KR100215090B1 - Process for preparing angiotensin converting enzyme inhibitor from plasma protein hydrolysate - Google Patents

Process for preparing angiotensin converting enzyme inhibitor from plasma protein hydrolysate Download PDF

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KR100215090B1
KR100215090B1 KR1019960046123A KR19960046123A KR100215090B1 KR 100215090 B1 KR100215090 B1 KR 100215090B1 KR 1019960046123 A KR1019960046123 A KR 1019960046123A KR 19960046123 A KR19960046123 A KR 19960046123A KR 100215090 B1 KR100215090 B1 KR 100215090B1
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송경빈
원미선
박은희
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8103—Exopeptidase (E.C. 3.4.11-19) inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types

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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract

본 발명은 소, 돼지 도축혈액의 혈장단백질의 가수분해물로부터의 엔지오텐신전환효소(angiotensin converting enzyme) 저해제를 제조하는 방법에 관한 것으로 상수원 오염등 환경 오염을 방지하고 폐수 처리 비용을 절감하며 또한 대부분이 폐기되고 있는 도축혈액으로 부터 고부가가치 신기능성 소재인 항고혈압성 펩타이드 (peptide)를 분리하는데 있다. 도축시 발생하는 도축혈액은 도축 즉시 저장탱크를 거쳐 혈액 응고방지 처리한 후 연속식 원심분리기를 거쳐 혈장과 혈구로 분리한다. 분리된 혈장에 2%에서 10% 되게 트리클로로아세트산 (trichloroacetic acid) 또는 50%에서 70%되게 황산암모늄염 또는 염산 0.6N에서 3N 처리하여 혈장단백질을 침전시켜 원심 분리하여 얻은후 상업용 단백분해효소를 첨가하여 가수분해시켜 한외여과(ultrafiltration), 겔여과크로마토그라피 (gel permeation chromatography)와 고속액체크로마토그라피(high pressure liquid chromatography)를 이용하여 분획하여 엔지오텐신 전환 효소 저해제를 제조하는 방법으로서 그 의의 및 효과가 크다 할 수 있다.The present invention relates to a method for producing an angiotensin converting enzyme inhibitor from the hydrolysates of plasma proteins of cattle and pig slaughter blood, to prevent environmental pollution such as pollution of a water source, to reduce waste water treatment costs, And to isolate antihypertensive peptides that are high-value-added new functional materials from slaughtered blood. The slaughter blood from the slaughter is immediately slaughtered, stored in a storage tank to prevent blood clotting, and then separated into plasma and blood cells through a continuous centrifuge. Separated plasma was treated with 2% to 10% trichloroacetic acid or 50% to 70% ammonium sulfate or hydrochloric acid (0.6N to 3N) to precipitate plasma proteins and centrifugation. Commercial protease was added And then fractionated by hydrolysis, ultrafiltration, gel permeation chromatography and high pressure liquid chromatography to produce a angiotensin converting enzyme inhibitor. It can be big.

Description

도축 혈액의 혈장단백질 가수분해물로부터 엔지오텐신전환효소 저해제의 제조방법Preparation of angiotensin converting enzyme inhibitor from plasma protein hydrolysates of slaughter blood

제 1 도 본 발명품의 제조 중 겔여과크로마토그람Figure 1 shows the gel filtration chromatogram

제 2 도는 본 발명품의 제조 중 고속액체크로마토그람FIG. 2 is a graph showing the results of measurement of high-performance liquid chromatogram

