Thermal and conformational stability of seed coat soybean peroxidase

Soybean peroxidase (SBP) obtained from the soybean seed coats belongs to class III of the plant peroxidase superfamily. Detailed circular dichroism and steady state fluorescence studies have been carried out to monitor thermal as well as denaturant-induced unfolding of SBP and apo-SBP. Melting of secondary and tertiary structures of SBP occurs with characteristic transition midpoints, T(m), of 86 and 83.5 degrees C, respectively, at neutral pH. Removal of heme resulted in greatly decreased thermal stability of the protein (T(m) = 38 degrees C). The deltaG degrees (H2O) determined from guanidine hydrochloride-induced denaturation at 25 degrees C and at neutral pH is 43.3 kJ mol(-1) for SBP and 9.0 kJ mol(-1) for apo-SBP. Comparison with the reported unfolding data of the homologous enzyme, horseradish peroxidase (HRP-C), showed that SBP exhibits significantly high thermal and conformational stability. We show that this enhanced structural stability of SBP relative to HRP-C arises due to the unique nature of their heme binding. A stronger heme-apoprotein affinity probably due to the interaction between Met37 and the C8 heme vinyl substituent contributes to the unusually high structural stability of SBP.     

Asymmetric glycosylation of soybean seed coat peroxidase

Reanalysis of the tryptic digests of soybean seed coat peroxidase (SBP) and its carboxyamidated peptide derivatives in the light of more complete sequence data has thrown light on the diglycosylated tryptic peptides, TP13 (Leu[183-205]Arg) and TP15 (Cys[208-231]Arg). Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analyses indicate that although all potential sites carry some glycan substituents, not all sites are fully occupied. Tryptic glycopeptide TP13, carrying two N-glycosylation consensus sequons (Asn185 and Asn197), occurs mainly (85-90%) as the diglycosylated species, the remainder (10-15%) being monoglycosylated. In contrast, tryptic peptide TP15, also with two N-glycosylation sites (Asn211 and Asn216), is primarily monoglycosylated (approximately 90%), with the remainder (10%) being diglycosylated. No non-glycosylated TP13 or TP15 was observed. Some artifacts are noted in the reactions of N-terminal cysteine residues and aspartate/asparagines residues in glycopeptide TP15. Mapping the glycans onto the crystal structure of SBP shows that these are asymmetrically distributed on the molecule, occurring primarily on the substrate-channel face of the enzyme. In contrast, the glycans of HRP, isozyme c, are more uniformly distributed over the enzyme surface.     

Covalent structure of soybean seed coat peroxidase

Peroxidase from soybean seed coat (SBP) is very stable at high temperature, extremes of pH, and in organic solvent. At the same time, it is highly reactive towards both organic and inorganic substrates, similar to horseradish peroxidase. SBP has a wide range of potential applications, and its structure is of particular interest for engineering purposes and as a model for stable heme peroxidases. The covalent structure of SBP has been determined by Edman sequencing and MALDI-TOF MS. SBP is a highly heterogeneous glycoprotein with MS determined masses from 39 to 41 kDa. The mature protein consists of 306 residues starting with pyrrolidone carboxylic acid. Seven glycosylation sites have been observed, although some sites were only partially glycosylated. No putative plant peroxidases were orthologous to SBP. However, SBP showed greater than 70% amino acid sequence identity to peroxidases from other legumes recruited in various defense responses.     

Structure of soybean seed coat peroxidase: a plant peroxidase with unusual stability and haem-apoprotein interactions

