Motoyasu Adachi

Comparison of degradation abilities of α- and β-amylases on raw starch granules.

The degradation abilities of α-amylase from Bacillus amyloliquefaciens and β-amylases from Bacillus cereus and soybean on raw starch granules from various botanical sources (potato, sweet potato, wheat, rice and corn) were examined by scanning electron microscopy. All the amylases showed different degradation patterns on starch granules. The α-amylase was more efficient than the β-amylases. α-Amylase showed both centrifugal and centripetal hydrolysis on corn, rice and wheat granules, but only centrifugal hydrolysis on potato granules. On the other hand, β-amylase moved very slowly on granules. The kinetic assays which explain the release of maltose were carried out at 12, 18 and 24 h. The rice granules were found to be the best substrate for enzymic hydrolysis by α and β-amylases. In addition, While Bacillus cereus β-amylase hydrolyzed corn granules efficiently at 45°C; soybean β-amylase was 60% less active than bacterial β-amylase at the same temperature.

Identification of the Bile Acid-binding Region in the Soy Glycinin A1aB1b Subunit

Soy glycinin has five major subunits which are classified into two groups according to their homology in amino acid sequences (group I, A1aB1b, A1bB2 and A2B1a; group II, A3B4 and A5A4B3). It has been reported that the peptide fragments derived from the A1a and A2 chains of the A1aB1b and A2B1a subunits had bile acid-binding ability and that the region of 114-161 residues of the A1a chain was responsible for this bile acid-binding ability. In this study, we constructed A1a, A3 and 9 deletion mutants of A1a lacking various numbers of residues at the C-terminus, and evaluated their bile acid-binding ability by a cholic acid-conjugated column and fluorescence analysis. The bile acid-binding ability of A1a was higher than that of A3 and there was a remarkable decrease in the bile acid-binding ability between the Δ[138-291] and Δ[130-291] mutants. The 130-138 region is rich in hydrophobic residues. In this regard, when we constructed the Δ[129-134] mutant lacking six contiguous hydrophobic residues (VAWWMY) and evaluated its bile acid-binding ability, a similar remarkable decrease in the bile acid-binding ability was observed. These results indicate that the 129-134 residue region (VAWWMY) with high hydrophobicity was important for bile acid-binding of A1a.

CO-OVEREXPRESSION OF FOLDING MODULATORS IMPROVES THE SOLUBILITY OF THE RECOMBINANT GUINEA PIG LIVER TRANSGLUTAMINASE EXPRESSED IN ESCHERICHIA COLI

ABSTRACT Transglutaminases (EC 2.3.2.13) catalyze the formation of ε-(γ-glutamyl)lysine cross-links and the substitution of primary amines for the γ-carboxamide groups of protein bound glutamine residues, and are involved in many biological phenomena. Transglutaminase reactions are also applicable in applied enzymology. Here, we established an expression system of recombinant mammalian tissue-type transglutaminase with high productivity. Overexpression of guinea pig liver transglutaminase in Escherichia coli, using a plasmid pET21-d, mostly resulted in the accumulation of insoluble and inactive enzyme protein. By the expression culture at lower temperatures (25 and 18°C), however, a fraction of the soluble and active enzyme protein slightly increased. Co-overexpression of a molecular chaperone system (DnaK-DnaJ-GrpE) and/or a folding catalyst (trigger factor) improved the solubility of the recombinant enzyme produced in E. coli cells. The specific activity, the affinity to the amine substrate, and the sensitivity to the calcium activation and GTP inhibition of the purified soluble recombinant enzyme were lower than those of the natural liver enzyme. These results indicated that co-overexpression of folding modulators tested improved the solubility of the overproduced recombinant mammalian tissue-type transglutaminase, but the catalytic properties of the soluble recombinant enzyme were not exactly the same as those of the natural enzyme.

Structure-function relationships of soybean proteins revealed by using recombinant systems

Abstract Glycinin and β-conglycinin are the major storage proteins of soybean and determine the functional properties of soybean proteins. Structure-function relationships of glycinin and β-conglycinin were investigated by using Escherichia coli expression systems. Examination of functional properties of various modified versions of proglycinin A1aB1b suggests that the hydrophobicity of the C-terminal region is probably important for a high emulsifying ability, that the topology of free SH residues is closely related to the heat-induced gel forming ability and that the structural factors suitable for gelation and emulsification properties are quite different. Mutual comparison of functional properties of β-conglycinin constituent subunits (α, α′ and β) and the core regions of α and α′ indicate that the core regions determine thermal stability and surface hydrophobicity, that the extension regions of α and α′ contribute to high solubility and emulsifying abilities and that the carbohydrate moieties inhibit the formation of heat-induced aggregates. Analyses by chimerization of glycinin and β-conglycinin suggest that structure-function relationships are different between glycinin and β-conglycinin.

