• BMB Reports
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• 제34권6호
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• pp.509-516
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• 2001

A genomic DNA fragment that contains the gene, which codes for a novel extracellular serine protease in Burkholderia pseudomallei, was cloned by using pQE40 as a vector. It was maintained in Escherichia coli JM109. The expression of the gene(s) resulted in the production of a 52 kDa protease. The recombinant protease was purified from the culture filtrate via ammonium sulfate fractionation, gel filtration, and anion-exchange chromatography. The purified protease had an optimum pH and temperature of pH 8.9 and $38^{\circ}C$, respectively. The protease activity was inhibited by EGTA, EDTA, and PMSF, but not 1,10-phenanthroline. The first 11 amino acid residues from the N-terminus of the purified protease were identified as LAPNDPYYYGY. PNDPYY was found to show homology to the Bacillus cereus microbial serine protease and B. subtilis PD498 serine protease. These results indicate that the protease that was purified in this study is an extracellular calcium-dependent serine protease. The purified protease was able to digest the human serum 19A, IgG, albumin, and transferrin, as well as bovine muscle actin and myosin. Furthermore, it was able to promote or cause dermonecrosis in experimental rabbits. These results propose the possible role of a novel B. pseudomallei extracellular calcium-dependent serine protease in the virulence of the pathogen.

• 한국수산과학회지
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• 제47권2호
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• pp.135-143
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• 2014

Exopeptidase active fractions from the hepatopancreas of the Argentina shortfin squid Illex argentinus, were obtained with acetone (AC 30-40%), ammonium sulfate (AS 60-70% saturation), anion exchange chromatography (AE-II, 0.2 M NaCl) and gel filtration chromatography (GF-I, 30-50 kDa) fractionation methods. A bitter peptide solution that has a bitterness equivalent to that of 2% glycylphenylalanine and prepared by tryptic hydrolysis of milk casein, was treated with the exopeptidase active fractions. The GF-I fraction was the best based on aminopeptidase activity (35.3 U/mg), percentage of recovery (30.7%) and a sensory evaluation (1.7). The amount of released amino acids increased as incubation time increased, and the bitterness of the enzyme reaction mixtures decreased. Incubation with the GF-I fraction for 24 h resulted in the hydrolysis of several peptides as revealed by the reverse-phase high performance liguid chromatography profile, with three peaks (3, 5 and 6) decreasing in area (%) and three peaks (1, 2 and 4) increasing in area (%). Therefore, the GF-I fraction appeared to be ideally suited to reduce bitterness in protein hydrolysates by catalyzing the hydrolysis of bitter peptides.

• Journal of Microbiology and Biotechnology
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• 제11권5호
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• pp.845-852
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• 2001

A microorganism producing fibrinolytic enzyme was isolated from Korean traditional soybean paste and identified as Bacillus sp. KDO-13. The fibrinolytic enzyme was purified to homogeneity by ammonium sulfate fractionation, ion-exchange chromatography on DEAE-celluose, and gel chromatography on Sephadex G-100 of the culture supernatant of Bacillus sp. KDO-13. The molecular weight of the purified enzyme was estimated to be 44,000 by SDS-PAGE. The optimum pH and temperature for the enzyme activity were pH 8.0 and $50{\circ}C$, respectively. The enzyme activity was relatively stable at pH 7.0-9.0 and temperature below $50{\circ}C$. the activity of the enzyme was inhibited by $AI^{3+}$ and $Hg^{2+}$, but activated by $Co^{2+}$\;and\;Ni^{2+}. In addition, the enzyme activity was potently inhibited by EDTA and 0-phenanthroline. The purified enzyme could completely hydrolyze a fibrin substrate within 6 h in vitro, and had a low $K_m$ value for fibrin hydrolysis. It was concluded that the purified enzyme was a metalloprotease with relatively high specificity for fibrinolysis, and thus, could be applied as an effective thrombolytic agent.

