Detection and quantification of leucyl aminopeptidase after native electrophoresis using leucine-p-nitroanilide

Božić, Nataša; Vujčić, Zoran

doi:10.1002/elps.200500047

Cited by 25 publications

(8 citation statements)

References 15 publications

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“…The gels were incubated in the buffer containing the respective pNA substrate until a yellow band appeared and were then washed with H 2 O dd . The bands were redyed, according to the method of Bozǐćand Vujcǐc, 35 for improved visibility. Therefore, the gels were incubated in 1 g/L NaNO 2 (dissolved in 1 M HCl) for 120 s and washed in urea buffer (10 g/L) for 30 s. The gels were incubated in 0.25 g/L N-(1-naphthyl)-ethylenediamine dihydrochloride dissolved in 22% (v/v) EtOH for 10 min for staining.…”

Section: ■ Experimental Proceduresmentioning

confidence: 99%

Flavourzyme, an Enzyme Preparation with Industrial Relevance: Automated Nine-Step Purification and Partial Characterization of Eight Enzymes

Merz

Eisele

Berends

et al. 2015

J. Agric. Food Chem.

168

104

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Flavourzyme is sold as a peptidase preparation from Aspergillus oryzae. The enzyme preparation is widely and diversely used for protein hydrolysis in industrial and research applications. However, detailed information about the composition of this mixture is still missing due to the complexity. The present study identified eight key enzymes by mass spectrometry and partially by activity staining on native polyacrylamide gels or gel zymography. The eight enzymes identified were two aminopeptidases, two dipeptidyl peptidases, three endopeptidases, and one α-amylase from the A. oryzae strain ATCC 42149/RIB 40 (yellow koji mold). Various specific marker substrates for these Flavourzyme enzymes were ascertained. An automated, time-saving nine-step protocol for the purification of all eight enzymes within 7 h was designed. Finally, the purified Flavourzyme enzymes were biochemically characterized with regard to pH and temperature profiles and molecular sizes.

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Section: ■ Experimental Proceduresmentioning

confidence: 99%

Flavourzyme, an Enzyme Preparation with Industrial Relevance: Automated Nine-Step Purification and Partial Characterization of Eight Enzymes

Merz

Eisele

Berends

et al. 2015

J. Agric. Food Chem.

168

104

View full text Add to dashboard Cite

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“…The protein was first separated on Native PAGE (4% w/v staking gel and 10% w/v separating gel) by electrophoresis at 4°C. Following this step, activity staining was performed [15]. Briefly, the gel was washed twice with distilled water for 10 min followed by 50 mmol/l Tris-HCl buffer pH 8.5 twice for 5 min.…”

Section: Sds-page and Activity Stainingmentioning

confidence: 99%

Purification and Biochemical Characterization of Methionine Aminopeptidase (MetAP) from Mycobacterium smegmatis mc2155

Narayanan

Ramanujan

Krishna

et al. 2008

Appl Biochem Biotechnol

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The methionine aminopeptidase (MetAP) catalyzes the removal of amino terminal methionine from newly synthesized polypeptide. MetAP from Mycobacterium smegmatis mc(2) 155 was purified from the culture lysate in four sequential steps to obtain a final purification fold of 22. The purified enzyme exhibited a molecular weight of approximately 37 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Activity staining was performed to detect the methionine aminopeptidase activity on native polyacrylamide gel. The enzyme was characterized biochemically, using L-methionine p-nitroanilide as substrate. The enzyme was found to have a temperature and pH optimum of 50 degrees C and 8.5, respectively, and was found to be stable at 50 degrees C with half-life more than 8 h. The enzyme activity was enhanced by Mg(2+) and Co(2+) and was inhibited by Fe(2+) and Cu(2+). The enzyme activity inhibited by EDTA is restored in presence of Mg(2+) suggesting the possible role of Mg(2+) as metal cofactor of the enzyme in vitro.

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“…The lanes of the native PAGE gel used for activity staining were excised and then incubated with the corresponding p NA solution at 37°C until yellow bands were observed. The yellow bands developed were redyed to violet-colored bands using the following protocol [21]. The lanes of the native PAGE gel containing yellow bands were incubated in a 0.1% (w/v) NaNO 2 /1 M HCl solution until the bands disappeared (approx.…”

Section: Methodsmentioning

confidence: 99%

Characterization of the Recombinant Exopeptidases PepX and PepN from Lactobacillus helveticus ATCC 12046 Important for Food Protein Hydrolysis

et al. 2013

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The proline-specific X-prolyl dipeptidyl aminopeptidase (PepX; EC 3.4.14.11) and the general aminopeptidase N (PepN; EC 3.4.11.2) from Lactobacillus helveticus ATCC 12046 were produced recombinantly in E. coli BL21(DE3) via bioreactor cultivation. The maximum enzymatic activity obtained for PepX was 800 µkatH-Ala-Pro-pNA L−1, which is approx. 195-fold higher than values published previously. To the best of our knowledge, PepN was expressed in E. coli at high levels for the first time. The PepN activity reached 1,000 µkatH-Ala-pNA L−1. After an automated chromatographic purification, both peptidases were biochemically and kinetically characterized in detail. Substrate inhibition of PepN and product inhibition of both PepX and PepN were discovered for the first time. An apo-enzyme of the Zn2+-dependent PepN was generated, which could be reactivated by several metal ions in the order of Co2+>Zn2+>Mn2+>Ca2+>Mg2+. PepX and PepN exhibited a clear synergistic effect in casein hydrolysis studies. Here, the relative degree of hydrolysis (rDH) was increased by approx. 132%. Due to the remarkable temperature stability at 50°C and the complementary substrate specificities of both peptidases, a future application in food protein hydrolysis might be possible.

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Detection and quantification of leucyl aminopeptidase after native electrophoresis using leucine-p-nitroanilide

Cited by 25 publications

References 15 publications

Flavourzyme, an Enzyme Preparation with Industrial Relevance: Automated Nine-Step Purification and Partial Characterization of Eight Enzymes

Flavourzyme, an Enzyme Preparation with Industrial Relevance: Automated Nine-Step Purification and Partial Characterization of Eight Enzymes

Purification and Biochemical Characterization of Methionine Aminopeptidase (MetAP) from Mycobacterium smegmatis mc2155

Characterization of the Recombinant Exopeptidases PepX and PepN from Lactobacillus helveticus ATCC 12046 Important for Food Protein Hydrolysis

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