• 한국식품조리과학회지
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• 제17권5호
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• pp.497-502
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• 2001

능이버섯 〔Sarcodon aspratus(Berk.) S.Ito〕으로부터 단백질 가수분해 효소를 추출하여 75%(NH$_4$)$_2$SO$_4$ 염석과 DE52 anion exchange column chromatography 와 sepharyl-S 200 column 및 Mono s column chromatography 에 의해 정제하였는데 조 효소의 특이 활성은 55.2U/mg protein으로 조효소액에 비하여 11.26배 증가하였고 수율은 49.5%로 나타났다. 정제된 효소는 전기영동을 행한 결과 단일 band를 나타내었으며 분자량은 29,300으로 추정되었다. pH의 안정성은 4$^{\circ}C$에서 48시간 보존하였을 때 pH 8.5에서 가장 안정하였고, pH 5.5~10.5까지 높은 활성을 유지하였다. 온도에 대한 안정성은 30분간 보존 한 후 활성을 검토한 결과 단백분해능은 5$0^{\circ}C$까지 비교적 처음활성을 유지하다가 6$0^{\circ}C$에서는 53%정도의 활성이 유지되었으나 그 이상의 온도에서는 급격히 실화하여 7$0^{\circ}C$에서는 완전히 실화하였다. 금속 이온에 의해서는 크게 저해를 받지 않았으나 PMSF 저해제에 대하여 저해되어 본 효소가 serine protease임을 시사하였다.

• Korean Membrane Journal
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• 제10권1호
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• pp.13-19
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• 2008

Water softening is a very promising field for membranes and especially ultra low pressure membranes. Nanofiltration membranes based on pore-filling technology was prepared by using a new technique: the in-situ cross-linking. This route involves introducing a pre-formed polymer into the pores of a host membrane and then locking the polymer in the pores by in-situ cross-linking with an appropriate reagent. By this way, it is possible to make robust and competitive, pore-filled, anion-exchange membranes with excellent control over the properties of the incorporated gel without affecting the host membrane. In this paper, the possibilities of tuning such membranes for ultra low pressure water softening was examined by altering pore-filling chemistry (by changing cross-linking and aminating reagents). The results showed that tuning the chemistry of the pore-filling has important effects. In particularly, it had been shown that the correct selection of cross-linking reagent was not only essential to get pore-filled membranes but it could control their properties. Moreover, the aminating reagent could improve membrane performance. It was found that an increase in hydrophobicity could improve the Darcy permeability.

• 미생물학회지
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• 제31권1호
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• pp.54-62
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• 1993

Intracellular adenosine deaminase (ADA) from Aspergillus oryzae was purified using ammonium sulfate fractionation, a DEAE-Sephadex A-50 anion exchange chromatography, an ultrafiltration using a PM 10 membrane and two times of Sephadex G-100 gel filtration chromatography. The enzyme was purified 151 fold with a 9% recovery. Purified enzyme gave a single protein band with a molecular weight of 105,000 delton. The enzyme was reasonably stable. The enzyme activity was kept even after 1 hr incubation at 55.deg.C, but decreased significantly at 60.deg.C. The pH optimum was found to be from 6.5 to 7.5. Among tested compounds, the substrate activity was found with adenosine, adenine arainofuranoside, formymcin A, 2'-deoxyadenosine, 3'-deoxyadenosine, 2', 3'-isopropylidene adenosine, 2,6-diaminopurine deoxyriboside, .betha.-nicotinamide adenine dinucleotide (reduced form), 6-chloropurine riboside, 2'-adenine monophosphate (AMP), 3'-AMP and 5'-AMP. The values of Km of adenosine and 2'-deoxyadenosine were calculated to be 500 and .$710\mu$m, respectively. ADA was sensitivite to $Zn^{2+}$, $^Cu{2+}$ and $Fe^{3+}$, p-chloromercuribenzoate and mersalyl acid inactivated the enzyme. The activity of enzyme was not changed when ADA was incubated with dithiothreititol, 2-mercaptoethanol, N-ethylmaleimide, iodoacetic acid and iodoacetamide.

