• Journal of Pharmaceutical Investigation
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• v.30 no.3
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• pp.159-166
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• 2000

Kunitz-type soybean trypsin inhibitor (SBTI) and chondroitin sulfate (A, and C type) were conjugated using sodium periodate method. And the physicochemical, pharmacokinetic properties and immunogenecity of the conjugates (Chon-A-SBTI or Chon-C-SBTI) were characterized. We expected the conjugation using chondroitin sulfate to reduce the immunogenecity and to improve the pharmacological effect. As the results, the mean molecular weight of the conjugate highly increased. After I.V. injection of the radiolabeled conjugates or native SBTI into mice, it was found that native SBTI showed rapid elimination from plasma, whereas Chon-A-SBTI and Chon-C-SBTI were slowly eliminated. Organ distribution of the two agents at 30 min after I.V. injection was different : Chon-A-SBTI or Chon-C-SBTI accumulated to a large extent in the liver (13% in Chon-A-SBTI and 16% in Chon-C-SBTI), whereas native SBTI was taken up more rapidly by the kidney (107% dose/g of tissue) and excreated into the urine (26%). In addition we evaluated the therapeutic value of the conjugates by using the sublethal septic shock model caused by pseudomonal elastase and tested the immunogenecity by passive cutaneous anaphylaxis shock (PCA). The conjugates were more effective than native SBTI against pseudomonal elastase induced septic shock in guinea pig. In case of the conjugates, the pharmacological and therapeutic effect lasted over 3 hours long. In immunogenecity test, both of the conjugates showed the reduction of their immunogenecity, especially Chon-A-SBTI looked most effective.

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.8
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• pp.1251-1257
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• 1999

Two experiments were conducted to determine the effects of roasting and extrusion on nutritional value of conventional and low-inhibitor soy beans for nurser-age pigs. In Exp. 1, 100 weaning pigs (7.5 kg average initial BW) were used in a 35-d growth assay to determine the effects of processing method (roasting in a Rast-A-Tron$^{TM}$ raster vs extrusion in an Insta-Pro$^{TM}$ extruder) on the nutritional value of Williams 82 soybeans with (+K) and without (-K) gene expression for the Kunitz trypsin inhibitor. Treatments were 48% soybean meal with added soybean oil, +K roasted, +K extruded, -K roasted and -K extruded. All diets were formulated to contain 3.5 Mcal DE/kg, with 0.92% lysine for d 0 to 14 and 0.76% lysine for d 14 to 35 of the experiment. The lysine concentrations were 80% of NRC (1988) recommendations to accentuate difference in response to protein quality and lysine availability. For d 0 to 14, pigs fed extruded soybeans (+K and -K) had greater ADG (p<0.001), ADFI (p<0.09) and gain/feed (p<0.01) than pigs fed roasted soybeans. For d 14 to 35 and overall, the same effects were noted, i.e., pigs fed extruded soybeans had greater ADG, ADFI and gain/feed than pigs fed roasted soybeans (p<0.03). Also, pigs fed -K soybeans were more efficient (p<0.008) than pigs fed +K soybeans. In Exp. 2, 150 weanling pigs (7.0 kg average initial BW) were used in a 35-d growth assay. All diets were formulated to contain 3.5 Mcal DE/kg, with 1.25% lysine for d 0 to 14 and 1.10% lysine for d 14 to 35 of the experiment. The lysine concentrations were formulated to be in excess of NRC recommendation to determine if differences in nutritional value of the soybean preparations could be detected in protein-adequate diets. For d 0 to 14 (p<0.06), 14 to 35 (p<0.03) and 0 to 35 (p<0.02), pigs fed extruded soybeans had greater ADG and gain/feed than pigs fed roasted soybeans. Apparent digestibilities of DM, N and GE were greater for diets with extruded soybeans than diets with roasted soybeans and diets with soybean meal and soybean oil were intermediate. The response to extrusion processing was greater with -K than +K soybeans, with pigs fed extruded -K soybeans having the greatest growth performance and nutrient digestibilities and lowest skin-fold thickness of any treatment. In conclusion, extrusion yielded a full-fat soy product of greater nutritional value than roasting. Also, selection against genetic expression of the Kunitz trypsin inhibitor improved nutritional value of the resulting soybean preparations.

