COMBINATION OF BLACK BEAN (PHASEOLUS VULGARIS) AMYLASE INHIBITOR WITH MODIFIED PANCREATIC α-AMYLASE

Several amino acid residues important for the action of porcine pancreatic α-amylase on starch were modified using specific reagents: histidine groups by photooxidation with rose bengal or by diethylpyrocarbonate; cysteine by dithiobis nitrobenzoic acid; tryptophan by N-bromosuccimide and tyrosine by hydroxyl acetylation with N-acetylimidazole. These modifications, with the exception of cysteine, reduced the amylase activity but none of them alone was able to alter significantly the inhibition of the enzyme by a purified black bean (Phaseolus vulgaris) amylase inhibitor. Only by photooxidation that can modify several groups at the same time was the inhibitor action eliminated. The results suggested that the histidine of the amylase active site and tyrosine of the substrate binding site are not important for binding to the bean inhibitor. The terminal sugars of the amylase inhibitor were identified as mannose and xylose. Periodate oxidation of the carbohydrate moiety caused total loss of activity. The treatment of the inhibitor with α-mannosidase did not alter its inhibitor action on α-amylase.     

KINETICS OF THE INTERACTION OF PANCREATIC α-AMYLASE WITH A KIDNEY BEAN (PHASEOLUS VULGARIS) - AMYLASE INHIBITOR

An amylase inhibitor isolated from black beans (Phaseolus vulgaris) can completely inhibit porcine pancreatic α-amylase forming a 1:1 stoichiometric complex. The kinetic pattern of complex formation is pH dependent. At pH 5.5 it follows a first order reaction with rate constant of 0.029 min^?1 and 0.017 min^?1 at 37°C and equimolar inhibitor and enzyme concentration, respectively, of 10^?8 M and 10^?9 M. At pH 6.9 it is a second order reaction, with a rate constant of 0.25 × 10⁶ M^?1 min^?1 at 37°C, with 4 × 10^?8 M concentrations of enzyme and inhibitor. The dissociation constants of the enzymeinhibitor complex are 1.7 × 10^?10 M at pH 5.5 and 4.4 × 10^?9 M at pH 6.9, at 37°C. The kinetic data obtained at pH 5.5 suggested the formation of an initial reversible complex followed by a conformational change step. The complex can be dissociated either in acid pH (4.3) or at pH values higher than 6, 5 with partial recovery of the amylase activity.     

PURIFICATION AND SOME PHYSICAL AND CHEMICAL PROPERTIES OF RED KIDNEY BEAN (PHASEOLUS VULGARIS) α-AMYLASE INHIBITOR

Red kidney bean contains more amylase inhibitor than do California white bean and cowpea while garbanzo bean and Westan and Westley lima beans do not contain inhibitor. Red kidney bean amylase inhibitor was purified to homogeneity by selective heat treatment (60°C) of a water extract at pH 4. 0, fractionation with ethanol and successive chromatography on DEAE- and CM-cellulose chromatography. The inhibitor has an apparent molecular weight of 49,000 by Sephadex gel filtration and contains 8. 6% carbohydrate probably covalently linked via an amide linkage to asparagine. The inhibitor probably contains four subunits perhaps of three different types. The inhibitor is high in aspartic acid, glutamic acid, serine, threonine and valine, low in cysteine/cystine and does not contain proline. Stable 1:1 complex formation between inhibitor and porcine pancreatic α-amylase was demonstrated by gel filtration on Sephadex G-100. The inhibitor has activity against porcine pancreatic α-amylase, human salivary α-amylase, and Tenebrio molitor (yellow corn meal worm) larval midgut α-amylase but is inactive against Bacillus subtilis α-amylase, Aspergillus oryzae α-amylase, barley α-amylase and red kidney bean α-amylase.     