• Journal of Applied Biological Chemistry
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• 제43권4호
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• pp.235-239
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• 2000

A polysaccharide with antithrombin activity in Manda$^{(R)}& (PAM) was purified via procedures comprising three major steps, i.e. fractional precipitation with ethanol, anion exchange chromatography, and gel permeation chromatography. PAM showed a symmetrical peak on size exclusion HPLC, as assessed by refractive index, and behaved as a single band on cellulose acetate electrophoresis. The average molecular mass was estimated to be 222 kDa by gel filtration. PAM was found to be a sulfated heteropolysaccharide that contains sulfate group (20.5%, w/w) and uronic acid moiety (7.1 %, w/w) in addition to neutral sugar consisting of fucose, xylose, mannose, galactose, and glucose in a molar ratio of 1.00 : 0.35 : 0.28: 0.22 : 0.15. This polysaccharide appeared to inhibit blood coagulation via the intrinsic pathway in a dose-dependent pattern. The clotting of fibrinogen by thrombin was also significantly mitigated by the presence of PAM.

• Journal of Microbiology and Biotechnology
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• 제18권11호
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• pp.1789-1791
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• 2008

This study was aimed (i) to develop an effective method for the purification of ginsenosides for industrial use and (ii) to compare the distribution of ginsenosides in cultured wild ginseng roots (adventitious root culture of Panax ginseng) with those of red ginseng (steamed ginseng) and white ginseng (air-dried ginseng). The crude extracts of cultured wild ginseng roots, red ginseng, and white ginseng were obtained by using a 75% ethanol extraction combined with ultrasonication. This was followed sequentially by AB-8 macroporous adsorption chromatography, Amberlite IRA 900 Cl anion-exchange chromatography, and Amberlite XAD16 adsorption chromatography for further purification. The contents of total ginsenosides were increased from 4.1%, 12.1%, and 11.3% in the crude extracts of cultured wild ginseng roots, red ginseng, and white ginseng to 79.4%, 71.7%, and 72.5% in the final products, respectively. HPLC analysis demonstrated that ginsenosides in cultured wild ginseng roots were distributed in a different ratio compared with red ginseng and white ginseng.

• Journal of Microbiology and Biotechnology
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• 제23권4호
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• pp.489-498
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• 2013

A new Bacillus strain degrading starch, named Bacillus sp. UEB-S, was isolated from a southern Tunisian area. Amylase production using solid-state fermentation on millet, an inexpensive and available agro-resource, was investigated. Response surface methodology was applied to establish the relationship between enzyme production and four variables: inoculum size, moisture-to-millet ratio, temperature, and fermentation duration. The maximum enzyme activity recovered was 680 U/g of dry substrate when using $1.38{\times}10^9$ CFU/g as inoculation level, 5.6:1 (ml/g) as moisture ratio (86%), for 4 days of cultivation at $37^{\circ}C$, which was in perfect agreement with the predicted model value. Amylase was purified by Q-Sepharose anion-exchange and Sephacryl S-200 gel filtration chromatography with a 14-fold increase in specific activity. Its molecular mass was estimated at 130 kDa. The enzyme showed maximal activity at pH 5 and $70^{\circ}C$, and efficiently hydrolyzed starch to yield glucose and maltose as end products. The enzyme proved its efficiency for digesting raw cereal below gelatinization temperature and, hence, its potentiality to be used in industrial processes.

• 농업과학연구
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• 제39권4호
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• pp.561-568
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• 2012

The aim of this study was to introduce a simple method for isolation of ${\alpha}$-lactalbumin, ${\beta}$-lactoglobulin and bovine serum albumin from cow's milk, and peptides produced by enzymatic hydrolysis of ${\alpha}$-lactalbumin, ${\beta}$-lactoglobulin and bovine serum albumin with alcalase. Whey protein were precipitated from whey by ammonium sulfate and, ${\alpha}$-lactalbumin and ${\beta}$-lactoglobulin were isolated using Hi Prep 26/60 Sephacryl S-100 column gel filtration chromatography. Bovine serum albumin and ${\beta}$-lactoglobulin were isolated by Mono-Q 5/50 GL column anion exchange chromatography of the 50% Ammonium Sulfate-supernatant. Isolated whey proteins were hydrolyzed by proteolytic alcalase. Tricine SDS-PAGE and reverse-phase HPLC analyses revealed that almost hydrolyzed all the ${\alpha}$-lactalbumin, ${\beta}$-lactoglobulin and bovine serum albumin with alcalase. Molecular weight of various peptides derived from alcalase hydrolysate were small molecular weight than 3.5 kDa.