Soybean seed coat peroxidase (SBP) is a peroxidase with extraordinary stability and catalytic properties. It belongs to the family of class III plant peroxidases that can oxidize a wide variety of organic and inorganic substrates using hydrogen peroxide. Because the plant enzyme is a heterogeneous glycoprotein, SBP was produced recombinant in Escherichia coli for the present crystallographic study. The three-dimensional structure of SBP shows a bound tris(hydroxymethyl)aminomethane molecule (TRIS). This TRIS molecule has hydrogen bonds to active site residues corresponding to the residues that interact with the small phenolic substrate ferulic acid in the horseradish peroxidase C (HRPC):ferulic acid complex. TRIS is positioned in what has been described as a secondary substrate-binding site in HRPC, and the structure of the SBP:TRIS complex indicates that this secondary substrate-binding site could be of functional importance. SBP has one of the most solvent accessible delta-meso haem edge (the site of electron transfer from reducing substrates to the enzymatic intermediates compound I and II) so far described for a plant peroxidase and structural alignment suggests that the volume of Ile74 is a factor that influences the solvent accessibility of this important site. A contact between haem C8 vinyl and the sulphur atom of Met37 is observed in the SBP structure. This interaction might affect the stability of the haem group by stabilisation/delocalisation of the porphyrin pi-cation of compound I.     

The N-glycosylation sites of soybean seed coat peroxidase

Tryptic digestion of apo-soybean peroxidase (apo-SBP), withand without acetamidation, chromatographic separation of thetryptic fragments and MALDI-TOF analysis of the major components,both before and after digestion with glycopeptidase A, demonstratedthe presence of six carbohydrate groups on five peptides. Fiveof the glycopeptides can be mapped with confidence to the peptidescontaining Asn16, Asn90, Asn104, Asn169, and Asn174. The sixthN-glycosylation site is not known and does not appear to beAsn145. It may be present on the N-terminus of SBP, which hasnot been sequenced. soybean seed coat peroxidase glycopeptides glycosylation sites MALDI-TOF MS     

Immobilization of soybean seed coat peroxidase on polyaniline: Synthesis optimization and catalytic properties

Soybean seed coat peroxidase (SBP) was immobilized on various polyaniline-based polymers (PANI), activated with glutaraldehyde. The most reduced polymer (PANIG₂) showed the highest immobilization capacity (8.2 mg SBP?g^?1 PANIG₂). The optimum pH for immobilization was 6.0 and the maximum retention was achieved after a 6-h reaction period. The efficiency of enzyme activity retention was 82%. When stored at 4°C, the immobilized enzyme retained 80% of its activity for 15 weeks as evidenced by tests performed at 2-week intervals. The immobilized SBP showed the same pH-activity profile as that of the free SBP for pyrogallol oxidation but the optimum temperature (55°C) was 10°C below that of the free enzyme. Kinetic analysis show that the K_m was conserved while the specific V_max dropped from 14.6 to 11.4 µmol min^?1 µg^?1, in agreement with the immobilization efficiency. Substrate specificity was practically the same for both enzymes. Immobilized SBP showed a greatly improved tolerance to different organic solvents; while free SBP lost around 90% of its activity at a 50% organic solvent concentration, immobilized SBP underwent only 30% inactivation at a concentration of 70% acetonitrile. Taking into account that immobilized HRP loses more than 40% of its activity at a 20% organic solvent concentration, immobilized SBP performed much better than its widely used counterpart HRP.     

Immobilization of soybean seed coat peroxidase on polyaniline: Synthesis optimization and catalytic properties

Soybean seed coat peroxidase (SBP) was immobilized on various polyaniline-based polymers (PANI), activated with glutaraldehyde. The most reduced polymer (PANIG₂) showed the highest immobilization capacity (8.2 mg SBP g^-1 PANIG₂). The optimum pH for immobilization was 6.0 and the maximum retention was achieved after a 6-h reaction period. The efficiency of enzyme activity retention was 82%. When stored at 4°C, the immobilized enzyme retained 80% of its activity for 15 weeks as evidenced by tests performed at 2-week intervals. The immobilized SBP showed the same pH-activity profile as that of the free SBP for pyrogallol oxidation but the optimum temperature (55°C) was 10°C below that of the free enzyme. Kinetic analysis show that the K_m was conserved while the specific V_max dropped from 14.6 to 11.4 µmol min^-1 µg^-1, in agreement with the immobilization efficiency. Substrate specificity was practically the same for both enzymes. Immobilized SBP showed a greatly improved tolerance to different organic solvents; while free SBP lost around 90% of its activity at a 50% organic solvent concentration, immobilized SBP underwent only 30% inactivation at a concentration of 70% acetonitrile. Taking into account that immobilized HRP loses more than 40% of its activity at a 20% organic solvent concentration, immobilized SBP performed much better than its widely used counterpart HRP.     

