Lipoxygenase 결여 콩 계통의 나물 특성 및 Lipoxygenase 활성

이형일(Heung Il Lee),김광철(Kwang Chul Kim),박의호(Eui-Ho Park) 한국작물학회 2005 한국작물학회지 Vol.50 No.S

본 연구는 lipoxygenase 결여 품종인 진품콩2호와 lipoxygenase가 존재하는 광안콩, 소백나물콩, 푸른콩의 교배조합 내의 각 계통의 lipoxygenase 유무를 확인하고 activity 차이를 규명함으로써 lipoxygenase 결여 나물콩 육성의 가능성을 알아보고자 수행되었다. 콩나물 재배일수별 lipoxygenase 여부, 재배일수별 lipoxygenase 활성도 등을 분석한 결과를 요약하면 다음과 같다. 1. 콩나물 발아율은 대비품종과 계통들간의 큰 차이는 없었지만 정상 발아 개체율 에서는 계통이 63%로 낮았으며 T??은 1일로 소백나물콩, 푸른콩(0.6일)보다는 느렸으나 진품콩2호, 광안콩보다는 빨랐다. 2 콩나물 생육은 치상 후 2일부터 콩나물 전체길이, 배축 길이, 배축 직경을 조사한 결과 진품콩2호와 푸른콩 교배 조합 계통의 4537계통(콩나물 전체길이13.9㎝, 배축 길이: 7.3㎝, 배축 직경 : 2.2 ㎜)이 대비품종들보다 높은 신장성을 보였다. 3. 콩나물의 물성은 치상 후 96시간 후에 hardness, cutting force, masticcation을 측정한 결과 lipoxygenase 결여 계통이 대비품종들보다 높게 측정되었다. 4. 콩나물 재배 일수 별 lipoxygenase band는 자엽부에서는 종실과 같이 lipoxygenase band를 확인 할 수 없었으며 배축부에서 모든 계통과 대비품종에서 확인 할 수 있었다. 5. 콩나물 재배 일수별 lipoxygenase 활성도는 2일차에 최고로 높았으며, 3일차에 최저치로 낮아진 후 다시 증가하는 양상을 나타내었다. 6. 각 교배 조합 내에서는 진품콩2호보다 활성도가 낮은 4510, 4522, 4537계통들이 lipoxygenase 결여 통나물로서의 육종 가능성이 높을 것으로 생각되었다. This study was conducted to see the feasibility of breeding for sprout soybean cultivar with minimum beany flavor using lipoxygenase-less lines. Lipoxygenase-less cultivar Jinpumkong2 was crossed by lipoxygenase containing Gwangankong, Sobaeknamulkong, and Pureunkong as paternal parent and 24 lipoxygenase-less lines derived from those 3 combinations were selected and those lines were evaluated with their parental cultivars. Germination rate showed no difference between lipoxygenase-less lines and their parental cultivars, however, rates of normal sprout of those and Jinpumkong2 were 63 and 56%, and were lower than that of paternal parents. Hypocotyl length of those was same as Jinpumkong2, however, shorter than paternal parents. Texture characteristics including hardness, cutting force and mastication of 96 hour-cultured sprout of lipoxygenase-less lines showed higher value than that of their parental cultivars. Lipoxtgenase isozyme was not detected in the sprout cotyledon of lipoxygenase-less lines, however it was observed in the sprout hypocotyl of all the used genotypes. Though lipoxygenase activity in the seed of lipoxygenase-less lines was lower than that of Jinpumkong2(0.477, △A 234 nm min-l ㎎ meal-1), 2 lines revealed more than 0.5 value. Lipoxygenase-activity of 2 day-cultured sprout(both cotyledon and hypocotyl) was the highest, decreased in 3 days after culture and re-increased thereafter, Several lipoxygenase-less lines with lower lipoxygenase activity of sprout than Jinpumkong2 were selected and this suggested the possibility of breeding lines for soy-sprout with low beany flavor.

