for 126 min. The enzyme is more thermostable than any other glucose isomerases of Streptomyces spp.

• Journal of Microbiology and Biotechnology
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• 제16권3호
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• pp.457-464
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• 2006

Three extracellular proteases (Vpr, peptidase T, and subtilisin) were identified from the culture supernatant of Bacillus subtilis KCTC 3014. All the proteins were partially purified as a mature form by using a DEAE-cellulose ion-exchange column chromatography. Their activities were determined by using zymography and densitometry. The relative molecular masses of Vpr and peptidase T (PepT) were determined to be 68 and 48 kDa by SDS-PAGE and zymography, respectively. However, subtilisin formed a 'binding mode' at the top of the separating gel. After denaturation by boiling at $100^{\circ}C$ for 5 min, its molecular mass was determined to be 29 kDa, whereas its activity was lost. The optimal pH of Vpr, PepT, and subtilisin were 9.0, 6.0-7.0, and 7.0-8.0, respectively. The optimal temperature of Vpr, PepT, and subtilisin was 40, 50, and $40^{\circ}C$, respectively. Inhibitor test revealed that Vpr and subtilisin were serine proteases and that PepT was a metalloprotease. Interestingly, we found that Vpr showed no enzyme activity on a 2DE zymogram gel. Three genes, vpr, pepT, and apr (encoding subtilisin protein), were cloned and their nucleotide and deduced amino acid sequences were determined.

• Applied Biological Chemistry
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• 제33권1호
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• pp.73-78
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• 1990

한국산 토마토로부터 endo-polygalacturonase를 gel filtration과 이온교환크로마토그라피에 의하여 약 24배 정제할 수 있었고, 최대 효소활성을 위한 PH는 5.0, 최적온도는 $50^{\circ}C$ 였으며 pH안정범위는 $4.0{\sim}5.0$, 열안정성은 $50^{\circ}C$에서 1시간 열처리 하였을 때 약 45 % 실활 되었다. 정제된 이 효소는 SDS-polyacrylamide gel 전기영동에 의하여 단일밴드로 확인되었으며, 그 분자량은 50,000정도였고, Km값은 $1.43{\times}10^{-1}\;mol/l이었다. 금속이온중 $Ag^+$, $Zn^{++}$이온이 효소의 활성을 촉진시켰으며, $Na^+$, $K^+$등의 이온에 의해서는 활성이 저해되었다.

• Journal of Microbiology and Biotechnology
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• 제19권9호
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• pp.951-959
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• 2009

Most, if not all, Basidiomycetes mushrooms have biologically active polysaccharides showing potent antitumor activity with immunomodulating properties. These polysaccharides have various chemical compositions and belong primarily to the $\beta$-glucan group. In this study, the crude water-soluble polysaccharide HEF-P, which was obtained from the fruiting body of Hericium erinaceus by hot water extraction and ethanol precipitation, was fractionated by DEAE-cellulose and Sepharose CL-6B column chromatographies. This process resulted in four polysaccharide fractions, named HEF-NP Fr I, HEF-NP Fr II, HEF-AP Fr I, and HEF-AP Fr II. Of these fractions, HEF-AP Fr II was able to upregulate the functional events mediated by activated macrophages, such as production of nitric oxide and expression ofcytokines (IL-1${\beta}$ and TNF-${\alpha}$). The molecular mass of HEF-AP Fr II was estimated by gel filtration to be 13 kDa. Its structural characteristics were investigated by a combination of chemical and instrumental analyses, including methylation, reductive cleavage, acetylation, Fourier transform infrared spectroscopy (FT-IR), and gas chromatography-mass spectrometry (GC-MS). Results indicate that HEF-AP Fr II is a low-molecular-mass polysaccharide with a laminarin-like triple helix conformation of a ${\beta}$-1,3-branched-${\beta}$-1,6-glucan.

• Journal of Microbiology and Biotechnology
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• 제20권7호
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• pp.1053-1060
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• 2010

Cordyceps militaris, an entomopathogenic fungus belonging to the class Ascomycetes, has been reported to have beneficial biological activities such as hypoglycemic, anti-inflammatory, antitumor, antimetastatic, hypolipidemic, immunomodulatory, and antioxidant effects. In this study, the crude water-soluble polysaccharide CMP, which was obtained from the fruiting body of C. militaris by hot water extraction and ethanol precipitation, was fractionated by DEAE-cellulose and Sepharose CL-6B column chromatographies. This process resulted in three polysaccharide fractions, termed CMP Fr I, CMP Fr II, and CMP Fr III. Of these fractions, CMP Fr II, with an average molecular mass of 127 kDa, was able to upregulate effectively the phenotypic functions of macrophages such as NO production and cytokine expression. The chemical property of the stimulatory polysaccharide, CMP Fr II, was determined based on its monosaccharide composition, which consisted of glucose (56.4%), galactose (26.4%), and mannose (17.2%). Its structural characteristics were investigated by a combination of chemical and instrumental analyses, including methylation, reductive cleavage, acetylation, Fourier transform infrared spectroscopy (FTIR), and gas chromatography-mass spectrometry (GCMS). Results indicated that CMP Fr II consisted of the (1${\rightarrow}$4) or (1${\rightarrow}$2) linked glucopyranosyl or galactopyranosyl residue with a (1${\rightarrow}$2) or (1${\rightarrow}$6) linked mannopyranosyl, glucopyranosyl, or galactopyranosyl residue as a side chain. The configuration of the ${\beta}$-linkage and random coil conformation of CMP Fr II were confirmed using a Fungi-Fluor kit and Congo red reagent, respectively.

• 한국미생물·생명공학회지
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• 제25권2호
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• pp.166-172
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• 1997

Aspergillus niger was selected as a strain producing the potent raw starch hydorlyzing enzyme. These experiments were conducted to investigate the conditions of the glucoa- mylase production, the purification of the enzyme, some characteristics of the purified enzyme and hydrolysis rate on various raw starches such as com, rice, potato, glutinous rice, sweet potato, wheat and barley. The optimum cultural temperature and time for the enzyme production on wheat bran medium were $30^{\circ}C$ and 96hrs, respectively. The respective addition of yeast extract and nutrient broth on wheat bran medium increased slightly the enzyme production. The enzyme was purified by ammonium sulfate fractionation and DEAE-cellulose column chromatography. The specific activity of the purified enzyme was 30.7u/mg-protein and the yield of enzyme activity was 25.8%. The purified enzyme showed a single band on polyacrylamide disc gel electrophoresis and its molecular weight was estimated to be 56,000 by SDS-polyacrylamide disc gel electrophoresis. The isoelectric point for the purified enzyme was pH3.7. The optimum temperature and pH were $65^{\circ}C$ and pH 4.0, respectively. The purified enzyme was stable in the pH range of pH 3.0-9.5 and below $45^{\circ}C$, and its thermal stability was slightly increased by the addition of $Ca^{2+}$. The purified enzyme was activated by $Co^{2+},\;Sr^{2+},\;Mn^{2+},\;Fe^{2+},\;Cu^{2+}$. Raw rice starch, raw corn starch, raw glutinous rice starch, raw sweet potato starch, raw wheat starch and raw barley starch showed more than 90% hydrolysis rate in 48hrs incubation. Even raw potato starch, most difficult to be hydrolyzed, showed 80% hydrolysis rate. The purified enzyme was identified as glucoamylase.