본 발명은 소, 돼지 도축혈액의 혈장단백질의 가수분해물로부터 엔지오텐신전환효소(angiotensin converting enzyme) 저해제를 제조하는 방법에 관한 것으로 도축혈액의 원심분리후 얻어진 혈장에 트리클로로아세트산(trichloroacetic acid) 또는 염산 (HC1) 또는 황산암모늄염 (ammonium sulfate) 등을 첨가하여 혈장단백질을 침전시킨후 얻어진 단백질을 단백분해효소 (protease)로 가수분해하여 가수분해물로부터 엔지오텐신전환효소 저해제를 제조하는 것이다. 일반적으로 엔지오텐신전환효소 저해제는 고혈압치료제로 사용하고 있는데 엔지오텐신전환효소는 데카펩타이드인 엔지오텐신I(angiotensin I)의 C-말단 (C-term)에서부터 디펩타이드(dipeptide)인 히스티딘-류신(His-leu)를 절단함으로써 강력한 혈관수축작용을 갖는 옥타펩타이드(octapeptide)인 엔지오텐신 II(angiotensin II)로 전환시키는 작용을 하는 효소이다. 또한 엔지오텐신전환효소는 혈관이완 작용을 하는 노나펩타이드(nonapeptide)인 브레디키닌(bradykinin)을 분해하여 불활성화시킴으로써 결과적으로 혈압 상승을 유발시킨다. 따라서 엔지오텐신전환효소 저해제는 엔지오텐신전환효소의 작용을 억제시켜 혈관 수축을 막고 혈압 강화 효과를 나타낼수 있으므로 현재 각종 식품단백질 가수분해물로부터 엔지오텐신전환효소 저해 펩타이드의 탐색에 관한 연구가 진행되고 있다. 그러나 식품단백질에 비해 경제성 및 효율성 등 면에서 본 발명에서 개발한 도축혈액의 가수분해물은 많은 장점을 갖는다. 특히 도축장에서 발생하는 상당량의 도축혈액은 현재 그 이용가치가 떨어져 폐수처리에 많은 어려움이 있다. 환경오염에 관한 인식이 높아지고 폐수방류수의 수질 기준이 엄격해지는 추세에 비추어 볼때 도축혈액의 처리 비용은 높아질 수 밖에 없다. 따라서 본 건은 상수원 오염 등 환경 오염을 방지하고 폐수 처리비용을 절감하며 또한 대부분이 폐기되고 있는 도축혈액으로부터 고부가가치 신기능성 소재인 항고혈압성 펩타이드인 엔지오텐신전환효소 저해제를 분리하는 데 그 목적이 있다.The present invention relates to a method for producing an angiotensin converting enzyme inhibitor from a hydrolyzate of plasma proteins of cattle and pig slaughter blood, and more particularly, to a method for producing an inhibitor of angiotensin converting enzyme (HC1) or ammonium sulfate to precipitate a plasma protein, and then hydrolyzing the obtained protein with a protease to prepare a angiotensin converting enzyme inhibitor from the hydrolyzate. In general, the angiotensin converting enzyme inhibitor is used as a therapeutic agent for hypertension. The angiotensin converting enzyme is derived from the C-terminus of the decapeptide angiotensin I to the histidine-leucine (His) (angiotensin II), which is an octapeptide having a strong vasoconstrictor action by cleavage of angiotensin II (-leu). In addition, angiotensin converting enzyme degrades and inactivates the nonapeptide bradykinin, which causes relaxation of blood vessels, resulting in an increase in blood pressure. Therefore, the angiotensin converting enzyme inhibitor can inhibit the action of angiotensin converting enzyme to prevent vasoconstriction and enhance blood pressure. Therefore, research on the discovery of angiotensin converting enzyme inhibitory peptide from various food protein hydrolysates is underway. However, the hydrolyzate of slaughter blood, which has been developed in the present invention, has many advantages in terms of economy and efficiency compared with food protein. Especially, a large amount of slaughter blood from slaughterhouses is currently inferior in its utility value, which makes it difficult to treat wastewater. In view of the increasing awareness of environmental pollution and the increasing stringency of water quality standards for wastewater effluents, the cost of treating slaughter blood is inevitable. Therefore, the purpose of this study is to isolate angiotensin converting enzyme inhibitor, which is an antihypertensive peptide which is high value-added new functional material, from slaughter blood which is mostly used for prevention of environmental pollution such as pollution of water source, have.