Lipoxygenase 결여 콩 계통의 나물 특성 및 Lipoxygenase 활성

이형일,김광철,박의호,Lee Heung Il,Kim Kwang Chul,Park Eui-Ho 한국작물학회 2005 한국작물학회지 Vol.50 No.suppl1

본 연구는 lipoxygenase 결여 품종인 진품콩2호와 lipoxygenase 가 존재하는 광안콩, 소백나물콩, 푸른콩의 교배조합 내의 각 계통의 lipoxygenase 유무를 확인하고 activity 차이를 규명함으로써 lipoxygenase 결여 나물콩 육성의 가능성을 알아보고자 수행되었다. 콩나물 재배일수별 lipoxygenase 여부, 재배일수별 lipoxygenase 활성도 등을 분석한 결과를 요약하면 다음과 같다. 1. 콩나물 발아율은 대비품종과 계통들간의 큰 차이는 없었지만 정상 발아 개체율 에서는 계통이 $63\%$로 낮았으며 $T_{50}$은 1일로 소백나물콩, 푸른콩(0.6일)보다는 느렸으나 진품콩2호, 광안콩보다는 빨랐다. 2. 콩나물 생육은 치상 후 2일부터 콩나물 전체길이, 배축길이, 배축 직경을 조사한 결과 진품콩2호와 푸른콩 교배 조합 계통의 4537계통(콩나물 전체길이 13.9cm, 배축 길이: 7.3cm, 배축 직경: 2.2mm)이 대비품종들보다 높은 신장성을 보였다. 3. 콩나물의 물성은 치상 후 96시간 후에 hardness, cutting force, masticcation을 측정한 결과 lipoxygenase 결여 계통이 대비품종들보다 높게 측정되었다. 4. 콩나물 재배 일수 별 lipoxygenase band는 자엽부에서는 종실과 같이 lipoxygenase band를 확인 할 수 없었으며 배축부에서 모든 계통과 대비품종에서 확인 할 수 있었다. 5. 콩나물 재배 일수별 lipoxygenase 활성도는 2일차에 최고로 높았으며, 3일차에 최저치로 낮아진 후 다시 증가하는 양상을 나타내었다. 6. 각 교배 조합 내에서는 진품콩2호보다 활성도가 낮은 4510, 4522, 4537계통들이 lipoxygenase 결여 콩나물로서의 육종 가능성이 높을 것으로 생각되었다. This study was conducted to see the feasibility of breeding for sprout soybean cultivar with minimum benny flavor using lipoxygenase-less lines. Lipoxygenase-less cultivar Jinpumkong2 was crossed by lipoxygenase containing Gwangankong, Sebaeknamulkong, and Pureunkong as paternal parent and 24 lipoxygenase-less lines derived from those 3 combinations were selected and those lines were evaluated with their parental cultivars. Germination rate showed no difference between lipoxygenase-less lines and their parental cultivars, however, rates of normal sprout of those and Jinpumkong2 were 63 and $56\%$, and were lower than that of paternal parents. Hypocotyl length of those was same as Jinpumkong2, however, shorter than paternal parents. Texture characteristics including hardness, cutting force and mastication of 96 hour-cultured sprout of lipoxygenase-less lines showed higher value than that of their parental cultivars. Lipoxtgenase isozyme was not detected in the sprout cotyledon of lipoxygenase-less lines, however it was observed in the sprout hypocotyl of all the used genotypes. Though lipoxygenase activity in the seed of lipoxygenase-less lines was lower than that of Jinpumkong2(0.477, ${Delta}A$ 234 nm min-1 mg meal-1),2 lines revealed more than 0.5 value. Lipoxygenase-activity of 2 day-cultured sprout(both cotyledon and hypocotyl) was the highest, decreased in 3 days after culture and re-increased thereafter. Several lipoxygenase-less lines with lower lipoxygenase activity of sprout than Jinpumkong2 were selected and this suggested the possibility of breeding lines for soy-sprout with low benny flavor.

