• Microbiology and Biotechnology Letters
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• v.17 no.2
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• pp.121-125
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• 1989

Actinomycetes GF 155-2, which produced an extracellular pepsin inhibitor, was isolated from soil samples. Optimal conditions of inhibitor production by flask-shacking culture were 2% glucose, 0.7% polypeptone, initial pH 1.0, culture time 60 hours and temperature 30%. Effect of in-organic salts was not observed. About 5mg of colorless crystalline inhibitor was obtained from 5L culture broth in jar tormentor by means of ammonium sulfate precipitation, methanol extraction, and column chromatographies on Amberlite IR-120, XAD-2 and silicagel 60.

• Microbiology and Biotechnology Letters
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• v.18 no.5
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• pp.496-500
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• 1990

When pepsin was used at a concentration of 8 mglml for hydrolysis of 0.02% casein, inhibitory activity of this inhibitor was proportional to a inhibitor concentration of 20 ${\mu}g$/ml, and fifty percent inhibition ($IC_{50}$) was observed to be 15 ${\mu}g$/ml. The inhibitor was pH-stable at pH range of 5-9 at $100^{\circ}C$ for 10 minutes and thermo-stable at pH 7.0 at $100^{\circ}C$ to give 100% activity for 20 minutes. The formation of pepsin-inhibitor complex was confirmed by sephadex 6-25 gel filtration and type of inhibition was determined as non-competitive inhibition by Lineweaver-Burk plot. The inhibitor strongly inhibited acid proteases such as pepsin and renin, and it was soluble in methanol very well. On TLC analysis of silicagel 60 using various sohent systems, the inhibitor gave a single spot at Rf range 0.4-0.6. From the result of IR spectrum and color reaction (Rydon-Smith, Biuret), this inhibitor was considered as peptide substance. Melting point and elemental contents were 220-$230^{\circ}C$, and C 50.61%-H 8.02%-N 9.34% (found), respectively.

• Microbiology and Biotechnology Letters
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• v.17 no.2
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• pp.115-120
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• 1989

For the purpose of obtaining microorganisms which produced an extracellular pepsin inhibitor, screening test was carried out. One strain of Actinomycetes (GF 155-2) isolated from soil samples showed a high inhibitory activity against porcine pepsin. The morphological, physiological and cultural characteristics of the strain GE 155-2 on various culture media were studied according to ISP methods and Bergey's manual of determinative bacteriology (8th ed.). This Actinomycetes GE 155-2 was found to be similar to the genus Microtetraspora.

• Journal of the Korean Chemical Society
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• v.14 no.2
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• pp.161-168
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• 1970

In order to obtain the more effective evidence, supporting the hypothesis which have been previously described by former report that pepsin (EC 3.4. 4.1) forms a hydrophobic bond with the nonpolar side chain of its substrate, the inhibitory effect of carboxylic acids(from formic acid to iso-butyric acid) on the activity of pepsin to the synthetic dipeptide, N-Carbobenzoxy-L-glutamyl-L-tyrosine, was discussed. The kinetic study showed that the inhibition by carboxylic acids was competitive. The Kidecreased with increasing size of the inhibitor molecule. The $-{\Delta}F^{\circ}$increased linearly with increasing number of carbon atoms in the hydrocarbon chain of the inhibitor. It was confirmed that the hydrophobic bond between more than one side chain of amino acid residues(phenylalanine) in the binding region of the active center of pepsin and the side chain of amino acid residues in the substrate was formed as the first step of its enzymic mechanism. The inhibitory effect of carboxylic acids was due to the competition of the hydrocarbon group of the carboxylic acids with the side chain of the substrate for the hydrophobic binding site(the side chain of phenylalanine) of the pepsin.

• YAKHAK HOEJI
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• v.37 no.2
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• pp.149-157
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• 1993

Protein methylase inhibitor which is a modulator of biological methylation has been purified and characterized from porcine liver soluble fraction by cell fractionation, Sephadex G25 chromatography, reverse phase HPLC, size exclusion HPLC. The results are summarized as follows. 1) The purified inhibitor shows apparent homogeneity, as judged by HPLC. 2) A molecular weight of the purified inhibitor which is composed of 18 amino acid residues is about 1,400 daltons. 3) A single absorption peak of ultraviolet spectrum was observed at 260nm. 4) The inhibitor was not inactivated by heating at $100^{\circ}C$ until 60min. and its activity was not influenced by treatment with digestive enzymes, such as trypsin, pepsin, pronase, chymotrypin, lysozyme, DNase, and RNase. 5) The purified inhibitor inhibited protein rnethylase I, II, III and phospholipid methyltransferase activities. 6) The purified inhibitor inhibited noncompetitively protein methylase II from porcine liver, spleen, and testis. 7) The $K_{i}$ values for protein methylase II from porcine liver, spleen, and testis were 300nM, 250nM, 297nM, respectively.

• Current Research on Agriculture and Life Sciences
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• v.32 no.3
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• pp.149-154
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• 2014

It is already known that adzuki beans (Vigna angularis) are able to control appetite. Therefore, this study tested the proteins isolated from adzuki beans for their protease resistance and interaction with the intestinal mucosa. The major proteins from adzuki beans were found to be resistant to the digestive enzymes pepsin and pancreatin, and were identified using 2D-SDS-polyacrylamide gel electrophoresis and mass spectrometry. The major adzuki proteins were easily fractionated by treating the soluble protein extract with 10mM $CaCl_2$, and were found to contain lactotransferrin, a homologous protein to the dynein light chain domain, proteinase inhibitor, and proteins with unknown functions. From a tissue binding assay using mouse intestinal tissue sections, the major protein fraction showed weak, yet significant and specific binding to the mucosa layer of the small intestine. Thus, the current results suggest that adzuki proteins are resistant to digestive enzymes, which enables them to survive protease digestion in the intestinal tract, plus they may interact with the intestinal mucosa layer. Therefore, the molecules responsible for controlling appetite in adzuki beans are presumably protease-resistant proteins that interact with the intestinal mucosa or delay digestion in the digestive tract.