Steady-state and picosecond time-resolved fluorescence studies on native and apo seed coat soybean peroxidase.

Seed coat soybean peroxidase (SBP) belongs to class III of the plant peroxidase super family. The protein has a very similar 3-dimensional structure with that of horseradish peroxidase (HRP-C). The fluorescence characteristics of the single tryptophan (Trp117) present in SBP and apo-SBP have been studied by steady-state and pico-second time-resolved fluorescence spectroscopy. Fluorescence decay curve of SBP was best described by a four exponential model that gave the lifetimes, 0.035 ns (97.0%), 0.30 ns (2.0%), 2.0 ns (0.8%), and 6.3 ns (0.2%). These lifetime values agreed very well with the values obtained by the model independent maximum entropy method (MEM). The three longer lifetimes that constituted 3% of the fluorophore population in the SBP sample are attributed to the presence of trace quantities of apo-SBP. The pico-second lifetime value of SBP is indicative of efficient energy transfer from Trp117 to heme. From fluorescence resonance energy transfer (FRET) calculations, the energy-transfer efficiency in SBP is found to be relatively higher as compared to HRP-C and is attributed mainly to the higher value of orientation factor, kappa(2) for SBP. Decay-associated spectra of SBP indicated that the tryptophan of SBP is relatively more solvent exposed as compared to HRP-C and is attributed to the various structural features of SBP. Linear Stern-Volmer plots obtained from the quenching measurements using acrylamide gave k(q) = 5.4 x 10(8) M(-1) s(-1) for SBP and 7.2 x 10(8) M(-1) s(-1) for apo-SBP and indicated that on removal of heme in SBP, Trp117 is more solvent exposed.     

Lignin content and peroxidase activity in soybean seed coat susceptible and resistant to mechanical damage

Mechanical damage is one of the causes of great loss in the quality of soybean seeds during harvest and processing. Considerable interest exists in the lignin since its deposition in the seed coat tissue provides mechanical resistance and protects the cell against microorganisms. In addition, peroxidases might be involved in the oxidation of cinnamyl alcohols prior to their polymerization during lignin formation. Thus, the aim of the present work was to analyze the lignin contents and peroxidases activities of six Brazilian soybean cultivars (Savana, Paranagoiana, FT-10, Santa Rosa, Doko and Paraná) and their relationships with the mechanical damage. Results showed that the lignin content and peroxidase activity in the seed coat significantly differed among the soybean cultivars. Cultivars Doko and Paraná had the highest contents of lignin and peroxidases activities while the other cultivars had lowest lignin contents and enzyme activities. Lignin content and peroxidase activity may be reasonable indicators of resistance to mechanical damage in soybean seeds.     

Immobilisation of soybean seed coat peroxidase on its natural support for phenol removal from wastewater

Soybean seed coat peroxidase (SBP; EC 1.11.1.7) was immobilised on its natural support, soybean seed coats, anticipating its use in phenol removal. Periodate and glutaraldehyde chemistries were assayed. Periodate failed to immobilise any SBP, whereas glutaraldehyde was effective. The optimum concentration of glutaraldehyde was found to be 1%. Immobilisation shifted the optimum pH for phenol removal from 4.0 to 6.0. Treated seed coat retained its activity over a 4-week period, and reusability assays showed that treated seed coats could be reused once for phenol removal. Polyethylene glycol (PEG) increased the stability of phenol degradation activity. In addition, the phenolic polymer was adsorbed on to seed coats, thus making removal of the polymeric product easier.     

Immobilisation of soybean seed coat peroxidase on its natural support for phenol removal from wastewater

Soybean seed coat peroxidase (SBP; EC 1.11.1.7) was immobilised on its natural support, soybean seed coats, anticipating its use in phenol removal. Periodate and glutaraldehyde chemistries were assayed. Periodate failed to immobilise any SBP, whereas glutaraldehyde was effective. The optimum concentration of glutaraldehyde was found to be 1%. Immobilisation shifted the optimum pH for phenol removal from 4.0 to 6.0. Treated seed coat retained its activity over a 4-week period, and reusability assays showed that treated seed coats could be reused once for phenol removal. Polyethylene glycol (PEG) increased the stability of phenol degradation activity. In addition, the phenolic polymer was adsorbed on to seed coats, thus making removal of the polymeric product easier.     