• 한국미생물·생명공학회지
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• 제24권1호
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• pp.50-58
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• 1996

Interspecific hybrids between Aspergillus oryzae var oryzae and A. nidulans 514 were obtained by nuclear transfer technique. Several autotrophic mutants isolated from conidiospores of the two strains were mutagenized with ultraviolet and N-methyl-N-nitrosoguanidine. Optimal conditions for formation of intergeneric hybrids were investigated. Frequencies of hybrid formation by nuclear transfer were $3{\times}10^{-5}{\sim}1{\times}10^{-5}$. From observation of genetic stability, conidial size, DNA content, and nuclear stain, it was suggested that their karyptypes are aneuploid. The hybrids showed 1.1~1.4 fold higher xylanase activities than parental strains did. The xylanase of Aspergiilus sp. TAVD514-3 was purified and some of it's enzymatc characteristics were investigated. The enzyme was purified about 85 fold with an overall yield of 17% from the culture medium by ammonium sulfate fractionation, Sephadex G-75 gel permeation chromatography, and CM-sephadex A-50 ion exchange chromatography. The purified enzyme functions optimally at pH 9.0 and 80$^{\circ}C$. The enzymatic activity was increased by the presence of $Mg^{2+}$ and $Mn^2$ ions.

• 한국광물학회지
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• 제19권2호
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• pp.123-128
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• 2006

딕카이트를 출발물질로 이용하여 이팔면체형 스멕타이트인 바이델라이트를 수열합성한 후, 바이델라이트의 층 내의 금속 양이온을 유기 양이온인 10-Carboxy-n-decyldimethylethyl-ammonium bromide와 교환 반응시켜 유기 [DEACOOH]-바이델라이트를 제조하였다. [DEACOOH]-바이델라이트는 고진공 상태 하에서 건조시킨 후 증류수, 메탄올, 에탄올, 아세톤, 에테르, 아세토니티릴 및 카프로락탐 등 상이한 팽윤용액과 반응시켜 팽윤거동에 대하여 연구하였다. 실험한 결과, 고진공하에서의 층간거리는 15.1 ${\AA}$이었고, 증류수, 메탄올, 에탄올, 아세톤, 에테르, 아세토니티릴, 카프로락탐에서 반응 후에는 각각 19.4, 29.9, 15.9, 16.8, 14.8, 26.5, 14.8 ${\AA}$층간거리가 얻어졌다. 또한 합성 바이델라이트와 자연산 몬모릴로나이트의 층간화합물 및 팽윤거동에 대하여 비교 분석하였다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권5호
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• pp.442-448
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• 2006

Human lactoferrin (hLf) is an iron-binding glycoprotein that has been considered to play many biological roles in the human, including the stimulation of the immune system, antimicrobial and anti-inflammatory effects, and regulation of iron absorption. We generated transgenic Siberian ginseng (Acanthopanax senticosus) cell cultures producing a functional hLf protein using the signal peptide sequence from the endoplasmic reticulum and driven by an oxidative stress-inducible SWPA2 promoter which is highly expressed in plant cell cultures. The production of hLf increased proportionally to cell growth and showed a maximal level (up to 3.6% of total soluble protein) at the stationary phase in suspension cultures. Full-length hLf protein was identified by immunoblot analysis in transgenic cell cultures of Siberian ginseng. Recombinant hLf (rhLf) was purified from suspension cells of Siberian ginseng by ammonium sulfate precipitation, cation-exchange and gel filtration chromatography. N-terminal sequences of rhLf were identical to native hLf (nhLf). The overall monosaccharide composition of rhLf showed the presence of plant specific xylose while sialic acid is absent. Antibacterial activity of purified rhLf was higher than that of nhLf. Taken together, we anticipate that medicinal Siberian ginseng cultured cells, as demonstrated by this study, will be a biotechnologically useful source for commercial production of functional hLf not requiring further purification.

• 한국식품영양과학회지
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• 제19권5호
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• pp.434-442
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• 1990

Aspergillus sp. CC-29 ws selected for its strong protease activity among various stains of molds found in soil. It was found that the production of alkaline protease reached to maximum when the wheat bran medium containing glucose as carbon source had been cultured for 4 days. Alkaline proteased was purified 36.10 fold from Aspergillus sp. CC-29 The purification procedures included ammonium sulfate fractunation gel filteration on Sepha-dex G-75 G-150 and DEAE-cellulose ion-exchange chromatography, The yield of the purified enzyme was 22.40% The purified enzyme was confirmed as a single band by the polyacryla-mide. When the purified enzyme was applied to SDS-PAGE the molecular weight was estima-ted 24000. The optimum pH for the enzyme activity was 9.0 and the optimum temperature was 4$0^{\circ}C$ The reaction of this enzyme followed typical Michaelis-Menten kinetics with the Km value of 2.10$\times$10-4M with the Vmax of 29.41 $\mu$g/min. The enzyme was reactively stable in alkalic condition and unstable by heat treatment. The activity of alkaline protease was increased by the addition of Ca2+ whereas it was inhibited by Hg2+ Zn2+ at concentration of 1$\times$10-3M.