콩 종실의 Lipoxygenase 활성이 발아특성에 미치는 영향

이석하 서울대학교 농업개발연구소 1999 농업생명과학연구 Vol.3 No.-

Three lipoxygenase isozymes in soybean [Glycine max (L.) Merr.] seeds are thought to be a major contributor to lipid peroxidation and the generation of free radicals which may result in seed deterioration. This study was conducted to understand the relationship between seed lipoxygenase activity and germination and seed vigor in soybean. Two cultivars, Jinpumkong lacking lipoxygenase-2, 3 and Jinpumkong 2 lacking lipoxygenase-1, 2, 3 were evaluated for major traits of seed and germination, and were compared with the normal soybean Taekwangkong containing lipoxygenase-1, 2, 3 isozymes in seed. Seed protein and lipid contents of Jinpumkong and Jinpumkong 2 were similar to those of Taekwangkong. Jinpumkong and Jinpumkong 2 had more palmitic acid, linoleic acid, and linolenic acid in soybean seed than Taekwangkong, but had less stearic acid and oleic acid than Taekwangkong. Type I lipoxygenase activity (pH 9.0) of Jinpumkong lacking lipoxygenase-2, 3 was higher than that of normal Taekwangkong. Germination percentages of Jinpumkong and Jinpumkong 2 were lower than that of Taekwangkong. Electric conductivity was not significantly different in all cultivars. Using two soybean populations derived from the cross between normal Taekwangkong (Lx1Lx2Lx3) and Jinpumkong 2 (lx1lx2lx3) lacking lipoxygenase-1, 2, 3, and between normal Pureunkong (Lx1Lx2Lx3) and Jinpumkong 2 (lx1lx2lx3) lacking lipoxygenase-1, 2, 3, the effects of presence or absence of seed lipoxygenase activity on germination characteristics were evaluated. The F2 derived from the cross between normal Taekwangkong(Lx1Lx2Lx3) and Jinpumkong 2 (lx1lx2lx3) lacking lipoxygenase-1, 2, 3 fitted to a expected segregation ratio of 9 : 3 : 3 : 1 (normal : lacking L-3 : lacking L-1 and L-2 : lacking L-1, L-2 and L-3), suggesting the tight linkage between the lx1 and lx2 loci, and F8 seeds derived from the cross between normal Pureunkong (Lx1Lx2Lx3) and Jinpumkong 2 (lx1lx2lx3) lacking lipoxygenase-1, 2, 3 fitted to a expected segregation ratio of 1 : 1(L-1, L-2 : l-1, l-2 and L-3 : l-3, respectively). Germination percentages showed wide ranges but didn't differ among lipoxygenase isozyme types of F3 and F8 seeds derived from the cross between normal Taekwangkong (Lx1Lx2Lx3) and Jinpumkong 2 (lx1lx2lx3) lacking lipoxygenase-1, 2, 3, and between normal Pureunkong(Lx1Lx2Lx3) and Jinpumkong 2 (lx1lx2lx3) lacking lipoxygenase-1, 2, 3. After accelerated aging, germination percentages showed wide ranges but didn't differ among lipoxygenase isozyme types of F3 and F8 seeds derived from the cross between normal Taekwangkong (Lx1Lx2Lx3) and Jinpumkong 2 (lx1lx2lx3) lacking lipoxygenase-1, 2, 3, and between normal Pureunkong (Lx1Lx2Lx3) and Jinpumkong 2 (lx1lx2lx3) lacking lipoxygenase-1, 2, 3. Presence or absence of lipoxygenase activity showed no effect on germination percentage.

Lipoxygenase 및 Kunitz Trypsin Inhibitor 단백질 결핍 콩 계통 육성

김명식 ( Myung Sik Kim ),성미경 ( Mi Kyung Sung ),서상배 ( Sang Bae Seo ),김경록 ( Kyung Roc Kim ),이경자 ( Kyoung Ja Lee ),박모세 ( Mo Se Park ),정종일 ( Jong Il Chung ) 한국콩연구회 2008 韓國콩硏究會誌 Vol.25 No.1