Expression and characterization of soybean seed coat peroxidase in Escherichia coli BL21(DE3)

Soybean seed coat peroxidase (SBP) is a valuable enzyme having a broad variety of applications in analytical chemistry, biochemistry, and food processing. In the present study, the sscp gene (Gene ID: 548068) was optimized based on the preferred codon usage of Escherichia coli, synthesized, and expressed in E. coli BL21(DE3). SDS-PAGE and western blot analysis of this expressed protein revealed that its molecular weight is approximately 39?kDa. The effects of induction temperature, concentration of isopropyl-β-D-thiogalactoside and hemin, induction time, expression time were optimized to enhance SBP production with a maximum activity of 11.23?U/mL (8.64?U/mg total protein). Furthermore, the kinetics of enzyme-catalyzed reactions of recombinant protein was determined. When 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) was used as substrate, optimum reaction temperature and pH of the enzyme were 85°C and 5.0, respectively. The effects of metal ions on the enzymatic reaction were also further investigated. The SBP was successfully expressed in E. coli BL21(DE3) which would provide a more efficient production strategy for industrial applications of SBP.     

Special secretory cells in the soybean seed coat

Summary The seed coat of soybean (Glycine max L. Merr.) is of physiological interest for synthesis and transport of amino acids and photosynthates during embryo development. A transmission and scanning electron microscopic study to elucidate the structure of the seed coat disclosed a specialized convex area (antipit) appressed to a concave pit in the center of the abaxial surface of the cotyledon. The antipit, which lies on the inner surface of the seed coat at a medial point in the anterior to posterior direction of the seed, contained specialized secretory cells bounded by loose multi-layered cell walls. These cells were rectangular in the developing seed, varied in length, and contributed directly to the convex morphology of the antipit seen on the ventral surface of the seed coat. At maturity these cells assumed the shape of a cone, extending from the aleurone layer in a perpendicular array. The aleurone and cone cells contained numerous Golgi apparatus, laminated rough endoplasmic reticulum, secretory vesicles, and amyloplasts. Secretory vesicles arose directly from tubules of fenestrated trans cisternae of the Golgi apparatus. Mitochondria were clustered with the amyloplasts; stacks of lamellar cisternae of rough endoplasmic reticulum were associated with the nucleus and Golgi apparatus. The cellular contents, the interconnections by plasmodesmata, and the close physical association with the cotyledon suggested that the aleurone and cone cells may be involved in symplastic transport of nutrients for use by the developing embryo.This paper is dedicated to the memory of my parents, Joseph and Theresa Yaklich, who by their example taught me the value of work and the enjoyment of simple things.     

Hourglass cell development in the soybean seed coat

Background and Aims

Hourglass cells (HGCs) are prominent cells in the soybean seed coat, and have potential use as ‘phytofactories’ to produce specific proteins of interest. Previous studies have shown that HGCs initiate differentiation at about 9 d post-anthesis (dpa), assuming their characteristic morphology by 18 dpa. This study aims to document the structural changes in HGCs during this critical period, and to relate these changes to the concurrent development of a specific soybean peroxidase (SBP) encoded by the Ep gene.

Methods

Pods were collected from plants at specific growth stages. Fresh material was processed for analysis of Ep peroxidase activity. Tissues were processed for scanning and transmission electron microscopy, as well as extracted for western blotting. A null variety lacking expression of Ep peroxidase was grown as a control.

Key Results and Conclusions

At 9 dpa, HGCs are typical undifferentiated plant cells, but from 12–18 dpa they undergo rapid changes in their internal and external structure. By 18 dpa, they have assumed the characteristic hourglass shape with thick cell walls, intercellular air spaces and large central vacuoles. By 45 dpa, all organelles in HGCs have been degraded. Additional observations indicate that plasmodesmata connect all cell types. SBP activity and SBP protein are detectable in the HGC before they are fully differentiated (approx. 18 dpa). In very early stages, SBP activity appears localized in a vacuole as previously predicted. These results increase our understanding of the structure and development of the HGC and will be valuable for future studies aimed at protein targeting to components of the HGC endomembrane systems.     