• Journal of Microbiology and Biotechnology
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• 제17권4호
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• pp.624-631
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• 2007

Thermostable protease is very effective to improve the industrial processes in many fields. Two thermostable extracellular proteases from the culture supernatant of the thermophilic fungus Chaetomium thermophilum were purified to homogeneity by tractional ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sepharose, and Phenyl-Sepharose hydrophobic interaction chromatography. By SDS-PAGE, the molecular mass of the two purified enzymes was estimated to be 33 kDa and 63 kDa, respectively. The two proteases were found to be inhibited by PMSF, but not by iodoacetamide and EDTA. The 33 kDa protease (PRO33) exhibited maximal activity at pH 10.0 and the 63kDa protease (PRO63) at pH5.0. The optimum temperature for the two proteases was $65^{\circ}C$. The PRO33 had a $K_m$ value of 6.6mM and a $V_{max}$ value of $10.31{\mu}mol/l/min$, and PRO63 l7.6mM and $9.08{\mu}mol/l/min$, with casein as substrate. They were thermostable at $60^{\circ}C$. The protease activity of PRO33 and PRO63 remained at 67.2% and 17.31%, respectively, after incubation at $70^{\circ}C$ for 1h. The thermal stability of the two enzymes was significantly enhanced by $Ca^{2+}$. The residual activity of PRO33 and PRO63 at $70^{\circ}C$ after 60min was approximately 88.59% and 39.2%, respectively, when kept in the buffer containing $Ca^{2+}$. These properties make them applicable for many biotechnological purposes.

• Journal of Microbiology and Biotechnology
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• 제23권6호
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• pp.811-817
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• 2013

The present study dealt with the isolation, identification and enzyme characterization of potential luminous bacteria from water, sediment, squid, and cuttle fish samples of the Karaikal coast, Bay of Bengal, India during the study period September 2007 - August 2008. Bioluminescent strains were screened in SWC agar and identified using biochemical tests. As Shewanella henadai was found to be the most common and abundant species with maximum light emission [69,702,240 photons per second (pps)], the optimum ranges of various physicochemical parameters that enhance the luciferase activity in Shewanella hanedai were worked out. The maximum luciferase activity was observed at the temperature of $25^{\circ}C$ (69,674,387 pps), pH of 8.0 (70,523,671 pps), salinity of 20 ppt (71,674,387 pps), incubation period of 16 h (69,895,714 pps), 4% peptone (70,895,152 pps) as nitrogen source, 0.9% glycerol (71,625,196 pps), and the ionic supplements of 0.3% $CaCO_3$ (73,991,591 pps), 0.3% $K_2HPO_4$ (73,919,915 pps), and 0.2% $MgSO_4$ (72,161,155 pps). Shewanella hanedai was cultured at optimum ranges for luciferase enzyme characterization. From the centrifuged supernatant, the proteins were precipitated with 60% ammonium sulfate, dialyzed, and purified using anion-exchange chromatography, and then luciferase was eluted with 500 mM phosphate of pH 7.0. The purified luciferase enzyme was subjected to SDS-PAGE and the molecular mass was determined as 78 kDa.

• 한국미생물·생명공학회지
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• 제20권2호
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• pp.225-232
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• 1992

효소면역측정법에 의한 aflatoxin $B_1(AFB_1)$의 정량법을 개발하기 위하여, 항체를 생산.정제하여 분석법을 확립하고 직접법과 간접법(direct/indirect competitive ELISA)의 특성 및 문제점을 비교. 검토하였다. Bovine serum albumin(BSA)을 carrier protein으로 한 $AFB_1$-1-(O-carboxymethyl)oxime -BSA를 토끼에 면역하여 항$AFB_1$항혈청을 생산하였다. 정량 침강반응에 의하여 항혈청으로부터 항BSA항체를 제거하고 황산암모늄 침전법 및 EDAE-Sephadex A-50 이온교환 크로마토그래피를 통하여 순도 높은 IgG항체를 정제하여 항$AFB_1$항체를 사용하였다.