Soybean Glycinin Research Articles

Evaluation of amino acid content and nutritional quality of transgenic soybean seeds with high-level tryptophan accumulation

Anthranilate synthase (AS) is a key regulatory enzyme in tryptophan (Trp) biosynthesis and is subject to feedback inhibition by Trp. The gene encoding a mutated feedback-resistant α subunit of rice AS (OASA1D) under the control of either a soybean glycinin gene promoter or the 35S promoter of cauliflower mosaic virus for seed-specific or constitutive expression, respectively, was introduced into soybean [Glycine max (L.) Merrill] by particle bombardment. A total of seven different transgenic lines that showed markedly increased accumulation of free Trp in their seeds were developed. The overproduction of free Trp was stably inherited in subsequent generations without any apparent detrimental effect on plant growth or reproduction. The total Trp content of transgenic seeds was also about twice that of nontransgenic seeds, whereas the amount of protein-bound Trp was not substantially affected by OASA1D expression. In spite of the marked increase in free Trp content, metabolic profiling by high-performance liquid chromatography coupled with mass spectrometry revealed little change in the amounts of other aromatic compounds in the transgenic seeds. We developed a rapid and feasible system based on farmed rainbow trout to evaluate the nutritional quality of a limited quantity of transgenic soybean seeds. Supplementation of fish food with OASA1D transgenic soybean seeds or with nontransgenic seeds plus crystalline Trp increased the growth rate of the farmed fish. These results indicate transformation with OASA1D is a reliable approach to improve the nutritional quality of soybean (or of other grain legumes) for human and animal food.

Linear IgE-epitope mapping and comparative structural homology modeling of hazelnut and English walnut 11S globulins

Allergic reactions to walnuts and hazelnuts can be serious. The 11S globulins (legumins) have been identified as important allergens in these and other nuts and seeds. Here we identify the linear IgE-binding epitopes of walnut and hazelnut 11S globulins, and generate 3D 11S globulin models to map the locations of the epitopes for comparison to other allergenic homologues. Linear IgE-epitope mapping was performed by solid-phase overlapping 15-amino acid peptides probed with IgE from pooled allergic human sera. Several walnut (Jug r 4) and hazelnut (Cor a 9) 11S globulin peptides with reactivity to patient IgE were identified. Comparative alignment with cashew (Ana o 2), peanut (Ara h 3), and soybean G1 (Gly m 6.0101) and G2 (Gly m 6.0201) allergenic homologues revealed several shared allergenic ‘hot spots’. Homology modeling was performed based on the atomic structure of the soybean glycinin. Surface map comparisons between the tree nut and peanut homologues revealed structural motifs that could be important for IgE elicitation and binding and show that, contrary to predictions, the reactive epitopes are widely distributed throughout the monomeric subunits, both internally and externally, including regions occluded by quaternary subunit association. These findings reveal structural features that may be important to allergenicity and cross-reactivity of this protein class.

Phosphorescence Probes of Molecular Mobility, Oxygen Permeability, and Dynamic Site Heterogeneity in Amorphous Soybean Glycinin

The physical properties of amorphous biomolecules are important to the stability of low-moisture foods and pharmaceuticals. In the amorphous solid state, slow molecular motions are suitable for study by phosphorescent techniques. We use phosphorescence of erythrosin B dispersed in soy glycinin films to characterize the molecular mobility, oxygen permeability, and dynamic heterogeneity of the protein matrix. Films are spread from concentrated solutions of probe/protein at mole ratios of 0.045/1. Measurements as a function of temperature are made of phosphorescence intensity decays, emission spectra, and time-resolved emission spectra. Decays are fit with a stretched exponential function and both lifetimes and stretching exponents decrease with temperature. Arrhenius analysis of non-radiative quenching constants suggests that the protein matrix undergoes a broad softening transition between 70 and 120C during which additional modes of molecular motion are activated. The stretching exponent, a measure of the breadth of distribution of lifetimes and hence probe site heterogeneity, decreases gradually with temperature up to 70C and more steeply at higher temperatures, providing evidence of the onset of softening at 70C. Oxygen quenching rates, calculated from a comparison between emission lifetimes in the presence and absence of oxygen, vary roughly linearly with collisional quenching rates, which suggests that the local molecular mobility responsible for collisional quenching also modulates oxygen permeability. Delayed emission spectra are fit to a double lognormal function that provides peak emission energy and bandwidth. Bandwidth increases dramatically above 70C, which reflects an increase in the width of the distribution of energetically distinct matrix environments and provides further evidence of softening. The emission spectra blue shift with increasing delay time providing strong evidence that probes reside in distinct sites that vary in molecular mobility. Research supported by CSREES.

