• BMB Reports
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• 제31권4호
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• pp.345-349
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• 1998

Aspartase (EC 4.3.1.1) from Hafnia alvei was purified to homogeneity by a combination of DEAE-cellulose, Red A-agarose, and Sepharose 6B chromatography. The purified enzyme appeared homogeneous on denatured SDS-polyacrylamide gel electrophoresis. The purified enzyme was a tetrameric protein composed of identical subunits with a molecular weight of 55,000 daltons. The optimum pH for the enzymatic reaction was 8.5 and the optimum temperature for maximum activity was $45^{\circ}C$. The enzyme has an absolute requirement of divalent metal ions ($Mg^{2+}$, $Mn^{2+}$) at the alkaline pH. The enzyme, however, was inactivated in the presence of other divalent cations such as $Zn^{2+}$, $Ca^{2+}$. The helical content of the purified enzyme was estimated by CD spectropolarimetry to be 61%.

• 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.

• 한국미생물·생명공학회지
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• 제49권2호
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• pp.181-191
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• 2021

The present study addresses isolation, optimization, partial purification, and characterization of a haloalkaline serine protease from a newly isolated haloarchaeal strain isolated from Wadi El Natrun in Egypt. We expected that a two-step sequential statistical approach (one variable at a time, followed by response surface methodology) might maximize the production of the haloalkaline serine protease. The enzyme was partially purified using Hiprep 16/60 sephacryl S-100 HR gel filtration column. Molecular identification revealed the newly isolated haloarchaeon to be Natrialba hulunbeirensis strain WNHS14. Among several tested physicochemical determinants, casamino acids, KCl, and NaCl showed the most significant effects on enzyme production as determined from results of the One-Variable-At-A-time (OVAT) study. The BoxBehnken design localized the optimal levels of the three key determinants; casamino acids, KCl, and NaCl to be 0.5% (w/v), 0.02% (w/v), and 15% (w/v), respectively, obtaining 62.9 U/ml as the maximal amount of protease produced after treatment at 40℃, and pH 9 for 9 days with 6-fold enhancement in yield. The enzyme was partially purified after size exclusion chromatography with specific activity, purification fold, and yield of 1282.63 U/mg, 8.9, and 23%, respectively. The enzyme showed its maximal activity at pH, temperature, and NaCl concentration optima of 10, 75℃, and 2 M, respectively. Phenylmethylsulfonyl fluoride (PMSF, 5 mM) completely inhibited enzyme activity.

• Journal of Microbiology and Biotechnology
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• 제24권11호
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• pp.1516-1524
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• 2014

Flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) can utilize a variety of external electron acceptors and also has stricter substrate specificity than any other glucose oxidoreductases, which makes it the ideal diagnostic enzyme in the field of glucose biosensors. A gene coding for a hypothetical protein, similar to glucose oxidase and derived from Aspergillus terreus NIH2624, was overexpressed in Pichia pastoris GS115 under the control of an AOX1 promoter with a level of 260,000 U/l in the culture supernatant after fed-batch cultivation for 84 h. After a three-step purification protocol that included isopropanol precipitation, affinity chromatography, and a second isopropanol precipitation, recombinant FAD-GDH was purified with a recovery of 65%. This is the first time that isopropanol precipitation has been used to concentrate a fermentation supernatant and exchange buffers after affinity chromatography purification. The purified FAD-GDH exhibited a broad and diffuse band between 83 and 150 kDa. The recombinant FAD-GDH was stable across a wide pH range (3.5 to 9.0) with maximum activity at pH 7.5 and $55^{\circ}C$. In addition, it displayed very high thermal stability, with a half-life of 82 min at $60^{\circ}C$. These characteristics indicate that FAD-GDH will be useful in the field of glucose biosensors.

• 한국식품영양과학회지
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• 제17권4호
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• pp.348-357
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• 1988

갈변반응에 관여하는 polyphenol oxidase(PPO : EC 1.10.3.1)를 한국산 고구마(Ipomoea batatas, val : Hong-mi)로부터 추출하여 ammoniun sulfate 분획 및 DEAE-cellulose column chromatography법에 의하여 정제한 결과, 효소활동도는 23.1배였으며 enzyme activity 수율은 41.5%이었다. 이 효소는 일반 전기 영동법에 의하여 8개의 isozymes 으로, 또한 isolectric focusing에 의하여 pI가 각각 다른 12개의 isozymes으로 분리되었고 그 pI의 범위는 3.2-9.6이었으며, Isoelectric focusing에 의하여 분리된 각 isozyme의 specific activity는 6,000-46,700U/mg protein의 범위에 있었다. 고구마 중의 PPO는 $65^{\circ}C$이하에서는 안정하였으며 $65^{\circ}C$ 에서는 1분 가열에 의하여 약 50%의 효소활성이 상실되었고, pH optimum은 6.0-6.5이었다. o-diphenol이 이 효소의 가장 좋은 기질로서, 이 효소는 o-diphenolase임이 확인되었고, catechol에 대한 Km치는 6.7mM로 나타났다. 또한 이 효소에 대한 저해작용은 dithiothreitol, cysteine 및 ascorbic acid 순으로 크게 나타났다.