SUBUNIT STRUCTURES AND ESSENTIAL AMINO ACID RESIDUES OF WHITE KIDNEY BEAN (PHASEOLUS VULGARIS) α-AMYLASE INHIBITORS

Both white kidney bean α-amylase inhibitors WKB 858A (MW 42,000) and WKB 858B (MW 20,000) were composed of two subunits as determined by N-terminal amino acid analysis by amino acid sequence, by SDS-PAGE and by separation on a chromatofocusing column in 8 M urea. N-Terminal amino acids for Inhibitor WKB 858A were alanine and glycine, with a sequence of H₂N-Ala-Glu-Asn-Ala-Gly-Thr-Tyr–COOH for deglycosylated 9,000 MW peptide and H₂N-Gly-Asn–COOH for deglycosylated 12,000 MW peptide. N-Terminal amino acids for Inhibitor WKB 858B were alanine and serine, with a sequence of H₂N-Ala-Thr-Glu-Thr-Ser–COOH for the deglycosylated 9,000 MW peptide and H₂N-Ser-Ala-Val-Gly-Leu-Asp-Phe-Val-Leu-Val-Pro-Val-Gin-Pro-Glu-Ser-Lys-Gly-Asp-Thr-VaVal-Glu-Phe-Asp–COOH for the deglycosylated 15,000 MW peptide. Chemical modification of 2 of 7 His residues with diethylpyrocarbonate resulted in 26% loss of inhibitory activity. Modification of 1.5 of 7 Trp residues with N-bromosuccinimide gave 60% loss of inhibitory activity. Modification of 2 of 6 Tyr residues with N-acetylimidazole gave 60% loss of inhibitory activity. Modification of 3.6 of 6 Arg residues with p-hydroxyphenylglyoxal gave 64% loss of inhibitor activity. These results indicate the possible importance of one or more His, Trp, Tyr and perhaps Arg residues for inhibitory activity against porcine pancreatic α-amylase.     

PURIFICATION AND PARTIAL CHARACTERIZATION OF WHITE KIDNEY BEAN (PHASEOLUS VULGARIS)β-AMYLASE INHIBITORS FROM TWO EXPERIMENTAL CULTIVARS

β-Amylase inhibitors WKB 858B and WKB 858B were purified to homogeneity from different cultivars of white kidney beans by extraction from the ground beans and by sequential heat treatment, ethanol fractionation, DEAE-cellulose chromatography, Sephadex G-75 gel chromatography and CM-cellulose chromatography. The inhibitors were homogeneous by 7.5% polyacrylamide gel electrophoresis; no isoinhibitors were found. Inhibitors WKB 858A and WKB 858B had isoelectric points of 5.0 and 4.65, respectively, and molecular weights of 42,000 and 20,000, respectively, by FPLC Superose 12 gel filtration chromatography. Inhibitor WKB 858A had molecular weights of 40,000 and 38,000 by Sephadex G-75 gel filtration chromatography and by native gel electrophoresis, respectively. Inhibitor WKB 858A contained 11.0% carbohydrate, N-linked to asparagine residues, with a composition of 1 fucose, 1 xylose, 4 galactose, 8 N-acetylglycosamine and 13 mannose residues per mol of inhibitor. Amino acid analysis of Inhibitor WKB 858A gave a high content of Asx, Glx, Ser, Thr and Val (combined total of 60% molar ratio) and low content of sulfur amino acids (0.8% molar ratio of Met and no 1/2 cystine). No-SH groups were found. The amino acid composition was similar to that of eight other a-amylase inhibitors from beans. Inhibitor WKB 858A formed a 1:1 stoichiometric complex with porcine pancreatic a-amylase with a Ki of 1.0 × 10^?11 M at pH 5.4 and 30C; it had no trypsin inhibitory activity. At pH 6.90 and 30C, the rate of complex formation between Inhibitor WKB 858A and porcine pancreatic β-amylase was 2.76 times faster at 1.385 vs 0.035 ionic strength (with Na₂SO₄), indicating hydrophobic bonds are most important in complex formation.     

STRUCTURAL FEATURES OF RED KIDNEY BEAN á-AMYLASE INHIBITOR IMPORTANT IN BINDING WITH á-AMYLASE

The structural features of the glycoprotein α-amylase inhibitor from red kidney bean (Phaseolus vulgaris) important for binding to α-amylase were examined. The inhibitor contained 13.0% carbohydrate. The carbohydrate portion of the inhibitor is composed of 25 mannose, 2 xylose, 1 fucose and 17 N-cetylglucosamine residues per mol. Seventy (70) percent of the neutral carbohydrates were removed from the inhibitor by treatment with endo-β- N -acetylglucosaminidase H. The carbohydrate remaining attached to the protein consisted of 7 mannose, 2 xylose, and 1 fucose residues (N-acetylogucosamine was not determined). The intact glyco chains obtained from the pronase digest of the inhibitor failed to inhibit β-amylase (3.3 times 10⁵ fold molar excess of glyco chains to enzyme) when preincubated with the enzyme at 30°C for 30 min at pH 6.9 before adding starch as substrate.
Oxidation of one tryptophan residue of the α-amylase inhibitor with N-bromosuccinimide at pH 6.0 led to a 50% loss in inhibitory activity. Periodate oxidation of α-amylase inhibitor led to less of two tyrosine and one methionine residues per mole of inhibitor within 15 min. There was not a direct correlation between modification of these residues and loss of inhibitory activity. Modification of three of the five histidine residues with diethylpyrocarbonate resulted in about 50% loss in inhibitory activity.     