Characterization of a Cellular Immunostimulating Peptide from a Soybean Protein Fraction Digested with Peptidase R

An immunostimulating glutamine-rich peptide was purified from a soybean protein fraction digested with Peptidase R produced by Rhizopus oryzae (Ro-digest) by a combination of SP-Sepharose column chromatography and reversed-phase high-performance liquid chromatography. The purified peptide was supposed to be located at or near the glutamine-rich region 202 to 222 of the glycinin G4 subunit. The peptide significantly increased the number of CD8(+), CD11b(+), and CD49b(+) cells in C3H/HeN mouse spleen cell cultures, while 2 chemically synthesized glutamine-rich peptides corresponding to residues 202 to 213 (QQQQQQKSHGGR) and residues 214 to 225 (KQGQHQQEEEEE) of the glycinin G4 subunit increased the number of interleukin (IL)-12(+)CD11b(+) cells. The peptide 202-213 also significantly increased the number of CD49b(+), IL-2(+)CD4(+), and interferon-gamma(+)CD4(+) cells and stimulated the cytotoxic activity of spleen cells toward the human erythroleukemia cell line K562. These results indicate that the glutamine-rich region of the soybean glycinin G4 subunit stimulates the cellular immune system in mouse spleen cell cultures.

Effect of Sodium Bisulfite on Properties of Soybean Glycinin

The objective of this work is to understand the function of glycinin in soy protein adhesive formation. Glycinin protein was treated with sodium bisulfite, and physicochemical, morphological, and adhesion properties of the modified soy glycinin were characterized. More disulfide bonds that associated acidic and basic polypeptides of glycinin broke as the sodium bisulfite concentration increased. The reduction of disulfide bonds did not decrease the thermal stability of glycinin. Instead, the denaturation temperature of modified glycinin increased as sodium bisulfite increased. Sodium bisulfite-induced disulfide bond cleavage increased the surface hydrophobicity of modified glycinin. Hydrophobic force is the main driving force for glycinin aggregation, and the balance between hydrophobic and electrostatic forces makes glycinin form chainlike aggregates. The adhesive strength and water resistance of glycinin dropped significantly at lower levels of sodium bisulfite and then increased as the amount of sodium bisulfite increased up to 24 g/L. The adhesive performance decreased again with further addition of sodium bisulfite. The adhesive strength of glycinin was not improved by sodium bisulfite modification in the studied range.

Vitamin C: An immunomodulator that attenuates anaphylactic reactions to soybean glycinin hypersensitivity in a swine model

To date, there is insufficient satisfactory therapeutic intervention for food-induced allergies caused by glycinin, the main storage globulin found in soybean protein. The purpose of this study was to evaluate the possible attenuation effects of oral supplementation with vitamin C on soybean glycinin-induced hypersensitivity using a swine model. Ten day old piglets that were intraperitoneally sensitised with purified soybean glycinin were orally challenged with a diet containing 4% glycinin. Vitamin C was administered starting one day prior to sensitisation and continuing everyday thereafter. Results showed that unsupplemented soybean glycinin sensitised piglets presented an increase in serum IgE and histamine release as well as occurrence of diarrhoea, but reduced performance. Vitamin C supplementation attenuated these indicators by increasing interferon-γ whilst decreasing interleukin-4 in the cultured primary splenocytes, which suppresses the Th2-type immune response. These results suggest that a mega-dose of vitamin C can be used to prevent soybean allergies.

