Soybean Β-amylase Research Articles

Structural titration of mobile components in the active site of soybean β-amylase with maltose

Structural titration of mobile components in the active site of soybean β-amylase with maltose

Kinetic monitoring of site-directed mutational β-amylase catalysis on a 27-MHz QCM

► A QCM was applied for site-directed mutagenesis of soybean β-amylase (SBA). ► Effects of mutations appeared on the time-course of each frequency change. ► Kinetic parameters of k on , k off , and k cat for mutants were obtained. A quartz-crystal microbalance (QCM) technique was applied to analyze the effects of site-directed mutagenesis of soybean β-amylase (SBA) by using an amylose-immobilized 27-MHz QCM in buffer solution. We followed directly and quantitatively the formation and decomposition of an enzyme-substrate (ES) complex as frequency changes (mass changes) on the QCM plate. It has been predicted from X-ray crystallography of SBA that two carboxyl groups, Glu186 and Glu380, are the crucial residues for the catalytic hydrolysis of α-1,4-glycoside linkage, and the side chain Thr342 on the inner loop structure bends the glycoside at subsites −1 and +1 of the substrate, thereby accelerating the hydrolysis. When SBA wild-type (WT) was employed, the frequency increased (mass decreased) simply because of the hydrolysis of the substrate on the QCM plate. When T342S and T342A mutants having small side chains with no hydrogen bond on the inner loop were employed, the frequency change showed a sigmoidal curve pointing to an increase in ES complex formation and hydrolysis of the substrate. When E186A and E380A mutants were employed, the frequency decreases (mass increases) showed only enzyme binding to the substrate ES complex formation, but not hydrolysis. From curve fitting of these different patterns of frequency changes, we obtained all kinetic parameters for each step of the enzyme binding to the substrate ( k on ), the enzyme release from the ES complex ( k off ), and the catalytic rate constant ( k cat ) for WT and mutants. From the frequency change patterns of the substrate-immobilized QCM we evaluated how mutations of the enzyme affect k on , k off , and k cat values for ES complex formation and decomposition. We also evaluated the effects of accelerators and inhibitors on enzymes from the frequency change pattern.

Structure and Function of Soybean β-Amylase

Structure and Function of Soybean β-Amylase

Structural titration of two mobile loops in trigonal soybean β-amylase crystal with maltose

Structural titration of two mobile loops in trigonal soybean β-amylase crystal with maltose

Structural analysis of Thr342 mutants of soybean β-amylase: the role of conformational changes of two loops in the catalytic mechanism

Structural analysis of Thr342 mutants of soybean β-amylase: the role of conformational changes of two loops in the catalytic mechanism

Structural Analysis of Threonine 342 Mutants of Soybean β-Amylase: Role of a Conformational Change of the Inner Loop in the Catalytic Mechanism,

Two different conformations of the inner loop (residues 340-346) have been found in the soybean beta-amylase structures. In the "product form", the Thr 342 residue creates hydrogen bonds with Glu 186 (catalytic acid) and with the glucose residues at subsites -1 and +1, whereas most of those interactions are lost in the "apo form". To elucidate the relationship between the structural states of the inner loop and the catalytic mechanism, Thr 342 was mutated to Val, Ser, and Ala, respectively, and their crystal structures complexed with maltose were determined together with that of the apo enzyme at 1.27-1.64 A resolutions. The k(cat) values of the T342V, T342S, and T342A mutants decreased by 13-, 360-, and 1700-fold, respectively, compared to that of the wild-type enzyme. Whereas the inner loops in the wild-type/maltose and T342V/maltose complexes adopted the product form, those of the T342S/maltose and T342A/maltose complexes showed the apo form. Structural analyses suggested that the side chain of Thr 342 in product form plays an important role in distorting the sugar ring at subsite -1, stabilizing the deprotonated form of Glu 186, and grasping the glucose residue of the remaining substrate at subsite +1. The third hypothesis was proved by the fact that T342V hydrolyzes maltoheptaose following only multichain attack in contrast to multiple attack of the wild-type enzyme.