• BMB Reports
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• 제35권2호
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• pp.220-227
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• 2002

Two aspartate aminoransferase (EC 2.6.1.1) isoenzymes (AAT-1 and AAT-2) from Lupinus albus L. cv Estoril were separated, purified, and characterized. The molecular weight, pI value, optimum pH, optimum temperature, and thermodynamic parameters for thermal inactivation of both isoenzymes were obtained. Studies of the kinetic mechanism, and the kinetics of product inhibition and high substrate concentration inhibition, were performed. The effect of some divalent ions and irreversible inhibitors on both AAT isoenzymes was also studied. Native PAGE showed a higher molecular weight for AAT-2 compared with AAT-1. AAT-1 appears to be more anionic than AAT-2, which was suggested by the anion exchange chromatography. SDS-PAGE showed a similar sub-unit molecular weight for both isoenzymes. The optimum pH (between 8,0 and 9.0) and temperature ($60-65^{\circ}C$) were similar for both isoenzymes. In the temperature range of $45-65^{\circ}C$, AAT-2 has higher thermostability than AAT-1. Both isoenzymes showed a high affinity for keto-acid substrates, as well as a higher affinity to aspartate than glutamate. Manganese ions induced an increase in both AAT isoenzymes activities, but no cooperative effect was detected. Among the inhibitors tested, hydroxylamine affected both isoenzymes activity by an irreversible inhibition mechanism.

• BMB Reports
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• 제40권1호
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• pp.107-112
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• 2007

To study the functioning of HSP70 in Escherichia coli, we selected NtHSP70-2 (AY372070) from among three genomic clones isolated in Nicotiana tabacum. Recombinant NtHSP70-2, containing a hexahistidine tag at the amino-terminus, was constructed, expressed in E. coli, and purified by $Ni^{2+}$ affinity chromatography and Q Sepharose Fast Flow anion exchange chromatography. The expressed fusion protein, $H_6NtHSP70$-2 (hexahistidine-tagged Nicotiana tabacum heat shock protein 70-2), maintained the stability of E. coli proteins up to 90$^{\circ}C$. Measuring the light scattering of luciferase (luc) revealed that NtHSP70-2 prevents the aggregation of luc without ATP during high-temperature stress. In a functional bioassay (1 h at 50$^{\circ}C$) for recombinant $H_6NtHSP70$-2, E. coli cells overexpressing $H_6NtHSP70$-2 survived about seven times longer than those lacking $H_6NtHSP70$-2. After 2 h at 50$^{\circ}C$, only the E. coli overexpressing $H_6NtHSP70$-2 survived under such conditions. Our NtHSP70-2 bioassays, as well as in vitro studies, strongly suggest that HSP70 confers thermo-tolerance to E. coli.

• Journal of Ginseng Research
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• 제19권3호
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• pp.237-243
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• 1995

Agmatine iminohydrolase (EC 3.5.3.12) catalyzes the hydrolysis of agmatine into putrescine. The enzyme seems to be one of the critical enzymes in putrescine biosynthesis. The enzyme was purified to homogeneity from Panax ginseng C.A. Meyer by combined method of ammonium sulfate 1 fractionation, DEAR anion exchange column, hydroxyapatite column and agmatine carboxyhexyl Sepharose 4B affinity column. The molecular weight estimated by native pore gadient polyacrylamide gel electrophoresis was 71, 000 Dalton, while that estimated by SDS-PAGE was 70, 000 Dalton, indicating a monomeric enzyme. The optimal pH and temperature were 9.0 and 37$^{\circ}C$, respectively. The Km and 1 Vmax for agmatine were 8.3 mM and 14.4 nmole/hr, respectively. Heat stability of this enzyme was high. The enzyme was observed to be inhibited by polyamines such as putrescine, cadaverine, spermidine and spermine. Especially, putrescine was a potent inhibitor of the purified enzyme. These results suggest that polyamines could be important in growth regulation of Panax ginseng C.A. Meyer.