Co-Expression of Soybean Glycinins A1aB1b and A3B4 Enhances Their Accumulation Levels in Transgenic Rice Seed

The soybean major storage protein glycinin is encoded by five genes, which are divided into two subfamilies. Expression of A3B4 glycinin in transgenic rice seed reached about 1.5% of total seed protein, even if expressed under the control of strong endosperm-specific promoters. In contrast, expression of A1aB1b glycinin reached about 4% of total seed protein. Co-expression of the two proteins doubled accumulation levels of both A1aB1b and A3B4 glycinins. This increase can be largely accounted for by their aggregation with rice glutelins, self-assembly and inter-glycinin interactions, resulting in the enrichment of globulin and glutelin fractions and a concomitant reduction of the prolamin fraction. Immunoelectron microscopy indicated that the synthesized A1aB1b glycinin was predominantly deposited in protein body-II (PB-II) storage vacuoles, whereas A3B4 glycinin is targeted to both PB-II and endoplasmic reticulum (ER)-derived protein body-I (PB-I) storage structures. Co-expression with A1aB1b facilitated targeting of A3B4 glycinin into PB-II by sequestration with A1aB1b, resulting in an increase in the accumulation of A3B4 glycinin.

Effects of soybean glycinin on performance and immune function in early weaned pigs

Glycinin, a major storage protein in soybean, has been identified as an important food allergen. The present study was conducted to investigate the effects of soybean glycinin on the local and systemic immune responses using a swine model specific for glycinin allergy. Performance, peripheral blood lymphocyte proliferation and CD4+ and CD8+ subsets in the plasma of naive and sensitised piglets were determined. In addition, the concentrations of IgA, IgG, IgM, interleukin-4 and interleukin-6 in the jejunum mucosa were measured. Our results showed that dietary supplementation of glycinin reduced piglet performance (p < 0.05), while increasing lymphocyte proliferation and CD4+/CD8+ ratio (p < 0.01). Intestinal mucosal immune responses to glycinin were enhanced with high levels of IgA (p < 0.01) as well as interleukin-4 and interleukin-6 in the jejunum mucosa in glycinin-treated piglets compared with the control (p < 0.01). There were no differences due to treatment in the densities of IgG and IgM. In conclusion, it was found that glycinin stimulated local and systemic immune responses in allergic piglets and had negative effects on piglet performance. The severity of the immune reactions depends on the dose of glycinin with higher doses causing more severe symptoms.

Oral allergy syndrome and anaphylactic reactions in BALB/c mice caused by soybean glycinin and β‐conglycinin

Soybean protein is used in a number of food products but is also a common cause of food allergy. Soybean glycinin and beta-conglycinin represent up to one-third of protein in the soybean. Many reports have indicated that glycinin and beta-conglycinin have been characterized as major soybean allergens involved in food hypersensitivity. To investigate oral allergy syndrome and anaphylactic reactions in BALB/c mice caused by soybean glycinin and beta-conglycinin with an intragastric feeding protocol without using an adjuvant. BALB/c mice were sensitized by gavages with glycinin and beta-conglycinin, and allergen-specific IgE and IgG1 responses were studied by a passive cutaneous anaphylaxis assay. Serum histamine release and blood pressure were measured according to other methods. Epithelium and mast cell dye used the method of light microscopy. Sensitization with soybean allergens induced high levels of antigen-specific IgE and IgG1 and increased serum histamine in BALB/c mice. Percentiles of intact mast cell of small intestine in mice sensitized with glycinin and beta-conglyinin significantly decreased for 28 days. Degranulation of mast cells and damage of the epithelium in the small intestine of mice sensitized with globulins were observed. The level of blood pressure in sensitized mice reached a minimum at 3 h. Soybean-specific IgE and IgG1 antibodies increased, with high levels of histamine release, severe degranulation of mast cells and damage of the epithelium of small intestine in mice sensitized with glycinin and beta-conglyinin.

Rheological properties of κ-carrageenan and soybean glycinin mixed gels

Abstract Low amplitude dynamic oscillatory measurements were used to investigate κ-carrageenan/soybean glycinin mixed gel formation and rheological properties in the presence of native and denatured glycinin. The elastic modulus of mixed gel (20 °C for 30 min) was decreased with the increase in concentration of soybean glycinin. However, during heating ramp (20–95 °C) the elastic modulus showed a biphasic profile. The first phase exhibited a decrease in the elastic modulus while the second phase involved an increase in the elastic modulus. Cooling from 95 to 20 °C and holding at 20 °C for 30 min induced consolidation of protein network and gelation of κ-carrageenan which also increased the elastic modulus with the increase in glycinin concentration. Gelation of κ-carrageenan in the presence of gelled glycinin induced formation of phase-separated network. Native and denatured glycinin have different functions on large deformation of mixed gel, which is of practical significance for the food processors.