THE MOLECULAR WEIGHT OF α-AMYLASE INHIBITOR FROM WHITE BEAN cv 858B (PHASEOLUS VULGARIS L.) IS 56 kDa, NOT 20 kDa

The molecular weights (M_rs) of α-amylase inhibitors (αAIs)fiom 18 (Ah) bean cultivars estimated by Superose 12 gelfiltration chromatography were 22–62% smaller than those determined by Sephadex G-100 gel filtration and by polyacrylamide gel electrophoresis (PAGE) methods. αAI-4 from WKB cultivar 858B was purified and the Mr was shown to be 51.0 kDa based on Sephadex G-100 gel filtration chromatography and by PAGE. A M_r for aAI-4 of 56.714 kDa was determined by laser-assisted time-of-flight mass spectrometry and appears to be the true M_r of the mature glycosylated active aAI-4. The results show that Superose 12 gelfiltration chromatography is not usefulfor M_r determination of some proteins. Sodium dodecyl sulfate electrophoresis (SDS-PAGE) showed that the 56.7 kDa aAI-4 molecule dissociated into 45.0, 33.6,15.2 and 12.4 kDa submolecules, with only the two small subunits, a and β, present at high SDS concentration. This provides evidence that the aAI-4 molecules composition is α₂β².     

STABILITY OF TERTIARY STRUCTURE OF PHASEOLIN OF RED KIDNEY BEAN (PHASEOLUS VULGARIS) AS LIMITING FACTOR IN PROTEOLYSIS

The major storage protein, phaseolin, of red kidney bean (Linden variety) was purified by ammonium sulfate fractionation between 3 and 4.1 M. It was composed of three subunits with MW 49,000, 45,000, and 42,000 and there were no disulfide bonds. Phaseolin was treated with heat (121°C, 15 min), pH (1.0, 7.5, 9.0), urea (2 to 10 M), guanidine (3 to 8.5 M) and alkali (0.02 N NaOH) at 35°C to destabilize the tertiary structure. Increase of in vitro proteolysis by the prior treatments was estimated with chymotrypsin, trypsin, pepsin and pronase. Native phaseolin was more resistant than Hammarsten casein to hydrolysis by chymotrypsin, trypsin, pepsin and pronase. An equimolar mixture of chymotrypsin and trypsin gave lower proteolysis than the sum of hydrolysis by each proteinase separately. Chymotrypsin hydrolysis products of phaseolin inhibited trypsin, and trypsin hydrolysis products of phaseolin inhibited chymotrypsin. All the treatments listed above enhanced in vitro proteolysis. Phaseolin treated with 0.02 M NaOH had higher hydrophobicity and higher absorbance between 240 and 350 nm than native phaseolin, indicating that its tertiary structure was destroyed. Autoclaving at pH 7.5, treatment with 8.5 M guanidine or with 0.02 M NaOH increased the extent of proteolysis of phaseolin to a similar or higher level than Hammarsten casein. Guanidine (8.5 M) destroyed the tertiary structure of phaseolin sufficiently that almost all susceptible bonds were hydrolyzed by pepsin, chymotrypsin and trypsin (and probably pronase).     

PURIFICATION AND CHARACTERIZATION OF AN α-AMYLASE INHIBITOR FROM MAISE (ZEA MAIZE)

α-Amylase inhibitor is presented in maize seeds. It is a protein as indicated by precipitation with ammonium sulfate and trichloroacetic acid, denaturation by heat, digestion with proteases and by dye-staining. It was purified to homogeneity by ammonium sulfate precipitation and Sephadex G-75 gel filtration. It had an apparent molecular weight of 29,600 and did not contain any carbohydrate. Its properties differed from those of previously reported α-amylase inhibitors, since it was active against α-amylase of maize, produced during germination as well as against Bacillus subtilis α-amylase. It was also active against α-amylase from the insects Tribolium castaneum, Sitophilus zeamais and Rhyzopertha dominica, but it was inactive against α-amylase from human saliva, hog pancreas, Aspergillus oryzae, wheat, rye, barley, triticale, and sorghum. It was stable for 5 min at 96°C at pH 7. Maximal inhibition required at least 10 min of preincubation with the enzyme at pH 6.8 and 257deg;C. Polyacrylamide gel electrophoresis gave three protein bands, but only one was obtained in S.D.S. and mercaptoethanol.     