Distorted sugar ring in the active site of soybean β-amylase

Distorted sugar ring in the active site of soybean β-amylase

The Roles of Glu186 and Glu380 in the Catalytic Reaction of Soybean β-Amylase

It has previously been suggested that the glutamic acid residues Glu186 and Glu380 of soybean β-amylase play critical roles as a general acid and a general base catalyst, respectively. In order to confirm the roles of Glu186 and Glu380, each residue was mutated to a glutamine residue and the crystal structures of the substrate (E186Q/maltopentaose) and product (E380Q/maltose) complexes were determined at resolutions of 1.6 Å and 1.9 Å, respectively. Both mutant enzymes exhibited 16,000- and 37,000-fold decreased activity relative to that of the wild-type enzyme. The crystal structure of the E186Q/maltopentaose complex revealed an unambiguous five-glucose unit at subsites −2 to +3. Two maltose molecules bind on subsites −2 to −1 and +2 to +3 in the E380Q/maltose complex, whereas they bind in tandem to −2 to −1 and +1 to +2 in the wild-type/maltose complex. The conformation of the glucose residue at subsite −1 was identified as a stable 4C 1 α-anomer in the E380Q/maltose complex, whereas a distorted ring conformation was observed in the wild-type/maltose complex. The side-chain movement of Gln380 to the position of a putative attacking water molecule seen in the wild-type enzyme caused the inactivation of the E380Q mutant and an altered binding pattern of maltose molecules. These results confirm the critical roles played by Glu186 in the donation of a proton to the glycosidic oxygen of the substrate, and by Glu380 in the activation of an attacking water molecule. The observed difference between the backbones of E186Q/maltopentaose and E380Q/maltose in terms of Thr342 suggests that the side-chain of Thr342 may stabilize the deprotonated form of Glu186 after the cleavage of the glycosidic bond.

Sequencing of soy oil and soy lecithin allergens

Rationale It has been shown that soy oil and soy lecithin may contain allergens. The aim of the present study is to characterize, by means of the physico-chemical and immunological methods, the allergenic proteins of soybean seed transferred to soy oil and soy lecithin during industrial refining. Methods Extraction of proteins was conducted using a buffer solution. The identification of allergens used specific IgE from a soybean allergic patient. Bi-dimensional electrophoresis and sequencing from N and C terminals were used to characterize the allergenic proteins. Results Two allergenic proteins present in soybean seed, soy oil and soy lecithin have been isolated and identified: one 56 kDa non glycosylated allergen, whose isoelectric point of 5.4 has been identified as soybean β-Amylase (7S). One 20 kDa non glycosylated allergen, whose isoelectric point of 4.6 has been identified as Kunitz Trypsin inhibitor [Glycine max] (2S). Conclusions We have sequenced two allergenic proteins present as hidden allergens in soy oil and soy lecithin. It is to note that these highly refining-resistant proteins are not the main proteins of soybean seed.

Structural and Enzymatic Analysis of Soybean β-Amylase Mutants with Increased pH Optimum

Comparison of the architecture around the active site of soybean beta-amylase and Bacillus cereus beta-amylase showed that the hydrogen bond networks (Glu380-(Lys295-Met51) and Glu380-Asn340-Glu178) in soybean beta-amylase around the base catalytic residue, Glu380, seem to contribute to the lower pH optimum of soybean beta-amylase. To convert the pH optimum of soybean beta-amylase (pH 5.4) to that of the bacterial type enzyme (pH 6.7), three mutants of soybean beta-amylase, M51T, E178Y, and N340T, were constructed such that the hydrogen bond networks were removed by site-directed mutagenesis. The kinetic analysis showed that the pH optimum of all mutants shifted dramatically to a neutral pH (range, from 5.4 to 6.0-6.6). The Km values of the mutants were almost the same as that of soybean beta-amylase except in the case of M51T, while the Vmax values of all mutants were low compared with that of soybean beta-amylase. The crystal structure analysis of the wild type-maltose and mutant-maltose complexes showed that the direct hydrogen bond between Glu380 and Asn340 was completely disrupted in the mutants M51T, E178Y, and N340T. In the case of M51T, the hydrogen bond between Glu380 and Lys295 was also disrupted. These results indicated that the reduced pKa value of Glu380 is stabilized by the hydrogen bond network and is responsible for the lower pH optimum of soybean beta-amylase compared with that of the bacterial beta-amylase.