Effect of maleylation on physicochemical properties of soybean glycinin

Soybean proteins appear to harbor a great deal of potential as functional ingredients due to the fact that they are composed of highly bioavailable peptides and amino acids. To develop drink- or gel-type foods formulated with soybean protein, the physicochemical properties of intact and chemically modified soy glycinin were assessed. Maleylation to soy glycinin altered the surface charges of glycinin via the modification of lysine residues, and subsequently generated the dissociation of glycinin subunits owing to the increase in charge repulsion. This modification thus improved the solubility of glycinin, particularly under acidic pH conditions. It is worthy of note that maleylation increased the susceptibility of the basic subunits of mTGase and the formation of a substantial quantity of molecules at a low protein solution concentration. The results of dynamic rheological studies indicated that the 5% intact glycinin progressively formed the gel with mTGase treatment in a concentration-dependent manner, but maleylated-glycinin did not.

A Circular Dichroism and Fluorescence Spectrometric Assessment of Effects of Selected Chemical Denaturants on Soybean (Glycine max L.) Storage Proteins Glycinin (11S) and β-Conglycinin (7S)

Soybean glycinin (11S) and beta-conglycinin (7S) were subjected to select chemical treatments at various concentrations and resulting changes in protein structures were investigated by circular dichroism (CD) and fluorescence spectrometry. Fluorescence quenching results indicated that urea >/=3 M caused significant unfolding of 11S, but not that of 7S. GuHCl was more effective than urea in denaturation of 11S. A two-step transition in 11S structure was observed with a possible existence of a folding intermediate at 2.5 M GuHCl. Sodium dodecyl sulfate (SDS) measurably altered secondary and tertiary structures of 11S and 7S below SDS critical micellar concentration (CMC), possibly due to formation of mixed peptide-SDS micelles. SDS treatment increased alpha-helical and unordered structures of both proteins at the expense of beta-sheet structure. NaCl and CaCl 2 caused a significant decrease in fluorescence intensity without shifting emission lambda max. Exposure of 7S and 11S to NaSCN respectively at >/=0.3 and >/=0.6 M NaSCN caused a significant increase in fluorescence intensity measured at the corresponding lambda max of the protein. beta-Mercaptoethanol (beta-ME), N-ethylmaleimide (NEM), and phytic acid caused variable red shifts, 2.5-4 nm, in the emission lambda max.

Engineering two mutants of cDNA-encoding G2 subunit of soybean glycinin capable of self-assembly in vitro and rich in methionine

The main goal of this work was to make the cDNA-encoding subunit G2 of soybean glycinin, capable of self-assembly in vitro and rich in methionine residues. Two mutants (pSP65/G4SacG2 and pSP65/G4SacG2HG4) were therefore constructed. The constructed mutants were successfully assembled in vitro into oligomers similar to those occurred in the seed. The successful self-assembly was due to the introduction of Sac fragment of Gy4 (the codons of the first 21 amino acid residues), which reported to be the key element in self-assembly into trimers. The mutant pSP65/G4SacG2HG4 included the acidic chain of Gy4 (HG4), which was previously molecularly modified to have three methionine residues. This mutant will be useful in the efforts to improve the seed quality.

Ionic Strength and pH-Induced Changes in the Immunoreactivity of Purified Soybean Glycinin and Its Relation to Protein Molecular Structure

This study examined the immunogenic response of glycinin under varying conditions of pH and ionic strength using enzyme-linked immunosorbent assay. Differential scanning calorimetric (DSC) analysis and Fourier transform infrared spectroscopy (FTIR) were used to investigate the conformational changes induced as a result of these conditions, and the correlation with the changes observed in glycinin immunoreactivity were determined. A highly purified glycinin obtained by isoelectric precipitation followed by native preparative continuous flow electrophoresis was used for these studies. Purity was confirmed by two-dimensional-polyacrylamide gel electrophoresis and mass spectroscopy. DSC and FTIR results suggest that glycinin immunoreactivity is affected by changes in the tertiary and secondary packing of the protein, when flexibility, stability, and accessibility of certain substructures are modified. Aggregation and/or increased compactness of glycinin subcomponents could have potentially prevented epitopes from reacting with the IgG antibodies.