PURIFICATION AND CHARACTERIZATION OF AN α-AMYLASE INHIBITOR FROM RYE (SECALE CEREALE) FLOUR

An α-amylase inhibitor from rye (Secale cereale) flour has been purified to homogeneity by extraction with 70% ethanol, ammonium sulfate fractionation and column chromatography on DEAE- and CM-cellulose. The isoelectric point was pH 5.8, and the molecular weight 28,000 by polyacrylamide gel electrophoresis with different gel concentrations and 27,000 by sedimentation equilibrium centrifugation. Under denaturating conditions the molecular weight was about 14,000, indicating two subunits identical in size. The inhibitor was active towards human salivary and hog pancreatic α-amylases but inactive towards Bacillus subtilis and Aspergillus oryzae α-amylases. The pH optimum for the reaction between the rye inhibitor and human salivary α-amylase was 6.0. The inhibitor did not change activity when exposed to pH 2 (0.01M HCl), but prolonged digestion by trypsin destroyed the inhibitor. The rye α-amylase inhibitor lost about 80% of its activity after 10 min at 100°C.     

ANTIOXIDANT ACTIVITY OF PHENOLIC FRACTIONS OF WHITE BEAN (PHASEOLUS VULGARIS)

An extract from white bean seeds was prepared using 80% (v/v) acetone. Four fractions (I-IV) -were separated from the crude extract on a Sephadex LH-20 column using methanol as the mobile phase. The antioxidant activity of fractions was investigated in a β-carotene-linoleate model system. For individual fractions, IV spectra were recorded and the content of total phenolics was determined. Fractions were also characterized based on the number of phenolic compounds, and their antioxidant activity determined by TLC analysis. The presence of caffeic, p-coumaric, ferulic, and sinapic acids in the form of free and estrified compounds was found in fraction IV. One dominant phenolic compound was present in fraction III after acid hydrolysis with a maximum absorption at 278 nm. Results of the β-carotene-linoleate model system indicated that antioxidant activity of separated fractions did not correlate exactly with their content of total phenolic compounds and were in the order of IV>III>II>I. Individual fractions contained several phnolic compounds as noted by TLC. Spots on the plates sprayed with a solution of p carotene-linoleate indicated that these compounds can act as natural antioxidants. Absorption maxima in the W spectra showed that jlavonoids, and not phenolic acids, were the main phenolic compounds present in the separated fractions.     

PREPARATION AND CHARACTERIZATION OF α-AMYLASE IMMOBILIZED ON COLLAGEN MEMBRANES

Crystalline pancreatic α-amylase was codispersed with hide collagen at pH 4.0 and tanned to form a membrane which degraded starch. The optimum pH for the codispersed membrane preparation was at pH 7.0 in contrast to the soluble enzyme which was as active at pH 8.0 as at pH 7.0. The immobilized enzyme responded maximally to 0.22M chloride whereas 0.02M chloride gave optimum rates for the soluble enzyme. The immobilized enzyme resisted thermal inactivation better than the soluble α-amylase. Raising the temperatures from 30 ° to 50 °C produced a 500% increase in rate for the bound enzyme. It was also demonstrated that membranes retained greater activity when stored in starch solution than in water. The effect of glutaraldehyde concentration on membrane activity was also studied.     

THE LECTINS AND LECTIN-LIKE PROTEINS OF TEPARY BEANS (PHASEOLUS ACUTIFOLIUS) AND TEPARYCOMMON BEAN (PHASEOLUS VULGARIS) HYBRIDS

High levels of lectin activity were found in sixty cultivated and ten wild tepary (Phaseolus acutifolius) accessions. No lectin deficient varieties were observed and all examples studied contained both the phytohemagglutinin-E and L-like lectins previously described (Pusztai et al. 1987). There appeared to be no obvious differences between the wild and cultivated forms of the tepary lectins and all teparies studied contained lectin-like proteins in addition to the tepary lectins. One of the lectin related proteins (40 Kdalton subunit) was present in all teparies and may be comparable to arcelin, a lectin found in certain wild accessions of Phaselus vulgaris. All wild teparies contained a lectin related polypeptide of about 34 Kdaltons which appears to distinguish the wild teparies from the cultivated forms. Three tepary-common bean hybrids were examined and one hybrid was found to be expressing both tepary and common bean lectin genes.     