• Journal of Plant Biotechnology
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• v.47 no.2
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• pp.118-123
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• 2020

The black seed coat of soybeans contain anthocyanins which promote health. However, mature soybean seeds contain anti-nutritional factors like lipoxygenase, lectin and Kunitz Trypsin Inhibitor (KTI) proteins. Furthermore, these seeds can be used only after the genetic elimination of these proteins. Therefore, the objective of this study was to develop novel soybean genotypes with black seed coat and triple recessive alleles (lx1lx1lx2lx2lx3lx3, titilele) for lipoxygenase, lectin, and KTI proteins. From a cross of parent1 (lx1lx2lx3/lx1lx2lx3, ti/ti, Le/Le) and parent2 (lx1lx2lx3/lx1lx2lx3, Ti/Ti, le/le), 132 F₂ seeds were obtained. A 3:1 segregation ratio was observed during F₂ seed generation for the inheritance of lectin and KTI proteins. Between a cross of the Le and Ti genes, the observed independent inheritance ratio in the F₂ seed generation was 9: 3 : 3 : 1 (69 Le_Ti_: 32 leleTi_: 22 Le_titi: 9 leletiti) (χ²=2.87, P=0.5 - 0.1). From nine F₂ seeds with triple recessive alleles (lx1lx1lx2lx2lx3lx3, titilele genotype), one novel strain posessing black seed coat, and free of lipoxygenase, lectin and KTI proteins, was selected. The seed coat color of the new strain was black and the cotyledon color of the mature seed was green. The weight of 100 seeds belonging to the new strain was 35.4 g. This black soybean strain with lx1lx1lx2lx2lx3lx3, titilele genotype is a novel strain free of lipoxygenase, lectin, and KTI proteins.

• Korean Journal of Breeding Science
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• v.42 no.1
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• pp.35-39
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• 2010

Soybean proteins are widely used for human and animal feed in the world. ${\beta}$-conglycinin protein exhibits poor nutritional and food processing properties and Kunitz trypsin inhibitor (KTI) protein is a main anti-nutritional factor in soybean seed. The objective of this research was to identify the inheritance of $cgy_1$ gene and ti gene for the improvement of soybean cultivar with no KTI proteins and low amount of ${\beta}$-conglycinin. $F_2$ population was made by crossing between "Gaechuck2ho" (${\alpha}^{\prime}$-subunit present $Cgy_1Cgy_1$, KTI protein absent titi) and PI506876 (${\alpha}^{\prime}$-subunit absent $cgy_1cgy_1$, KTI protein present TiTi) parent. A total of 434 $F_2$ seeds were obtained and analyzed for the segregation of ${\alpha}^{\prime}$-subunit protein and KTI protein using SDS-PAGE. The segregation ratio of 3 : 1 for $Cgy_1$ locus (310 $Cgy_1$_ : 124 $cgy_1cgy_1$) and Ti locus (339 Ti_ : 95 titi) were observed. Segregation ratios of 9 : 3 : 3 : 1 (241 $Cgy_1$_Ti_: 69 $Cgy_1$_titi: 98 $cgy_1cgy_1$Ti_: 26 $cgy_1cgy_1titi$) between $Cgy_1$ gene and Ti gene in $F_2$ seeds were also observed (${\chi}^2= 5.367$, P = 0.10 - 0.20). This data showed that $Cgy_1$ gene was inherited independently with the Ti gene in soybean. These results will be useful in breeding program for selecting the line that does not exhibit or lacks both ${\alpha}^{\prime}$-subunit protein and KTI protein in soybean.

• Journal of Plant Biotechnology
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• v.45 no.4
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• pp.328-332
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• 2018