Maltogenic Amylase from Thermomonospora viridis TF-35 Is Well Suited for the Production of Extremely High G2-syrup

Thermomonospora viridis TF-35 maltogenic amylase (TVA) and Taka-amylase A (TAA) converted about 66-68% of soluble starch into maltose (G2) as a main product. High G2 syrup containing 89% G2 (G2-89 syrup) was converted to extremely high G2 syrup containing 94.2% G2 in the presence of TVA. In the case of TAA, G2-89 syrup was converted to G2 syrup containing 86% G2. We compared the transfer action of TVA and TAA. It is suggested that the transglycosyl activity of TVA is higher than that of TAA in the decomposition of G3, and that although TAA degrades G2, TVA does not degrade G2-89 syrup at a high concentration. Because of the above differences in transfer action, the current industrial production of extremely high G2 syrup containing more than 94% G2 can be produced by a cooperative 2-step reaction with the enzyme after obtaining starch hydrolyzates containing about 89% G2 by the simultaneous actions of soybean β-amylase and Flavobacterium odoratum isoamylase from a high concentration of starch liquefite.

Mechanisms of aggregation of alpha- and beta-amylases in aqueous dispersions

A lot of attention has been paid to elucidate the mechanisms of enzyme inactivation and aggregation so that stabilization strategies can be specifically applied to prevent thermal and/or physical denaturation of enzymes in industrial processes. In the present study, we have investigated the mechanisms of aggregate formation of two model enzymes, α-amylase from Bacillus subtilis and soybean β-amylase, under different experimental conditions. Aggregation can be observed among α-amylase particles after 1 week of aging either through physical contacts or through the intermolecular disulfide bond formed by the surface Cys residues from two enzyme monomer. In contrast, β-amylase does not form any aggregation probably due to the electrostatic repulsion and steric hindrance. Both surfactant sodium n-dodecyl sulfate and reducing agent beta-mercaptoethanol (BME) dramatically reduce the aggregate size, especially for α-amylase, by destroying the intermolecular disulfide linkages. Sonication appears to improve the formation of aggregates only for α-amylase. The average aggregate sizes are usually the largest at the isoelectric points of these two enzymes. A protein having more free Cys residues on its surface does not guarantee disulfide bond-induced aggregation.

Automated docking of alpha-(1-->4)- and alpha-(1-->6)-linked glucosyl trisaccharides and maltopentaose into the soybean beta-amylase active site.

The Lamarckian genetic algorithm of AutoDock 3.0 was used to dock alpha-maltotriose, methyl alpha-panoside, methyl alpha-isopanoside, methyl alpha-isomaltotrioside, methyl alpha-(6(1)-alpha-glucopyranosyl)-maltoside, and alpha-maltopentaose into the closed and, except for alpha-maltopentaose, into the open conformation of the soybean beta-amylase active site. In the closed conformation, the hinged flap at the mouth of the active site closes over the substrate. The nonreducing end of alpha-maltotriose docks preferentially to subsites -2 or +1, the latter yielding nonproductive binding. Some ligands dock into less optimal conformations with the nonreducing end at subsite -1. The reducing-end glucosyl residue of nonproductively-bound alpha-maltotriose is close to residue Gln194, which likely contributes to binding to subsite +3. In the open conformation, the substrate hydrogen-bonds with several residues of the open flap. When the flap closes, the substrate productively docks if the nonreducing end is near subsites -2 or -1. Trisaccharides with alpha-(1-->6) bonds do not successfully dock except for methyl alpha-isopanoside, whose first and second glucosyl rings dock exceptionally well into subsites -2 and -1. The alpha-(1-->6) bond between the second and third glucosyl units causes the latter to be improperly positioned into subsite +1; the fact that isopanose is not a substrate of beta-amylase indicates that binding to this subsite is critical for hydrolysis.