소, 돼지의 도축시 발생하는 도축혈액은 도축 즉시 저장탱크를 거쳐 혈액 응고 방지 처리한 후 연속식 원심분리기를 거쳐 혈장과 혈구로 분리된다. 분리된 혈장에 최종농도가 2%에서 10% 되게 트리클로로아세트산 (trichloroacetic acid) 또는 50%에서 70%되게 황산암모늄염 또는 염산 0.6N에서 3N 처리하여 혈장단백질을 침전시켜 원심 분리하여 얻은후 상업용 단백분해효소를 첨가하여 가수분해시켜 한외여과(ultrafiltration), 겔여과크로마토그라피 (gel permeation chromatography)와 고속액체크로마토그라피(high prlessure liquid chromatography)를 이용하여 분획한후 분획물의 엔지오텐신 전환 효소 (angiotensin converting enzyme, ACE)의 역가를 기질로 히퓨릴히스티딜류신 (hippuryl-L-histidyl-L-leucine)을 사용하여 저해(inhibition) 정도를 측정한후 저해능이 높은 항고혈압성 펩타이드를 제조함에 그 주된 내용이 있다.The slaughter blood from the slaughter of cows and pigs is immediately slaughtered and stored in a storage tank to prevent blood clotting and then separated into plasma and blood cells through a continuous centrifuge. Separated plasma was treated with trichloroacetic acid or 50% to 70% ammonium nitrate or hydrochloric acid (0.6N) at a final concentration of 2% to 10%, followed by centrifugation to precipitate plasma proteins, followed by commercial protein degradation After hydrolysis by adding enzyme, fractionation was performed by ultrafiltration, gel permeation chromatography and high prismure liquid chromatography. The fractions of angiotensin converting enzyme, L-histidine-L-leucine) as a substrate to measure the inhibition of the activity of ACE (ACE) as a substrate, and then to prepare an anti-hypertensive peptide having high ability .

본 건은 활용도가 매우 낮은 폐기물의 일부인 도축혈액으로 부터 고부가가치 기능성소재로서 항고혈압성 펩타이드인 엔지오텐신 전환 효소 저해제 (ACE inhibitor)를 제조하는 방법으로서 그 의의 및 효과가 크다 할 수 있다. 본 발명을 실시예에 의하여 좀더 상세하게 설명하면 다음과 같다.This is a method for producing angiotensin converting enzyme inhibitor (ACE inhibitor) which is an antihypertensive peptide as a high-value-added functional material from slaughter blood, which is a part of waste with very low utilization, and its significance and effect can be great. The present invention will be described in more detail with reference to the following examples.

[실시예 1][Example 1]

소, 돼지의 도축시 발생하는 도축혈액은 도축 즉시 저장탱크를 거쳐 혈액 응고 방지제인 에틸렌디아민테트라아세트산 (EDTA, 2g/L)을 첨가한후 원심분리를 하여 혈장을 분리한다. 분리된 혈장에 최종농도가 4% 되게 트리클로로아세트산(trichloroacetic acid)를 처리하여 혈장단백질을 침전시켜 얻은후 혈장단백질 대비 단맥분해효소인 플라보자임(Flavozyme) 1%를 첨가하여 50℃에서 8시간 가수분해 하여 원심분리하여 얻어진 상등액을 취하여 한외 여과한후 세파덱스 (Sephadex G25,1.8X120 cm) 겔여과크로마토그라피 (gel permeation chromatography)를 이용하여 분획한후 (참조 제1도), 분획물의 엔지오텐신전환효소 (angiotensin converting enzyme, ACE)의 역가를 기질로 히퓨릴히스티딜류신(hippuryl-L-histidyl-L-leucine)을 사용하여 저해 정도가 높은 분획물을 얻었는데 이때 저해정도(IC50, ACE 저해 50%에 필요한 저해제 농도)는 F2가 32 μM이었다. 이 분획물을 다시 역상고속액체크로마토그라피(reverse-phase high pressure liquid chromatography) C8컬럼을 사용하여 (참조 제2도) 분리 정제한 결과 저해정도가 20 μM인 분획물인 엔지오텐신 전환효소 저해제를 제조했다.Slaughter blood from cattle and pigs slaughtered is slaughtered immediately after storage, added with ethylenediaminetetraacetic acid (EDTA, 2g / L), a blood coagulation inhibitor, and centrifuged to separate the plasma. After separating the plasma with 4% final concentration of trichloroacetic acid to precipitate the plasma protein, 1% of Flavozyme, a venomolytic enzyme compared with plasma protein, was added and incubated at 50 ° C for 8 hours The supernatant obtained by hydrolysis and centrifugation was taken and ultrafiltered and fractionated using Sephadex G25 and 1.8X120 cm gel permeation chromatography (see FIG. 1), and the fraction of angiotensin The activity of angiotensin converting enzyme (ACE) was determined by using hippuryl-L-histidyl-L-leucine as a substrate. The degree of inhibition (IC 50 , ACE inhibition 50% inhibitor concentration) required F2 to be 32 [mu] M. This fraction was further purified by reverse-phase high pressure liquid chromatography on a C 8 column (see FIG. 2) to obtain a fraction having a degree of inhibition of 20 μM to produce a angiotensin converting enzyme inhibitor .