Soybean [Glycine max (L.) Merr.] seed is an important dietary source of protein, oil, carbohydrate, isoflavone and other various nutrients for humans and animals. However, there are anti-nutritional factors in the raw mature soybeans. Kunitz trypsin inhibitor (KTI) protein and stachyose are the main anti-nutritional factors in soybean seed. The ${\alpha}^{\prime}$-subunit of ${\beta}$-conglycinin protein exhibit poor nutritional and food processing properties. The genetic removal of the KTI and ${\alpha}^{\prime}$-subunit proteins will improve the nutritional value of the soybean seed. The objective of this research was to develop a new soybean strain with KTI and ${\alpha}^{\prime}$-subunit protein free ($titicgy_1cgy_1$ genotype) and proper agronomic traits. A breeding population was developed from the cross of the Bl-1 and 15G1 parents. A total of 168 $F_2$ seeds from the cross of the BL-1 and 15G1 parents were obtained. The segregation ratios of 9: 3: 3: 1 ($104Ti\_Cgy_{1\_}:\;30Ti\_cgy_1cgy_1:\;21cgy_1cgy_1Ti\_:\;13titicgy_1cgy_1$) between the Ti and $Cgy_1$ genes in the $F_2$ seeds were observed (${\chi}^2=5.12$, P=0.5-0.10). Two $F_4$ plant strains with proper agronomical traits and $titicgy_1cgy_1$ genotype (free of both KTI and ${\alpha}^{\prime}$-subunit protein) were selected and harvested. 2 strains (S1 and S2) had yellow seed coats and hilum. The plant height of the S1 strain was 65 centimeters. The 100-seed weight was 29.2 g. The plant height of the S2 strain was 66 centimeters and 100-seed weight was 26.2 g. The two strains selected in this research will be used to improve the new cultivar that will be free of the KTI and ${\alpha}^{\prime}$-subunit proteins.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.57 no.1
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• pp.78-82
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• 2012

Soybean [$Glycine$ $max$ (L.) Merr.] protein is a high quality source for food and feed. But, antinutritional factors in the raw mature soybean are exist. Kunitz trypsin inhibitor (KTI) protein is a main antinutritional factor in soybean seed. Also, P34 protein, referred as $Gly$ $m$ Bd 30K, has been identified as a predominant immunodominant allergen. Genetic relationship between KTI protein and P34 protein could be useful in soybean breeding program for the genetic elimination or reduction of these factors. The objective of this study was to determine the independent inheritance or linkage between KTI protein and P34 protein in soybean seed. A total of 479 $F_2$ seeds were obtained from the cross of 07B1 and PI567476 parents. KTI protein and relative amount of P34 protein were analysed from $F_2$ seeds harvested from the F1 plants by using SDS-PAGE and Western blot analysis. The segregation ratios of 3 : 1 for KTI protein (353 KTI protein present : 126 KTI protein absent) and relative amount of P34 protein (363 normal amount of P34 protein : 116 low amount of P34 protein). The segregation ratio of 3 : 1 suggested that KTI protein and relative amount of P34 protein in mature soybean seed were controlled by a single major gene. The segregation ratios of 9 : 3 : 3 : 1 (266 KTI protein present, normal amount of P34 protein: 88 KTI protein present, low amount of P34 protein: 102 KTI protein absent, normal amount of P34 protein: 23 KTI protein absent, low amount of P34 protein) and Chi-square value (${\chi}^2$=3.31, P=0.346) were observed in $F_2$ seeds. This data showed that KTI protein was inherited independently with relative amount of P34 protein in soybean. These results will be helpful in breeding program for selecting the line with lacking KTI protein and reduced amount of P34 protein in soybean.

• Journal of Life Science
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• v.29 no.2
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• pp.147-151
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• 2019

Soybean [Glycine max (L.) Merr.] is grown worldwide for its high protein and oil content. Anthocyanins from black soybean seed coats are known to have many pharmaceutical effects. Soybean cultivars with large seed sizes and black seed coats are needed by soybean farmers. However, antinutritional factors, like protein, stachyose, and Kunitz trypsin inhibitor (KTI) exist in raw mature soybeans. Genetic elimination or reduction of these components is needed in soybean breeding. The objective of this research was to develop new a soybean strain with black seed coats and green cotyledons that was KTI protein free and low in stachyose. Six parents were used. The presence or absence of KTI protein was detected using the Western blot technique. The content of stachyose in mature seeds was detected using HPLC. One new strain was selected from 11 $F_2$ plants with black seed coats and green cotyledons that lacked KTI protein. The new strain had black seed coats and green cotyledons and was KTI protein free and low in stachyose. The plant height of the new strain was 66 cm, and its 100-seed weight was 28.4 g. The stachyose content of the new strain was 2.59 g/kg. The new strain developed in this research will be used to develop new cultivars that are KTI protein free and low in stachyose.