• 한국식품영양과학회지
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• 제29권4호
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• pp.719-725
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• 2000

본 연구는 70여종의 국내산 해조류중 가장 높은 ACE 저해 활성을 보였던 김(Porphyra yezoensis, 서천)의 산가수분해물로부터 ACE 활성 저해 펩타이 드를 분리하여 그 특성을 조사하였다. ACE 저해물질의 분획은 균일하게 파쇄한 김을 2.5 N HCl로 산 가수분해한 후 중화하여 한외여과로 분자크기 3 kDa 이하의 물질로 분리하였다. 분자크기 3 kDa이하의 물질에 대하여 column chromatography(Amberlite XAD 8, DEAE-Toyopearl, Sephadex LH-20)와 reverse phase HPLC(C18)를 순차적으로 수행하여 ACE 저해제인 PY3--II-b-h5물질을 분리하였다. PY30-II-b-h5는 분자크기는 약 580 dalton으로 glycine(24.5%), arginine(56.8%), proline(18.8%)의 아미노산 조성을 갖는 저분자 펩타이드였으며, ACE의 저해양상은 경쟁적 저해작용을 하였고, IC50 값은 10.6$\mu\textrm{g}$/mL 이었다.

• 한국미생물·생명공학회지
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• 제16권6호
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• pp.526-531
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• 1988

방선균의 단백질 분해효소를 황산 암모늄분획, Sephadex G-75-50 gel filtration, DEAE-Sephadex A-50 ion-exchange chromatography, ultrafiltration 등의 과정을 통해 정제하였다. 염기성 단백질 분해 효소의 분자량은 SDS 전기영동에 의해 23,500 dalton 이었으며 Hammarsten casein에 대한 Km값은 0.8g/l였고 이때 Vmax값은 15.1 $\mu$mole/min/mg 이었다. 효소반응 최적 pH는 9.0이었고 최적 반응온도는 5$0^{\circ}C$였다. pH에 대한 안정성은 9.0-10.0 에서 최대로 안정하였고 5$0^{\circ}C$ 이상에서는 효소가 불활성화되었다. 중성단백질 분해효소의 분자량은 38900 dalton 이었으며 Hammarsten casein에 대한 Km값은 0.54g/l였고 이때 Vmax값은 12.4 $\mu$mole/min/mg이었다. 효소반응 최적 pH는 7.0이었고 최적 반응온도는 35$^{\circ}C$였다. pH 7.0-9.0에서는 안정하였으나 4$0^{\circ}C$ 이상에서는 신속하게 불활성화되었다.

• Journal of Microbiology
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• 제41권3호
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• pp.207-211
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• 2003

Extracellular protease, from Aeromonas hydrophila Ni 39, was purified 16.7-fold to electrophoretic homogeneity with an overall yield of 19.9％, through a purification procedure of acetone precipitation, and Q Sepharose and Sephacryl S-200 chromatographies. The isoelectric point of the enzyme was 6.0 and the molecular mass, as determined by Sephacryl S-200 HR chromatography, was found to be about 102 kDa. SDS/PAGE revealed that the enzyme consisted of two subunits, with molecular masses of 65.9 kDa. Under standard assay conditions, the apparent $K_{m}$ value of the enzyme toward casein was 0.32 mg/ml. About 90％ of the proteolytic activity remained after heating at 60$^{\circ}C$ for 30 min. The highest rate of azocasein hydrolysis for the enzyme was reached at 60$^{\circ}C$, and the optimum pH of the enzyme was 9.0. The enzyme was inhibited by the serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), by about 87.9％, but not by E64, EDTA, pepstatin or 1,10-phenanthroline. The enzyme activity was inhibited slightly by Ca$\^$2＋/, Mg$\^$2＋/ and Zn/supb 2＋/ ions.

• 한국미생물·생명공학회지
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• 제46권1호
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• pp.51-58
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• 2018

본 연구에서는 극지에서 유래한 Janthinobacterium sp. PAMC25641로부터 분리한 리파아제 유전자를 클로닝 하고 과발현시켜 정제하였으며, 이 분리한 재조합 리파아제 효소의 생화학적 특성에 대해 분석하였다. 이 효소는 $15^{\circ}C$ 이하의 온도에서 장시간 활성을 유지하는 효소로서 산업적으로 활용될 가능성이 높을 것으로 기대된다.

• Preventive Nutrition and Food Science
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• 제7권1호
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• pp.22-27
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• 2002

The cellulase from internal organs of edible snails was purified by fractionation with ammonium sulfate, DEAE-Sephadex chromatography and gel filtration on Sephacryl S-200 and Superose 12 HR 10/30. The specific activity of the purified cellulase was 85.1 units/mg protein with 24.3 purification fold from crude extract. Molecular weight of the enzyme was estimated to be approximately 74,000 dalton by gel filtration chromatography and SDS-PAGE eletrophoresis. T7e isoelectric point of the enzyme was determined to be pH 4.6. The optimum temperature and pH of the enzyme were 5$0^{\circ}C$ and pH 6.0, respectively. The enzyme was stable at 30~5$0^{\circ}C$ and pH 6.0~10.0. It was activates by Mn$^{2+}$, but inhibited by Li$^{2+}$, Zn$^{2+}$, Ag$^{2+}$ and Hg$^{2+}$./TEX> 2+/.