Structure of 8Sα globulin, the major seed storage protein of mung bean

The 8S globulins of mung bean [Vigna radiata (L.) Wilczek] are vicilin-type seed storage globulins which consist of three isoforms: 8Sα, 8Sα′ and 8Sβ. The three isoforms have high sequence identities with each other (around 90%). The structure of 8Sα globulin has been determined for the first time by X-ray crystallographic analysis and refined at 2.65 A resolution with a final R factor of 19.6% for 10–2.65 A resolution data. The refined 8Sα globulin structure consisted of 366 of the 423 amino-acid residues (one subunit of the biological trimer). With the exception of several disordered regions, the overall 8Sα globulin structure closely resembled those of other seed storage 7S globulins. The 8Sα globulin exhibited the highest degree of sequence identity (68%) and structural similarity (a root-mean-square deviation of 0.6 A) with soybean β-conglycinin β (7S globulin). Their surface hydrophobicities are also similar to each other, although their solubilities differ under alkaline conditions at low ionic strength. This difference seems to be a consequence of charge–charge interactions and not hydrophobic interactions of the surfaces, based on a comparison of the electrostatic potentials of the molecular surfaces. The thermal stability of 8Sα globulin is lower than that of soybean β-conglycinin β. This correlates with the cavity size derived from the crystal structure, although other structural features also have a small effect on the proteins thermal stability.

Improved bile acid-binding ability of soybean glycinin A1a polypeptide by the introduction of a bile acid-binding peptide (VAWWMY).

We have previously identified a potential bile acid-binding peptide sequence (VAWWMY) in acidic polypeptide A1a of the soybean glycinin A1aB1b subunit (Choi, S. K., et al., Biosci. Biotechnol. Biochem., 66, 2395–2401 (2002)). In this study, we introduced the nucleotide sequence encoding this peptide in the coding DNA which corresponds to amino acids between 251 and 256, and 282 and 287 into the A1a polypeptide by replacement to respectively give modified versions A1aM1 and A1aM2. A fluorescence analysis demonstrates that their bile acid-binding ability was improved compared to A1a. Moreover, modified proglycinin A1aB1b with the VAWWMY sequence at the same sites as those of A1aM1 and A1aM2 was judged to assume the correct conformation. These results suggest the possibility of developing transgenic crops to accumulate the modified glycinin.

N-Glycosylation does not affect assembly and targeting of proglycinin in yeast

Glycinin, a simple protein, and beta-conglycinin, a glycoprotein, are the dominant storage proteins of soybean and are suggested to be derived from a common ancestor. To investigate why glycinin does not require glycosylation for its maturation, we attempted N-glycosylation of proglycinin A1aB1b using site-directed mutagenesis and yeast expression system. An N-glycosylation consensus sequence Asn-X-Ser/Thr was created at positions 103, 183, 196, 284 and 457 in the variable regions being strongly hydrophilic revealed from the alignment of amino acid sequences of various glycinin-type proteins. Among five mutant proglycinins (Q103N, H183N, G198T, S284N, N459T), Q103N was fully glycosylated, and H183N and N459T were partly (around 20% of the expressed proteins), whereas others were barely or not glycosylated. The glycosylated proglycinin was susceptible to endo-beta-N-acetylglucosamidase and N-glycanase cleavages. N-glycosylation did not cause inconveniences to processing of signal peptide, assembly into trimers and targeting into the vacuoles. Thermal and trypsin sensitivity analyses of the glycosylated proglycinin suggested that N-linked glycan prevents protein-protein interaction but does not stabilize the protein conformation. The reason why glycinin does not require N-glycosylation for its maturation is discussed.

Cloning and expression of rapeseed procruciferin in Escherichia coli and crystallization of the purified recombinant protein.

Two rapeseed cruciferin cDNAs (cru2/3a and cru2/3b) were cloned and sequenced. A comparison of their DNA and protein sequences with other cruciferins, indicated cru2/3b to be a novel clone and, among them, an inherent and highly conserved sequence of twelve amino acids was identified. Procruciferin 2/3a and 2/3b were expressed in Eschericha coli, and procruciferin 2/3a was obtained in a soluble form. The expressed procruciferin 2/3a has a trimeric structure and formed crystals although the quality was not good, suggesting that this expression system is useful for protein engineering of procruciferin 2/3a.

Soybean Glycinin A1aB1b Subunit Has a Molecular Chaperone-like Function to Assist Folding of the Other Subunit Having Low Folding Ability

Soybean (Glycine max L.) glycinin is composed of five subunits which are classified into two groups (group I: A1aB1b, A1bB2, and A2B1a; group II: A3B4 and A5A4B3). All the common soybean cultivars contain both group I and II subunits (Maruyama, N. et al., Phytochemistry, 64, 701–708 (2003)). The biosynthesis of group I starts earlier compared with that of the A3B4 subunit during seed development (Meinke, D.W. et al., Planta, 153, 130–139 (1981)). We have revealed that group I A1aB1b was mostly expressed as a soluble protein, but that A3B4 was expressed mainly as an insoluble protein in Escherichia coli under the same expression conditions; namely, A1aB1b had higher folding ability than A3B4. We therefore assumed that A1aB1b assists folding of group II subunits like a molecular chaperone does. In order to ascertain this, A1aB1b and A3B4 were co-expressed in E. coli. All of the expressed proteins of A3B4 were recovered in a soluble fraction. To confirm this result, we also co-expressed A1aB1b with modified A3B4 versions having extremely low folding ability. All expressed modified A3B4 versions were soluble. These results clearly suggest that A1aB1b has a molecular chaperone-like function in their folding.