Oyxgen and temperature effects on soybean seed coat respiration rates

Soybean (Glycine max (L.) Merr) seed coat respiration rates in response to changing O₂ concentration and temperature were examined experimentally and with a mathematical analysis. The experimental observations showed seed coat respiration rates were sensitive to O₂ concentration below 0.25 micromole O₂ cm⁻³. There was a steady decline in respiration rates from the saturating O₂ concentration down to about 0 to 0.03 micromole O₂ per cubic centimeter. Seed coat respiration rates were found to change linearly with temperature between 8 and 28°C. The explanation for these results was sought by examining the diffusion of O₂ into the vascular bundles of the soybean seed coat. Differential equations describing O₂ uptake in two distinct zones of the vascular bundle were solved. The outer zone was assumed to be O₂ saturated and respiration proceeded at a constant rate per unit volume. The inner zone was assumed to have respiration rates which were linearly dependent on O₂ concentration. The solution of this mathematical model showed considerable similarity with the experimental results. Respiration rates were predicted to saturate at about 0.31 micromole O₂ per cubic centimeter and to decrease curvilinearly below that concentration. While the mathematical model predicted an exponential response in respiration rate to temperature, it was found that the exponential response is difficult to distinguish from a linear response in the temperature range studied experimentally. Consequently, both the experimental and theoretical studies showed the importance of O₂ diffusion into soybean seed coat vascular bundles as a potential restriction on respiration rates. In particular, it was suggested that increases in the total length of the vascular bundles in the soybean seed coat was the major option for increasing the total respiratory capability.     

A class I chitinase from soybean seed coat.

Protein extracts from soybean (Glycine max [L.] Merr) seed hulls were fractionated by isoelectric focusing and SDS-PAGE analysis and components identified by peptide microsequencing. An abundant 32 kDa protein possessed an N-terminal cysteine-rich hevein domain present in class I chitinases and in other chitin-binding proteins. The protein could be purified from seed coats by single step binding to a chitin bead matrix and displayed chitinase activity by an electrophoretic zymogram assay. The corresponding cDNA and genomic clones for the chitinase protein were isolated and characterized, and the expression pattern determined by RNA blot analysis. The deduced peptide sequence of 320 amino acids included an N-terminal signal peptide and conserved chitin-binding and catalytic domains interspaced by a proline hinge. An 11.3 kb EcoRI genomic fragment bearing the 2.4 kb chitinase gene was fully sequenced. The gene contained two introns and was flanked by A+T-rich tracts. Analysis by DNA blot hybridization showed that this is a single or low copy gene in the soybean genome. The chitinase is expressed late in seed development, with particularly high expression in the seed coat. Expression was also evident in the late stages of development of the pod, root, leaf, and embryo, and in tissues responding to pathogen infection. This study further illustrates the differences in protein composition of the various seed tissues and demonstrates that defence-related proteins are prevalent in the seed coat.     

Unstable expression of a soybean gene during seed coat development

Summary The R gene of soybean is involved in anthocyanin synthesis in the seed coat, and its r-m allele conditions a variegated distribution of black spots and/or concentric rings of pigment superimposed on an otherwise brown seed coat. We describe an unusual feature of r-m that causes expression at the R locus to switch between active and inactive phases both somatically and germinally. Non-heritable somatic changes of the allele produce single plants containing mixtures of seed with different coat colors (black+striped or brown+striped). Heritable changes of the r-m allele are manifested in progeny plants which produce all black seed or all brown seed. Surprisingly, subsequent generations from revertant sublines show continued instability of the allele such that brown revertants (r*/r*) or homozygous black seed revertants (R*/R*) can give rise to striped or striped+black-seeded plants. Thus, the revertants produced by the r-m allele are not stable but interconvert between all three forms (R*, r*, and r-m) at detectable frequencies. Mutability of the r-m allele in a different genetic background has also been found after inter-crossing various soybean genotypes.