• BMB Reports
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• v.40 no.4
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• pp.604-609
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• 2007

An Asp/His catalytic site of 10-formyltetrahydrofolate dehydrogenase (FDH) was suggested to have a similar catalytic topology with the Asp/His catalytic site of serine proteases. Many studies supported the hypothesis that serine protease inhibitors can bind and modulate the activity of serine proteases by binding to the catalytic site of serine proteases. To explore the possibility that soybean trypsin inhibitor (SBTI) can recognize catalytic sites of FDH and can make a stable complex, we carried out an SBTI-affinity column by using rat liver homogenate. Surprisingly, the Rat FDH molecule with two typical liver proteins, carbamoyl-phosphate synthetase 1 (CPS1) and betaine homocysteine S-methyltransferase (BHMT) were co-purified to homogeneity on SBTI-coupled Sepharose and Sephacryl S-200 followed by Superdex 200 FPLC columns. These three liver-specific proteins make a protein complex with 300 kDa molecular mass on the gel-filtration column chromatography in vitro. Immuno-precipitation experiments by using anti-FDH and anti-SBTI antibodies also supported the fact that FDH binds to SBTI in vitro and in vivo. These results demonstrate that the catalytic site of rat FDH has a similar structure with those of serine proteases. Also, the SBTI-affinity column will be useful for the purification of rat liver proteins such as FDH, CPS1 and BHMT.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.54 no.3
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• pp.299-306
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• 2009

In nitrogen-limited conditions, rhizobia lead to formation of nitrogen-fixing nodules on the roots of leguminous plants. The process of nodulation is autoregulated by pre-existing nodules in the same root system. The altered profile of sap proteins by inoculation with B. japonicum may indicate presence of a signal responsible for autoregulation transferred through stem. The 20 kDa protein enhanced by innoculation significantly decreased in intensity from 2.5 to 7 days after inoculation (DAI). However 6 kDa protein did increase during such a transition period. Western blot analysis showed that both 20 kDa and 6 kDa were cross-reacted with the SKTI antiserum. This suggests that SKTI may be involved in soybean nodulation by specific induction and degradation in stem sap during early stage of nodulation. RNAi technique and Agrobacterium rhizogenes-mediated transformation were applied to investigate the function of SKTI in nodulation. We have found that the number of rhizobium-induced nodule was much less in SKTIi-silenced hairy roots than the non-silenced. Indeed the quantitative RT-PCR showed that the expression level of SKTI gene was reduced over 40% in the transgenic hairy roots compared to the non-transgenic. It appears that the observed early induction of SKTI and degradation into small peptide in a specific time manner may be involved in autoregulation of nodulation in soybean and the specific mechanism of such regulation remains to be investigated.

• Asian-Australasian Journal of Animal Sciences
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• v.13 no.2
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• pp.200-206
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• 2000

Ninety nursery pigs were used in two metabolism experiments to determine the effects of roasting and extruding on the nutritional value of Williams 82 soybeans with (+K) and without (-K) gene expression for the Kunitz trypsin inhibitor. Treatments for both experiments were: 1) soybean meal; 2) +K roasted; 3) +K extruded; 4) -K roasted; and 5) -K extruded. Diets were the soybean preparations (96.5% of the diet) with only vitamins and minerals added as needed to meet or exceed NRC recommendations. Daily feed allowance was 5% of initial BW given as three equal meals. In Exp. 1, 50 weanling pigs (4.7 kg average BW and 21 d average age) were used. Apparent values for N digestibility (p<0.001), biological value (p<0.09), percentage N retention (p<0.05), GE digestibility (p<0.001) and percentage ME (p<0.001) were greater for pigs fed extruded soybeans than pigs fed roasted soybeans. Also, N digestibility (p<0.05), biological value (p<0.03) and percentage N retention (p<0.04) were greater for pigs fed -K soybeans than those fed +K soybeans. In Exp. 2, 40 pigs (9.7 kg average BW and 35 d average age) were allowed to adjust to the nursery environment before use in the experiment. In general, the pigs in Exp. 2 (i.e., the older pigs) had greater utilization of nutrients from all of the soybean products than the younger pigs used in Exp. 1. Digestibilities of DM, N and GE were greater (p<0.001) for pigs fed -K soybeans than those fed +K soybeans and extruded soybeans had greater digestibilities of DM, N and GE than roasted soybeans (p<0.001). Also, percentage N retention (p<0.01) and percentage ME (p<0.001) for pigs fed extruded soybeans were greater than for pigs fed roasted soybeans. In conclusion, extruded and -K soybeans were greater nutritional value than roasted and +K soybeans for 4.7 and 9.7 kg nursery pigs.