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.

Automated docking of maltose, 2-deoxymaltose, and maltotetraose into the soybean beta-amylase active site.

In this study, products and substrates were docked into the active site of beta-amylase using the simulated annealing algorithm AutoDock. Lowest-energy conformers reproduced known crystallographic atom positions within 0.4 to 0.8 A rmsd. Docking studies were carried out with both open and closed configurations of the beta-amylase mobile flap, a loop comprising residues 96 to 103. Ligands with two rings docked within the cleft near the active site when the flap was open, but those with four rings did not. The flap must be closed for alpha-maltotetraose to adopt a conformation allowing it to dock near the crystallographically determined subsites. The closed flap is necessary for productive but not for nonproductive binding, and therefore it plays a essential role in catalysis. The gain in total binding energy upon closing of the flap for alpha-maltose docked to subsites -2, -1 and +1, +2 is about 22 kcal/mol, indicating more favorable interactions are possible with the flap closed. Larger intermolecular interaction energies are observed for two alpha-maltose molecules docked to subsites -2, -1 and +1, +2 than for one alpha-maltotetraose molecule docked from subsites -2 to +2, suggesting that it is only upon cleavage of the alpha-1,4 linkage that optimal closed-flap binding can occur with the crytallographically determined enzyme structure.

The Crystal Structure of the Sevenfold Mutant of Barley β-Amylase with Increased Thermostability at 2.5 Å Resolution

The three-dimensional structure of the sevenfold mutant of barley β-amylase (BBA-7s) with increased thermostability was determined by X-ray crystallography. The enzyme was purified as a single component and crystallized by a hanging drop method in the presence of 14 % PEG 6000. The crystals belong to space group P 4 32 12 with cell dimensions a= b=72.11 Å, c =250.51 Å. The diffraction data up to 2.5 Å were collected after soaking the crystal in 100 mM maltose with R symof 8.6 %. The structure was determined by a molecular replacement method using soybean β-amylase (SBA) as a search model and refined to an R -factor of 18.7 %. The final model included 500 amino acid residues, 141 water molecules and three glucose residues, which were located at subsites 1-2 and 4 in the active site. The r.m.s. distance of 485 C αatoms between BBA-7s and SBA was 0.62 Å. Out of the seven mutated amino acids, four (Ser295Ala, Ile297Val, Ser351Pro and Ala376Ser) were substitutions from the common residues with SBA to the thermostable forms. A comparison of the structures of BBA-7s and SBA indicated that the side-chain of Ser376 makes new hydrogen bonds to the main-chain of an adjacent β-strand, and that the side-chains of Val297 reduce an unfavorable interaction between the side-chains of Ala314. The mutation of Ser295Ala breaks the hydrogen bond between Ser295 OG and Tyr195 OH, which seems to be the reason for the unoccupied glucose residue at subsite 3. The tandem mutations at 350-352 including substitutions to two Pro residues suggested the reduction of main-chain entropy in the unfolded structure of this solvent-exposed protruded loop.

An Isoelectric Separation of Soybean β-Amylase Isoforms and Their Enzymic Characteristics.