• Journal of Microbiology and Biotechnology
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• v.14 no.6
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• pp.1318-1323
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• 2004

This study describes the purification and characterization of a novel antihypertensive angiotensin 1­converting enzyme (ACE) inhibitory peptide from Saccharomyces cerevisiae. Maximal production of the ACE inhibitor from Saccharomyces cerevisiae was obtained from 24 h of cultivation at $30^{\circ}C$ and its ACE inhibitory activity was increased by about 1.5 times after treatment of the cell-free extract with pepsin. After the purification of ACE inhibitory peptides with ultrafiltration, Sephadex G-25 column chromatography, and reverse-phase HPLC, an active fraction with an $IC_{50}$ of 0.07 mg and $3.5\%$ yield was obtained. The purified peptide was a novel decapeptide, showing very low similarity to other ACE inhibitory peptide sequences, and its amino acid sequence was Tyr-Asp-Gly-Gly-Val-Phe-Arg-Val-Tyr-Thr. The purified inhibitor competitively inhibited ACE and also showed a clear antihypertensive effect in spontaneously hypertensive rats (SHR) at a dosage of 1 mg/kg body weight.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.44 no.2
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• pp.118-125
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• 2011

In this study, an angiotensin I-converting enzyme (ACE) inhibitor from squid skin was purified and characterized. Squid (Todarodes pacificus) skin protein isolates were hydrolyzed using six commercial proteases: alcalase, ${\alpha}$-chymotrypsin, neutrase, papain, pepsin, and trypsin. The peptic hydrolysate had the highest ACE inhibitory activity. The ACE inhibitory peptide was purified using Sephadex G-25 column chromatography and reverse phase high-performance liquid chromatography (HPLC) with a $C_{18}$ column. The purified ACE inhibitory peptide was identified and sequenced, and found to consist of seven amino acid residues: Ser-Ala-Gly-Ser-Leu-Val-Pro (657Da). The $IC_{50}$ value of the purified ACE inhibitory peptide was 766.2 ${\mu}M$, and Lineweaver-Burk plots suggested that the purified peptide acts as a noncompetitive ACE inhibitor. These results suggest that the ACE inhibitory peptide purified from the peptic hydrolysate of squid skin may be of benefit in developing antihypertensive drugs and functional foods.

• Microbiology and Biotechnology Letters
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• v.15 no.2
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• pp.129-134
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• 1987

Aspergillus sp. (MK-24) producing a biological active substance that inhibited the venom proteinase activity was isolated from soil. The substance also inhibited the activity of trypsin and coagulation of blood, but did not inhibit papain, $\alpha$-chymotrypsin and pepsin. The substance was partially purified from culture filtrate by precipitaion with acetone, and by chromatography of DEAE-Sepadex A-50 column and Amberlite IRC-50 ion exchange. The inhibitory substance was stable in the wide pH range from 2.0 to 12.0 at 37$^{\circ}C$, but not stable at $65^{\circ}C$ in the alkaline pH. Only 12% of the activity was decreased by the heat treatment at 10$0^{\circ}C$ for two hours. The inhibition on venom proteinase (Agkistrodon bromohoffi brevicaudus) was a mixed type. The inhibitory activity depended on the preincubation time and completely depressed by cupric, zinc and cobalt ions. The inhibition on the venom proteinase was appeared strongly on casein but not on ovalbumin or hemoglobin as a substrate.

• Food Science and Biotechnology
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• v.14 no.6
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• pp.727-731
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• 2005

Synthesized Ile-Ala-Val-Pro (IAVP) peptide, which has the highest hypocholesterolemic effect among a number of synthesized derivatives of Ile-Ala-Val-Pro-Gly-Glu-Val-Ala (IAVPGEVA) isolated from 11S globulin of soy protein by pepsin digestion, was selected for investigation in the present study. Using a recombinant Syrian hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), we studied in detail the inhibition of this enzyme by IAVP and compared the action of this peptide to that of lovastatin, a known competitive inhibitor of this enzyme. The concentration of IAVP required for 50% inhibition ($IC_{50}$) of HMGR activity in given experimental conditions was $340\;{\mu}M$. Kinetic analysis revealed that the studied peptide is a competitive inhibitor of HMGR with respect to both 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) and nicotinamide adenine dinucleotide phosphate (NADPH), with an equilibrium constant of inhibitor binding ($K_i\;=\;[E][I]/[EI]$) of $61{\pm}1.2\;{\mu}M$ and $157{\pm}4.4\;{\mu}M$, respectively. At the same conditions, $K_i$ and $IC_{50}$ for lovastatin were $2.2{\pm}0.1\;nM$ and 12.5 nM, respectively. Thus, the given peptide interacts with HMGR as a bisubstrate, consequently blocking access of both substrates to the active sites. The achieved results suggest the design of new peptide sequences having a higher relative affinity to binding sites of this enzyme and an enhancement of their hypocholesterolemic properties.