Claims (4)

소, 돼지 도축혈액의 혈장에서 4% 트리클로로아세트산으로 침전시킨 후 얻어진 혈장 단백질에 상업용 단백분해효소를 사용하여 단백질가수물을 제조하여 한외여과, 겔여과크로마토그라피, 고속액체크로마토그라피의 분획물로 부터 제조된 엔지오텐신 전환 효소 저해제를 제조하는 방법Protein hydrolyzate was prepared from plasma proteins obtained by precipitation of 4% trichloroacetic acid in blood plasma of cattle and pig slaughtered blood using commercial proteolytic enzymes and then fractionated from ultrafiltration, gel filtration chromatography and high-performance liquid chromatography fractions Method for producing the prepared angiotensin converting enzyme inhibitor 제 1 항에 있어서, 혈장단백질을 2%에서 10% 범위의 트리클로로아세트산으로 침전하는 방법The method according to claim 1, wherein the plasma protein is precipitated with trichloroacetic acid ranging from 2% to 10% 제 1 항에 있어서, 혈장단백질을 50%에서 70% 범위의 황산암모늄으로 침전하는 방법The method according to claim 1, wherein the plasma protein is precipitated with ammonium sulfate ranging from 50% to 70% 제 1 항에 있어서, 혈장단백질을 0.6N에서 3N범위의 염산으로 침전하는 방법.The method according to claim 1, wherein the plasma protein is precipitated with hydrochloric acid ranging from 0.6N to 3N.
KR1019960046123A 1996-10-14 1996-10-14 Process for preparing angiotensin converting enzyme inhibitor from plasma protein hydrolysate KR100215090B1 (en)

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
KR100370272B1 (en) * 2000-08-03 2003-01-30 송경빈 Manufacture of functional drink using bovine and porcine blood plasma protein hydrolysates
KR20160051479A (en) 2014-11-03 2016-05-11 (주)퓨젠바이오농업회사법인 Blood pressure reducing composition comprising exopolysaccharide produced by ceriporia lacerata as an active ingredient
KR20200120886A (en) * 2020-05-18 2020-10-22 주식회사 삼다 Method for Preparing Fermented Porcine Blood and a Feed Composition for poultry Using the Same

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Publication number Priority date Publication date Assignee Title
KR101390516B1 (en) * 2013-02-18 2014-04-30 주식회사 케이엠에프 High quality amino acid composition using waste blood of the slaughtered livestock and manufacturing method thereof

Non-Patent Citations (1)

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Biochem. Biophy. Res. Commun.., vol.139, pp.52-55 (1986) *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100370272B1 (en) * 2000-08-03 2003-01-30 송경빈 Manufacture of functional drink using bovine and porcine blood plasma protein hydrolysates
KR20160051479A (en) 2014-11-03 2016-05-11 (주)퓨젠바이오농업회사법인 Blood pressure reducing composition comprising exopolysaccharide produced by ceriporia lacerata as an active ingredient
KR20200120886A (en) * 2020-05-18 2020-10-22 주식회사 삼다 Method for Preparing Fermented Porcine Blood and a Feed Composition for poultry Using the Same
KR102187522B1 (en) 2020-05-18 2020-12-08 주식회사 삼다 Method for Preparing Fermented Porcine Blood and a Feed Composition for poultry Using the Same

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