Lipoxygenase and Kunitz trypsin inhibitor proteins in mature soybean seed are well known as major anti-nutritional factors. Lipoxygenase is responsible for the beany flavor and soybean Kunitz trypsin inhibitor (KTI) protein is responsible for the inferior nutritional quality of unheated or incompletely heated soybean meal. Anthocyanins from black soybean seed coat are known to have many pharmaceutical effects. The objective of this study was to breed soybean genotype of black coated-seed lacking lipoxygenase-2,3 and KTI protein and to breed soybean genotype of yellow coated-seed lacking lipoxygenase-2,3 and KTI protein. Two breeding populations were developed using four different parents. In F2 seed generations of both populations, F2 seeds deficient in lipoxygenase-2,3 and KTI protein were selected by SDS-PAGE. Selected F2 seeds were planted to advance F2 plant. Traits for seed coat and mature embryo colors were observed on the F3 seeds harvested from each F2 plant. F3 lines were advanced from F3 seeds selected. Plant type, height, and maturity date for each F3 plant within line were observed. After single plant harvesting, seed color and absence of lipoxygenase-2,3 and KTI protein were confirmed on random F4 seed of each F3 plant. Selected lines were advanced to the F6 generation. Maturity date, stem type, seed coat and cotyledon color, seed weight were observed on the new soybean lines with absence of lipoxygenase-2,3 and KTI protein.

Lipoxygenase, Kunitz Trypsin inhibitor, 7S α′-subunit 단백질이 결핍된 콩 계통의 선발

성미경,김경록,박정수,정종일,한은희,남진우 경상대학교 농업생명과학연구원 2010 농업생명과학연구 Vol.44 No.5

Soybean [Glycine max (L.) Merr.] seed is the main source of protein and oil for human and animal. The use of soybean protein has been expanded in the food industry due to their excellent nutritional benefits. But, antinutritional and allergenic factors are exist in the raw mature soybean. Lipoxygenase, Kunitz trypsin inhibitor (KTI) protein, and α′-subunit of 7S globulin are main antinutritional factors in soybean seed. Breeding of a new soybean strain with lacking these components is needed. The objective of this research was to select new soybean line with lipoxygenase-free, KTI-free, and α′-subunit free (lx1lx1lx2lx2lx3lx3titicgy1cgy1 genotype). Total 434 F2 seeds were obtained from the cross of cultivar, "Gaechuck#2" and PI506876. Presence and absence of lipoxygenase, KTI protein, and α′-subunit of 7S globulin was tested by SDS electrophoresis using a partial seed of each F2 seed. Only one F2 seed with lacking these three components was selected and was planted to F2 plant. Absence of lipoxygenase, KTI, and α′-subunit protein was confirmed on the F3 seeds harvested. Selected line with lx1lx1lx2lx2lx3lx3titicgy1cgy1 genotype might be useful for soybean breeding. 콩은 식물성 단백질 및 지방의 주요 공급원이고 콩 종실에는 기능성 성분이 많이 함유되어져 있어 소비가 점차 증가하고 있지만 Lipoxygenase, Kunitz Trypsin inhibitor, 7S α′-subunit 단백질과 같은 성분들이 존재하는데 이는 품질과 영양가치를 떨어뜨리고 섭취시 알러지를 일으키기도 한다. 콩에서 유전적으로 이러한 성분이 결핍되어져 있는 유전자형의 선발은 품질이 우수한 콩 육종의 기초단계이다. “개척2호”와 PI506876의 교배로부터 434개의 F2 종자를 얻어 F2 종자의 일부를 사용하여 SDS-PAGE로 각각의 종자를 분석한 결과 Lipoxygenase와 Kunitz Trypsin inhibitor 및 7S의 α′-subunit 단백질이 모두 결핍되어져 있는 lx1lx1lx2lx2lx3lx3titicgy1cgy1 유전자형을 가진 종자를 선발하여 F2 식물체로 길러 성숙 후 F3 종자를 수확하였다. F3 종자로부터 Lipoxygenase, Kunitz Trypsin inhibitor, 7S α′-subunit 단백질이 모두 결핍되어져 있음을 재확인하였으며 선발된 종자는 고품질 콩 품종 육성에 유용하게 활용될 것으로 기대된다.