Adhesion Performance of Amino Groups (ε-NH2) of Soybean Glycinin Proteins

Amino groups of a protein have been considered as a major factor contributing to adhesion strength and water absorption. Positively charged amino groups (−NH2+) of soybean glycinin protein were replaced with negative charged carboxyl groups (−COO−) by using succinylation modification. The amino groups also were replaced with hydrophobic methyl groups (−CH3) by using methylation modification. Adhesion strength and water resistance were reduced when the −NH2 groups were substituted with −COOH groups at pH 7.6. But adhesive performance at the same pH environment was improved when the −NH2 group was replaced with −CH3, which suggests that hydrophobic groups of protein contribute to adhesion and that hydrophilic groups reduce adhesive strength. Both methylation and succinylation modifications caused the fluorescence reading (λmax) of the protein samples to shift to a higher wavelength. Native glycinin protein λmax was 344.84 nm. Methylated glycinin at 6% substitute degree (SD) had a λmax of 350.92 nm, and at 8% SD of succinylated glycinin λmax was 354.92 nm. The native glycinin has a denaturation temperature (Tp) of 165.7 °C and enthalpy (ΔH) of 16.3 J/g of glycinin. The Tp of methylated glycinin was 152 °C and ΔH was 13.9 J/g of glycinin at 4% SD. At 6% SD, Tp was 147.4 °C and the ΔH was 11.3 J/g of glycinin. The Tp for succinylated glycinin was 165.6 °C and the ΔH was −2.5 J/g of glycinin when SD = 3%. At 8% SD, the Tp was 173.6 °C and ΔH was −4.8 J/g of glycinin (SD = 8%).

Soybean Glycinin Subunits: Characterization of Physicochemical and Adhesion Properties

Soybean proteins have shown great potential for applications as renewable and environmentally friendly adhesives. The objective of this work was to study physicochemical and adhesion properties of soy glycinin subunits. Soybean glycinin was extracted from soybean flour and then fractionated into acidic and basic subunits with an estimated purity of 90 and 85%, respectively. Amino acid composition of glycinin subunits was determined. The high hydrophobic amino acid content is a major contributor to the solubility behavior and water resistance of the basic subunits. Acidic subunits and glycinin had similar solubility profiles, showing more than 80% solubility at pH 2.0-4.0 or 6.5-12.0, whereas basic subunits had considerably lower solubility with the minimum at pH 4.5-8.0. Thermal analysis using a differential scanning calorimeter suggested that basic subunits form new oligomeric structures with higher thermal stability than glycinin but no highly ordered structures present in isolated acidic subunits. The wet strength of basic subunits was 160% more than that of acidic subunits prepared at their respective isoelectric points (pI) and cured at 130 degrees C. Both pH and the curing temperature significantly affected adhesive performance. High-adhesion water resistance was usually observed for adhesives from protein prepared at their pI values and cured at elevated temperatures. Basic subunits are responsible for the water resistance of glycinin and are a good starting material for the development of water-resistant adhesives.

Design of genetically modified soybean proglycinin A1aB1b with multiple copies of bioactive peptide sequences

The peptide IIAEK derived from β-lactoglobulin has a hypocholesterolemic activity greater than that of β-sitosterol. To create food proteins with multiple copies of this valuable peptide sequence, we introduced tandem multimers of the nucleotide sequence encoding the peptide into DNA regions corresponding to the five variable regions of soybean glycinin A1aB1b subunit, and expressed the mutants in Escherichia coli. The expression level and solubility of the five mutants, each containing four IIAEK sequences in each of the variable regions, were compared. Overall, the expression level and solubility of the mutants with four IIAEK sequences in the variable regions IV and V were the best followed by II > III > I. Further, introduction of the fifth IIAEK sequence to the variable region IV did not decrease expression level and solubility. Increasing the number of IIAEK to 7 and 10 slightly decreased expression level, while their solubility decreased to as low as 40 and 1%, respectively. Various mutations were combined to get a mutant containing as many IIAEK sequences as possible. Some of the resulting mutants were expressed in the soluble form. The mutant containing eight IIAEK from the combination of variable regions IV and V (IV-4 + V-4) showed the best balance of the expression level and solubility, followed by the combination of variable regions II and III (II-4 + III-4). The soluble fractions of these mutants were purified by hydrophobic, gel filtration and ion-exchange column chromatography. Yields of IIAEK peptide released by in vitro digestion with trypsin from both mutants were around 80%. This is the first report that a large amount of a physiologically active peptide could be introduced into food protein.