Authentic soybean β-amylase preparation, purified to homogeneity as judged by SDS-PAGE by using an affinity purification step, was composed of four pI-differing isoforms. By chromatofocusing, these isoforms were separated into three fractions, designated as fractions 1-3 in the order of elution. Fraction 1 contained two isoforms having the same molecular mass (55,989 Da), as measured by mass spectrometric analysis, with different pIs, 5.32 (Isoform I) and 5.22 (Isoform II). Fraction 2 showed a single isoform having a molecular mass of 55,994 Da and having a pI of 5.09. This component, named Isoform III, existed rather in excess in a mixture of the authentic enzyme isoforms. The remainder (fraction 3) also contained a single component (Isoform IV) which has a molecular mass of 56,310 Da with a pI of 4.97. Chemical analyses indicated that the N-termini and the C-terminal tripeptides of four pI-separated isoforms mentioned are similar to one another, and are blocked and are NH2-Val-Asp-Gly-COOH, respectively. Moreover, enzymic properties involving specific activity and the value of kcat/Km for the above three fractions are almost the same, and also agreed completely with those of an unfractionated authentic β-amylase preparation.

Maltal Binding Mechanism and a Role of the Mobile Loop of Soybean β-Amylase.

The inhibition of hydration of maltal (α-D-glucopyranosyl-(l→4)-2-deoxy-D-glucal) catalyzed by soybean ²-amylase with 4-0-α-D-glucopyranosyl-(l→4)-l-deoxynojirimycin (GDN) was investigated at 25°C and at pH 5.4. As the concentrations of GDN used were comparable to that of the enzyme, Henderson's treatment was applied to this system. It was found that two maltal molecules bind to the enzyme according to a random mechanism and GDN inhibits the hydration of maltal competitively at subsites 1 and 2, and noncompetitively at the other site. On the basis of this result, it was inferred that the role of the mobile loop of this enzyme is to create a convenient catalytic environment for the hydration, and the closing of the active site by the mobile loop is induced by the binding of maltal.

Affinity Purification of β-Amylases Originating from Plant Using Cyclomaltohexaose-Immobilized Sepharose 6B in the Presence of Ammonium Sulfate

This paper reports a novel method of affinity purification of soybean and barley β-amylase on cyclomaltohexaose-immobilized Sepharose. Until now, it has been shown that sweet potato β-amylase can be purified using the above absorbent but β-amylases from soybean and barley seeds cannot. We found that soybean and barley β-amylase becomes adsorbed specifically on the above absorbent if it is in solution with 1 to 2 M ammonium sulfate, and the adsorbed enzyme can be easily eluted with a buffer containing no ammonium sulfate. Employing this procedure, soybean β-amylase was demonstrated to be purified about 10-fold to homogeneity as judged from analysis of both a sodium dodecyl sulfate-gel electrophoresis and the specific activity, using a crude enzyme preparation (sp act 95 U/mg) as a starting material. The specific activity of this highly purified enzyme (950 U/mg) was almost the same as that of crystallized soybean β-amylase at 37°C.

Biochemical and physiological studies of soybean β-amylase

Soybean ( Glycine max L. Merr.) seed β-amylase has been identified among the ethanol-soluble proteins, purified and several cDNA clones isolated [Ren et al. (1993) Phytochemistry 33, 535]. Both soybean and barley ( Hordeum vulgare L.) β-amylases were soluble in ethanol solutions up to 60%. The solubility of the enzyme decreased with increasing ethanol concentration and dropped sharply between 50 and 60% ethanol. The results showed that β-amylase is an enzyme which can tolerate extensive ethanol treatment at room temperature without losing activity. In soybean seeds, β-amylase appeared to be located neither in protein bodies nor in a membrane fraction. The β-amylase accumulated in the developing seed and disappeared from the germinating cotyledons at a rate similar to that of the total seed protein. Soybean β-amylase was resistant to digestion by some proteases, such as trypsin, chymotrypsin and a protease from Staphylococcus aureus V8. Very little of the total amylase activity in soybean leaves and roots was ethanol soluble. In eight other legume seeds tested, amylase activity was very low compared to the activity present in soybean seeds.