산업적 응용을 위한 Lipoxygenase 생산 세균의 분리 및 특성

김예린,박규림,김예담,이오미,손홍주 한국환경과학회 2022 한국환경과학회지 Vol.31 No.3

Lipoxygenase is an enzyme, mainly produced by plants, capable of converting unsaturated fatty acids to fatty acids. It has vast application potential in the food, pharmaceutical and agricultural industries. The aim of this study was to isolate novel lipoxygenase-producing bacteria from the environment and to investigate the lipoxygenase enzymatic properties for industrial production. The strain, NC1, isolated from cultivation soils, was identified as Bacillus subtilis based on the phenotypic characteristics and 16S rRNA gene sequencing. This strain formed a pink color around the colony when cultured on indamine dye formation plates. The production of lipoxygenase by B. subtilis NC1 was influenced by the composition of the medium and linoleic acid concentrations. The optimum temperature and pH for lipoxygenase activity was determined to be 40 °C and pH 6, respectively. The enzyme showed relatively high stability at temperatures ranging from 20–50 °C and acid-neutral regions. In addition, the lipoxygenase produced by B. subtilis NC1 was able to degrade commercially available oils including sunflower seed oil and Perilla oil. In this study, a useful indigenous bacterium was isolated, and the fundamental physicochemical data of bacterial lipoxygenase giving it industrial potential are presented.

Chemical Modification of 5-Lipoxygenase from the Korean Red Potato

Kim,Kyoung-Ja The Korea Science and Technology Center 2000 BMB Reports Vol.33 No.2

The lipoxygenase was purified 35fold to homogeneity from the Korean red potato by an ammonium sulfate precipitation and DEAE-cellulose column chromatography. The simple purification method is useful for the preparation of pure lipoxygenase. The molecular weight of the enzyme was estimated to be 38,000 by SDS-polyacrylamide gel electrophoresis and Sepharose 6B column chromatography. The purified enzyme with 2 M (NH₄)₂SO₄ in a potassium phosphate buffer, pH 7.0, was very stable for 5 months at-20。C. Because the purified lipoxygenase is very stable, it could be useful for the screening of a lipoxygenase inhibitor. The optimal pH and temperature for lipoxygenases purified from the red potato were found to be pH 9.0. and 30。C, respectively. The Km and Vmax values for linoleic acid of the lipoxygenase purified from the red potato were 48μM and 0.03 μM per minute per milligram of protein, respectively. The enzyme was insensitive to the metal chelating agents tested (2 mM KCN, 1 and 10 mM EDTA, and 1 mM NaN₃), but was inhibited by several divalent cations, such as Cu??, Co?? and Ni??. The essential amino acids that were involved in the catalytic mechanism of the 5-lipoxygenase from the Korean red potato were determined by chemical modification studies. The catalytic activity of lipoxygenase from the red potato was seriously reduced after treatment with a diethylpyrocarbonate (DEPC) modifying histidine residue and Woodward’s reagent (WRK) modifying aspartic/glutamic acid. The inactivation reaction of DEPC (WRK) proceeded in the form of pseudo-first-order kinetics. The double-logarithmic plot of the observed pseudo-first-order rate constant against the modifier concentration yielded a reaction order 2, indicating that two histidine residues (carboxylic acids) were essential for the lipoxygenase activity from the red potato. The linoleic acid protected the enzyme against inactivation by DEPC(WRK), revealing that histidine and carboxylic amino acids residues were present at the substrate binding site of the enzyme molecules.

Chemical Modification of 5-Lipoxygenase from the Korean Red Potato

Kim, Kyoung-Ja 생화학분자생물학회 2000 Journal of biochemistry and molecular biology Vol.33 No.2

The lipoxygenase was purified 35 fold to homogeneity from the Korean red potato by an ammonium sulfate precipitation and DEAE-cellulose column chromatography. The simple purification method is useful for the preparation of pure lipoxygenase. The molecular weight of the enzyme was estimated to be 38,000 by SDS-polyacrylamide gel electrophoreses and Sepharose 6B column chromatography. The purified enzyme with 2 M $(NH_4)_2SO_4$ in a potassium phosphate buffer, pH 7.0, was very stable for 5 months at $-20^{\circ}C$. Because the purified lipoxygenase is very stable, it could be useful for the screening of a lipoxygenase inhibitor. The optimal pH and temperature for lipoxygenase purified from the red potato were found to be pH 9.0. and $30^{\circ}C$, respectively. The Km and Vmax values for linoleic acid of the lipoxygenase purified from the red potato were $48\;{\mu}M$ and $0.03\;{\mu}M$ per minute per milligram of protein, respectively. The enzyme was insensitive to the metal chelating agents tested (2 mM KCN, 1 and 10mM EDTA, and 1 mM $NaN_3$), but was inhibited by several divalent cations, such as $Cu^{++}$, $Co^{++}$ and $Ni^{++}$. The essential amino acids that were involved in the catalytic mechanism of the 5-lipoxygenase from the Korean red potato were determined by chemical modification studies. The catalytic activity of lipoxygenase from the red potato was seriously reduced after treatment with a diethylpyrocarbonate (DEPC) modifying histidine residue and Woodward's reagent (WRK) modifying aspartic/glutamic acid. The inactivation reaction of DEPC (WRK) processed in the form of pseudo-first-order kinetics. The double-logarithmic plot of the observed pseudo-first-order rate constant against the modifier concentration yielded a reaction order 2, indicating that two histidine residues (carboxylic acids) were essential for the lipoxygenase activity from the red potato. The linoleic acid protected the enzyme against inactivation by DEPC(WRK), revealing that histidine and carboxylic amino acids residues were present at the substrate binding site of the enzyme molecules.

