• Applied Biological Chemistry
• /
• v.50 no.4
• /
• pp.308-315
• /
• 2007

For the effective use of squid processing by-products as food resources, the distribution and the extraction condition of endoprotease and exopeptidase from viscera of Illex argentinus were investigated. Crude protein and lipid contents of viscera of Illex argentinus were 17.2% and 16.9%, respectively. Regardless of kinds of extraction solution (water, 1% NaCl, 1% KCl and 1% NaCl- KCl) and extraction times (1-20 h), endoprotease activities from viscera of Illex argentinus on Hb, casein and azocasein (pH 6.0) were higher than those on casein and azocasein of the other pHs, thus indicating that the distribution of protein hydrolysing protease is distinctive in the weak acid pH range. Exopeptidase activities against LeuPNA and ArgPNA at pH 7.5 were relatively higher than endoprotease activity of the same pH. The results suggested that exopeptidase among proteases from viscera of Illex argentinus was reasonable for application in food industry compared to endoprotease. The activity in enzymes from viscera of Illex argentinus was the highest in the exopeptidase extracted with deionized distilled water at room temperature for 6-8 h. The optimal reaction conditions of crude enzyme from viscera of Illex argentinus were 7.5 for pH and $50-55^{\circ}C$ for temperature.

• Fisheries and Aquatic Sciences
• /
• v.15 no.4
• /
• pp.283-291
• /
• 2012

This study examines the optimal fractionation method and conditions for the isolation of endoprotease- and exopeptidase-active fractions from crude extracts of cuttlefish hepatopancreas (HP) using four fractionation methods: ammonium sulfate fractionation (ASF), polyethylene glycol fractionation (PGF), ion exchange chromatography (IEC), and gel filtration chromatography (GFC). Total endoprotease activity highest in the fraction II (concentrate of fractions 34-42; 842.60 U) of GFC, followed by fraction III (40-60% ammonium sulfate fraction; 670.25 U) of ASF, fraction I (concentrate of fractions 8-12; 436.89 U) of IEC, and fraction II (10-20% polyethylene glycol; 307.31 U) of PGF. Total exopeptidase activity of these fractions was highest in fraction II (2,704.70 U) of GFC, fraction III (2,110.50 U) of ASF, fraction III (1,605.60 U) of PGF, and fraction II (concentrate of fractions 38-44; 1,196.22 U) of IEC. These results showed that fraction II of GFC had the highest activity toward both exopeptidase and endoprotease, with exopeptidase activity being 3.21 times higher than of endoprotease. These results suggest cuttlefish HP could be used as a potential source for the extraction of exopeptidase, an enzyme capable of catalyzing the cleavage of N- and C-terminal amino acids in polypeptides, Like endoprotease, the most efficient method for separating exopeptide-active fractions was GFC.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.37 no.8
• /
• pp.1009-1017
• /
• 2008

For the effective use of exopeptidase from squid viscera as food processing aids, the viscera of Argentina shortfin squid (Illex argentinus) were fractionated by various methods such as acetone treatment, ammonium sulfate treatment, anion exchange chromatography, and gel filtration. The positive exopeptidase fractions were obtained from the fraction II treated by cold acetone ($30{\sim}40%$, w/w), the fraction V by ammonium sulfate ($60{\sim}70%$ saturation), the fraction II (0.2 M NaCl) by anion exchange chromatography, and the fraction I ($30{\sim}50\;kDa$) by gel filtration. The specific activities of positive fractions from viscera of I llex argentinus against substrates were higher to LeuPNA than to ArgPNA. Total activity and recovery against LeuPNA of positive fraction by gel filtration were 1,867 U and 30.69%, respectively, which were the highest among those of positive fraction. The results suggested that the gel filtration chromatography method was the most efficient method for the fractionation of exopeptidase from viscera of Illex argentinus.

• Fisheries and Aquatic Sciences
• /
• v.17 no.2
• /
• pp.181-187
• /
• 2014

This study was performed to identify the optimum fractionation method and conditions to obtain exopeptidase-active fractions from octopus hepatopancreas (HP) crude extracts (CEs) using four techniques: solid ammonium sulfate fractionation, polyethylene glycol (PEG) fractionation, anion exchange chromatography, and gel filtration chromatography. The fractions with the highest total activity toward L-leucine-p-nitroanilide (Leu-pNA) were fraction IV from the ammonium sulfate and PEG fractionation, and fraction II in ion exchange and gel filtration chromatography. The total exoprotease activity of these fractions was highest in fraction IV (4,050.20 U) of ammonium sulfate fractionation, followed by fraction II (3,600.28 U) from gel filtration chromatography, fraction IV (2,861.30 U) from PEG fractionation, and fraction II (2,576.28 U) from ion exchange chromatography. These results suggest that ammonium sulfate fractionation using 60-80% ammonium sulfate was the most efficient method for separating the exoprotease active fractions from CEs of octopus HP.

• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.47 no.2
• /
• pp.135-143
• /
• 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
• /
• v.20 no.3
• /
• pp.460-466
• /
• 2010

A mutant of green fluorescent protein ($GFPmut3^*$) from the jellyfish Aequorea victoria was cyclized in vitro and in vivo by the use of a naturally split intein from the dnaE gene of Synechocystis species PCC6803 (Ssp). Cyclization of $GFPmut3^*$ was confirmed by amino acid sequencing and resulted in an increased electrophoretic mobility compared with the linear $GFPmut3^*$. The circular $GFPmut3^*$ was $5^{\circ}C$ more thermostable than the linear form and significantly more resistant to proteolysis of exopeptidase. The circular $GFPmut3^*$ also displayed increased relative fluorescence intensity. In addition, chemical stability of $GFPmut3^*$ against GdnHCl revealed more stability of the circular form compared with the linear form.

