• Applied Biological Chemistry
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• 제50권4호
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• pp.308-315
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• 2007

오징어 가공 부산물인 오징어 내장을 효소제재와 같이 식품가공소재로 이용하기 위하여 오징어 내장의 endoprotease와 exopeptidase의 분포 특성에 대하여 살펴보았다. 오징어 내장은 조효소가 함유되어 있으리라 추정되는 단백질이 17.2%를 차지하였고, 또한, 이물질로 제거하여야 되는 조지방의 경우도 16.9%로 다량 차지하였다. 오징어 내장 조효소의 활성을 천연기질(azocasein)과 합성기질(LeuPNA 및 ArgPNA)로 나누어 살펴 본 결과 추출 용매(탈이온수, 1% NaCl, 1% KCl 및 1% NaCl-KCl 혼합용액)및 추출 시간(1-20시간)에 관계없이 전 조 효소가 동일 pH에서 천연기질의 분해활성에 비하여 상대적으로 합성기질의 분해활성(pH7.5)이 높아, 오징어 내장으로부터 효소를 분리하여 이용하고자 하는 경우 exopeptidase를 분리하여 이용하는 것이 적절하리라 판단되었다. 오징어 내장으로부터 exopeptidase를 분리하여 이용하고자 하는 경우 탈이온수를 이용하여 6-8시간 동안 추출하는 것이 가장 적절하리라 판단되었다. 오징어 내장 조효소의 최적 pH와 온도는 pH 7.5 및 $50-55^{\circ}C$범위로 판단되었다.

• Fisheries and Aquatic Sciences
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• 제15권4호
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• pp.283-291
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• 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.

• 한국식품영양과학회지
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• 제37권8호
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• pp.1009-1017
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• 2008

원양산 오징어 내장 조효소로부터 아세톤법, 황산암모늄법, 음이온교환 크로마토그래피법 및 겔 여과법을 이용하여 exopeptidase를 분획한 다음, 각 분획방법별 fraction들의 효소 활성(총활성 및 비활성), 정제도 및 회수율 등을 검토하여 최적 분획조건을 구명하고자 하였다. 아세톤 분획법의 경우 아세톤의 최종농도가 $30{\sim}40%$가 되도록 분획하는 것이, 황산암모늄 분획법의 경우 황산암모늄을 $60{\sim}70%$의 포화농도가 되도록 분획하는 것이, 음이온교환 크로마토그래피의 경우 0.2 M NaCl을 이용하여 분획하는 것이, 그리고, 겔 여과의 경우 분자량 $30{\sim}50\;kDa$ 범위의 것을 분획하는 것이 효율적이었다. 분획방법별 최적 활성 fraction간의 상호비교에서 LeuPNA 및 ArgPNA에 대한 총활성 및 회수율은 겔 여과가, 비활성 및 정제도는 황산암모늄에 의한 분획법이 가장 우수한 것으로 나타났다. 원양산 오징어 내장으로부터 탈 이온수로 조효소를 추출한 다음, 겔 여과로 분획한 exopeptidase의 식품가공소재로서 산업적 이용을 위해서는 겔 여과법의 특성을 기초로 하여 추출 조효소로부터 연속 및 대량처리를 위한 공정개발이 필요할 것으로 사료된다.

• Fisheries and Aquatic Sciences
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• 제17권2호
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• pp.181-187
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• 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.

• 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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• 제20권3호
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• pp.460-466
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• 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
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• 제34권4호
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• pp.334-338
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• 1991

고상법으로 새로운 반응기구에 의한 bradykinin 및 $(D-Phe7\;-Leu^8)$ bradykinin을 합성하였다. Coupling은 N, N'-dicyclohexylcarbodiimide로 행하였으며 HBr 용액으로 cleavage한 후 조펩티드는 high pressure liquid chromatography로 정제하였다. 이들 펩티드의 순도는 paper chromatography, thin layer chromatography, paper electrophoresis, 융점측정기 및 아미노산기분석기에 의하여 분석하였다. Endopeptidase인 ${\alpha}-chymotrypsin$과 trysin, exopeptidase인 carboxypeptidase A와 leucine aminopeptidase를 사용하여 in vitro 상에서 이들 펩티드의 분해실험을 하였다. ${\alpha}-Chymotrypsine$ 및 carboxypeptidase A에 의하여 이들 펩티드는 빠르게 분해하였으나 leucine aminopeptidase는 N-말단의 2번 위치에 proline의 imino결합 때문에 분해하지 않았다.

• Fisheries and Aquatic Sciences
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• 제15권3호
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• pp.197-202
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• 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
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• 제9권6호
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• pp.861-869
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• 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.

• 미생물과산업
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• 제17권2호
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• pp.28-30
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• 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.