Relationship of Seed Germination and Lipoxygenase Activity in Soybean

Lee, Suk-Ha,Son, Beom-Young,Lee, Yeong-Ho,Lee, Hong-Suk The Korean Society of Crop Science 2002 한국작물학회지 Vol.47 No.2

Lipoxygenase might be associated with seed deterioration by catalyzing the incorporation of molecular oxygen into fatty acids and generating free radicals. This study was performed to determine whether seed lipoxygenase activity would alter soybean seed longevity. In this study, germination percentage of lipoxygenase-lacking cultivar Jinpumkong2 (lx1lx1lx2lx2lx3lx3) was lower than that of Taekwangkong (Lx1Lx1Lx2Lx2Lx3Lx3). Segregation ratio for the three lipoxygenase isozymes of the F2-derived from the cross between Taekwangkong and Jinpumkong2 was fitted to 9 (Lx1Lx2Lx3) : 3 (Lx1Lx2lx3) : 3 (lxllx2Lx3) : 1 (lx1lx2lx3), suggesting the tight linkage between the Lx1 and Lx2 loci. Germination percentages varied widely but not differed among lipoxygenase isozyme types of F$_3$ seeds before and after accelerated aging. Seed coat of Jinpumkong2 was damaged severely following accelerated aging, whereas that of Taekwangkong was not. Thus, seed of lipoxygenase-lacking soybean cultivar, Jinpumkong2 showed greater deterioration compared with that of the normal Taekwangkong. However, the presence or absence of lipoxygenase activity had no effect on soybean germination.

Development and Utilization of KASP Markers Targeting the Lipoxygenase Gene in Soybean

신서영,강세희,강병희,Sreeparna Chowdhury,이원호,이정동,이성우,최유미,하보근 한국작물학회 2023 한국작물학회지 Vol.68 No.4

Lipoxygenase gives soybeans their grassy flavor, which can disrupt food processing efficiency. This study aimed to identify soybean genotypes with lipoxygenase deficiency among 1,001 soybean accessions and to develop kompetitive allele specific PCR (KASP) markers that can detect lipoxygenase mutations. Three lipoxygenase isozymes (Lox1, Lox2, and Lox3) were analyzed using a colorimetric assay based on a substrate-enzyme reaction. Among the 1,001 accessions examined, two (IT160160 and IT276392) exhibited a deficiency solely in Lox1, and one (IT269984) lacked both Lox1 and Lox2. IT160160 had a 74-bp deletion in exon 8 of Lox1 (Glyma13g347600), whereas IT276392 displayed a missense mutation involving the change of C to A at position 2,880 of Lox1. Moreover, we successfully developed four KASP markers that specifically target Lox1, Lox2, and Lox3 mutations. To validate the Lox1 KASP markers, we used two F2:3 populations generated through a cross between Daepung 2 (lipoxygenase wild type, maternal parent), IT160160, and IT276392 (null Lox1, paternal parent). The results revealed that the Daepung 2 × IT160160 group followed the expected 3:1 ratio according to Mendel’s law, whereas the Daepung 2 × IT276392 group did not. Furthermore, a comparison between the colorimetric and KASP marker analyses results revealed a high agreement rate of 96%. KASP markers offer a distinct advantage by allowing the distinction of heterozygous types independent of other variables. As a result, we present an opportunity to expedite the lipoxygenase-deficient cultivar development.