• Applied Biological Chemistry
• /
• v.34 no.4
• /
• pp.334-338
• /
• 1991

Synthesis of $(D-Phe^7\;-Leu^8)$ bradykinin and bradykinin by solid phase method using a new reaction vessel was carried out. Coupling was performed by dicyclohexylcarbodiimide. After cleavage with dried HBr the peptides were purified by high pressure liquied chromatography. Their purify was assayed by paper and thin layer chromatography, melting point and amino acid analysis. $(D-Phe^7\;-Leu^8)$ bradykinin and bradykinin were incubater in vitro endopeptidase $({\alpha}-chymotrysis)$ and exopeptidase(carboxypeptidase A, leucine aminopeptidase) in order to study the degradation pattern of peptides. $(D-Phe^7\;-Leu^8)$ bradykinin and bradykinin were rapidly degradated by ${\alpha}-chymotrypsin$ and carboxypeptidase A $(D-Phe^7\;-Leu^8)$ bradykinin and bradykinin coution$(D-Phe^7\;-Leu^8)$ bradykinin and bradykinin contain imino peptide bound from proline at N-terminal and therefore they were not attacted by leucine aminopeptidase.

• Fisheries and Aquatic Sciences
• /
• v.15 no.3
• /
• pp.197-202
• /
• 2012

This study examined and compared the exopeptidase and endoprotease activities of the hepatopancreas (HP) of cuttlefish, squid, and octopus species. The protein concentration in crude extract (CE) from octopus HP was 3,940 mg/100 g, lower than those in CEs from squid HP (4,157 mg/100 g) and cuttlefish HP (5,940 mg/100 g). With azocasein of pH 6 as a substrate, the total activity in HP CE of octopus was 31,000 U, lower than the values for cuttlefish (57,000 U) and squid (69,000 U). Regardless of sample type, the total activities of the CEs with azocasein as the substrate were higher at pH 6 (31,000-69,000 U) than at pH 9 (19,000-34,000 U). With L-leucine-p-nitroanilide (LeuPNA) of pH 6 as the substrate, the total activity of the HP CE from octopus was 138,000 U, higher than values from both cuttlefish HP (72,000 U) and squid HP (63,000 U). Regardless of sample type, the total activities of the CEs with LeuPNA as the substrate were higher at pH 6 (63,000-138,000 U) than at pH 9 (41,000-122,000 U). With LeuPNA as the substrate, the total activities of the CEs from octopus HP and cuttlefish HP were higher at pH 6 than at pH 9. However, there was no difference in total activity between pH 6 and 9 for squid HP CE with LeuPNA as the substrate. These results suggest that the octopus HP is superior to the cuttlefish HP and squid HP as a potential resource for extracting exopeptidases. Exopeptidases from octopus HP have potential industrial applications and their use might aid in reducing pollution related to the octopus industry.

• Journal of Microbiology and Biotechnology
• /
• v.9 no.6
• /
• pp.861-869
• /
• 1999

An extracellular protease was purified to homogeneity from the culture supernatant of psychrotrophic bacteria Bacillus sp. JH 108 using procedures including ammonium sulfate fractionation, anion exchange chromatography, gel filtration chromatography, and cation exchange chromatography. The enzyme exhibited a molecular weight of 36 kDa, an optimum pH of 8 to 9, and optimum temperature of $60^{\circ}C$. The enzyme preferentially hydrolyzed leucine at the N-terminus of peptides and thus can be classified as an aminopeptidase. It was strongly inhibited by metal chelating agents such as EDTA and l, l0-phenanthroline. The activity lost by EDTA was restored with $Zn^{2+}{\;}or{\;}Co^{2+}$. These divalent cations also stimulated the native enzyme. This suggests that the enzyme is a metalloprotease acting as a leucine aminopeptidase.

• The Microorganisms and Industry
• /
• v.17 no.2
• /
• pp.28-30
• /
• 1991

APM의 화학적, 효소적 합성방법의 선택은 각기의 장단점을 비교 검토한 후 결정해야 할 문제로서, 수율, 공정의 효율성, 작업환경, 경제성 등의 여러 요인이 영향을 줄 수 있으나, 최근의 연구동향 및 산업적 생산의 추이는 효소를 이용한 bioconversion process에 의한 방식으로 나아가는 듯 하다. 결론적으로 bioconversion process에 의한 APM의 생산은 반응매질로써 유기용매의 사용이 불가피하므로 효소의 안정성을 증가시켜 장기간 사용할 수 있는 신기술의 개발이 필요하며 기존의 고정화 기술은 그 좋은 예가 될 수 있다. 또한 보호기의 도입과 제거과정이 보다 용이해야하며 더 나아가서 보호기의 부착없이도 반응을 가능케하는, 기질에 대한 특이성이 높은 새로운 효소(예를 들어 exopeptidase를 사용하면 기질에 보호기를 붙일 필요가 없으므로 화학적 방법에 비해 훨씬 유리하다)의 screening이 절실하다. 아울러 유기용매로 인한 효소의 deactivation mechanism의 규명과 반응기 운전 system의 개발이 요구된다 하겠다.