EFFECT OF EMULSIFIERS AND FUNGAL α-AMYLASE ON RHEOLOGICAL CHARACTERISTICS OF WHEAT DOUGH AND QUALITY OF FLAT BREAD

Teftoon, a flat bread made of whole wheat flour, is prepared by hand sheeting of dough, followed by baking. Different emulsifiers, like lecithin, E471 (distilled monoglyceride) and E472 (diacetylated tartaric acid esters of mono- and digelycerid of fatty acids), were added to the flour at various levels ranging between 0.25 and 1.0% w/w, and it was observed that they improved the dough characteristics. Improvement in bread quality parameters, such as force to tear and sensory acceptability, were monitored. Fungal α -amylase was also incorporated into the flour at 5–20 g/100 kg flour basis alone and in combination with the emulsifier. The force required to tear the fresh bread was decreased with emulsifier and enzyme addition; however, E472 addition at 0.75% w/w of whole wheat flour gave the softest bread. The tear force of stored bread significantly increased with storage; however, bread containing E472 showed a less increase in tear force up to a period of 3 days. The sensory acceptability was found to be higher than that of the control bread for emulsifiers, and lower for enzyme at a concentration higher than 10 g/kg flour.

PRACTICAL APPLICATIONS

Flat bread is normally consumed fresh, but the staling phenomenon starts immediately after baking this kind of bread. Today, large-scale production and increased consumer demands for high-quality bread with long shelf life have created the need for functional food additives such as emulsifiers and α -amylase enzyme. Incorporation of emulsifiers and enzyme decreased the hardness of Taftoon bread. Emulsifiers and α -amylase enzyme enhanced the flat bread dough quality. The sensory acceptability also improved with the addition of emulsifiers. Optimizing the amount of emulsifiers and enzyme required for reduction of bread hardness is vital because the quality and price of the final product depend on this parameter.     

MINERAL CONTENTS IN SEED COAT AND CANNING QUALITY OF SELECTED CULTIVARS OF DARK RED KIDNEY BEANS (PHASEOLUS VULGARIS L.)

Three different dark red kidney bean (Phaseolus vulgaris L.) cultivars (cvs. 85, 453 and Nickols) grown in Wisconsin were studied to investigate the correlations of the mineral contents in the seed coat and seed coat splits in the canned beans. In the canned product, highly significant differences ( P ≤0.01) in percentage of split seed coats were observed among the three cultivars studied. Canned cultivar 85 significantly showed fewer seed coat splits than the other two cultivars. Significant negative correlations were observed between the percentage of seed coat splits and sodium (r = ? 0.89, P ≤ 0.01), calcium ( r = ? 0.74, P ≤ 0.01) and iron (r = ? 0.79, P ≤ 0.05) contents in the seed coat. This study suggested that the mineral content of the seed coat of kidney beans may play important roles in the integrity of the seed coat during thermal processing.     

NUTRITIONAL EVALUATION OF BEAN (PHASEOLUS VULGARIS) PROTEIN. IN VIVO VERSUS IN VITRO PROCEDURE

Digestibility (D), Biological Value (BV) and Net Protein Utilization (NPU) of whole bean flour and of the protein fractions glutelin (GLU), globulin (GLO), globulin GI (GI), albumin (ALB) and a protease inhibitor/lectin rich fraction (PIL) were determined, after autoclaving (121C, 15 min). For the whole bean flour both in vivo and in vitro procedures were used. For the in vivo assay a nitrogen balance with rats was performed. In vitro evaluation was based on the mean essential amino acid index (MEAAI) and in vitro protein digestibility. Results for the whole bean flour (BF) showed no statistical differences (p ≤ 0.01) between in vivo and in vitro techniques. In vitro digestibility ranged from 75–93% for the protein fractions and was 76% for the whole bean protein; biological value for the protein fractions ranged from 62–79%, and was 85% in the whole bean protein; calculated NPU ranged from 47–76% for the fractions, and was 65% for the whole bean protein.     

PURIFICATION AND PARTIAL CHARACTERIZATION OF A PROTEINASE INHIBITOR FROM TEPARY BEAN (PHASEOLUS ACUTIFOLIUS) SEEDS

In a qualitative screening of 36 accessions of tepary beans seeds, all the accessions inhibit the activity of bovine trypsin and trypsin-like proteinases from the insect P. truncatus, and the majority of them inhibit α-amylase activity of several important insect pests. A protein proteinase inhibitor was purified from accession L-242-45, using fractional precipitation, gel filtration, ion exchange chromatography and reverse-phase HPLC. The protein showed an apparent molecular weight of 7,100 by PAGE. However, contrary to other inhibitors previously reported, the inhibitory activity was only present in the trimeric form. The protein was characterized as a serine-proteinase inhibitor that recognized trypsin, chymotrypsin and trypsin-like proteinases, but it also recognized aspartic acid proteinases from different insects. It contained no carbohydrate residues and showed a high stability at 96C at low pH.