Identification of stool proteins in C57BL/6J mice by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry

Gastrointestinal disease is a major cause of mortality in humans and animals, and the detection of disease-associated protein in stool is an established diagnostic method in this context. Yet, no data currently exists about the protein composition of mammalian faeces. Using a newly developed two-dimensional (2D) gel method, 28 of the most abundant proteins in murine faeces were identified. Mammalian faeces contains protein from multiple species (from the individual, from gastrointestinal bacteria, from food, etc.). Yet, it was found that the majority of mouse stool proteins were of mouse origin, with a minority of proteins being derived from food (in particular soybean glycinin and conglycinin) and bacteria (flagellin). Most mouse proteins were proteases and saccharidases derived from the exocrine pancreas. In addition, two unexpected mouse proteins were identified: one was a newly described mucin-like protein from intestinal goblet cells (FcγBP); the other was the secreted form of carbonic anhydrase (type VI) from salivary gland. The data suggest that 2D analysis of faecal protein is likely to provide meaningful information about the physiological stage of the gastrointestinal tract. Compared with studies based on biopsies, faecal protein analysis may reduce the number of laboratory animals, and might also allow quicker bridging from animal studies to humans, where biopsy material is more difficult to obtain and is less relevant for general practice use.

Structure−Physicochemical Function Relationships of Soybean Glycinin at Subunit Levels Assessed by Using Mutant Lines

Glycinin is a hexameric protein composed of five kinds of subunits. The subunits are classified into two groups, group I (A1aB1b, A1bB2, and A2B1a) and group II (A3B4 and A5A4B3). We purified four mutant glycinins composed of only group I subunits (group I-glycinin), only group II subunits (group II-glycinin), only A3B4 (A3B4-glycinin), and only A5A4B3 (A5A4B3-glycinin) from mutant soybean lines. The physicochemical properties of these glycinin samples were compared with those of the normal glycinin (11S) composed of five kinds of subunits. The thermal stabilities (as measured by thermal denaturation midpoint temperatures) of 11S, group I-glycinin, and group II-glycinin were similar to each other, although that of A3B4-glycinin was significantly lower than those of the others. The orders of aromatic and aliphatic surface hydrophobicities were the same: A3B4-glycinin > group II-glycinin > A5A4B3-glycinin > 11S > group I-glycinin. The solubility of 11S as a function of pH at mu = 0.5 was governed by that of group I-glycinin and followed this order at acidic pH: 11S = group I-glycinin > A3B4-glycinin > group II-glycinin = A5A4B3-glycinin. The order of emulsifying abilities was A5A4B3-glycinin > group II-glycinin > A3B4-glycinin > 11S > group I-glycinin. This order was consistent with that of the length of their hypervariable regions. Except for this relationship, there was no significant relationship among the other physicochemical properties of the mutant glycinins.

Effects of Protein Engineering of Canola Procruciferin on Its Physicochemical and Functional Properties

The primary structure of Brassica napus procruciferin 2/3a was engineered to elucidate structure-function relationships and to improve the functionality of cruciferin. The following mutants were constructed: (1) C287T, (2) DeltaII, variable region II was deleted; (3) C287T/DeltaII, mutation involving (1) and (2); (4) DeltaIV + A1aIV; and (5) DeltaIV + A3IV, variable region IV was replaced with variable region IV containing many charged residues from soybean glycinin A1aB1b and A3B4 subunits. Differential scanning calorimetry analysis revealed that the A1aIV region has a more favorable interaction with the procruciferin molecule than does A3IV as well as the original regions. On the basis of heat-induced precipitation analysis, it was concluded that replacement of the free cysteine residue with threonine (C287T) and insertion of charged regions (DeltaIV + A1aIV and DeltaIV + A3IV) could lead procruciferin to form soluble aggregates after heating. Low solubility was observed in mutants DeltaIV + A3IV, DeltaII, and C287T/DeltaII, especially between pH 4 and 6 at mu = 0.08, but not in DeltaIV + A1aIV, indicating that the number of acidic amino acid residues and the high number of glutamine residues are important factors for solubility at mu = 0.08. None of the mutants showed any improvements in emulsifying ability, indicating that destabilization and addition of the hydrophilic region are not effective for emulsification. The insertion of the A1aIV region in procruciferin made the molecule more susceptible to alpha-chymotrypsin.