The relationship between the amount of dietary fiber and particle morphology of an (bean paste) prepared from starchy pulses (Adzuki beans, tebou variety of kidney beans and kintoki variety of kidney beans) was clarified. Dietary fiber content in the three beans decreased with prolonged heating during an preparation. Differences in heating time did not affect the proportions of the general components of the prepared ans and had no effect on the distribution of an particle size. However, it was shown by scanning electron microscopy that many lesions existed on the surfaces of an particles heated for long periods of time. In fact prolonged heating increased the proportion of damaged particles and decreased the proportion of intact particles. There was a direct relationship between the amount of dietary fiber and the proportion of intact an particles. These results indicate that the principal reason why the amount of dietary fiber increased when the starchy pulse was heated was the production of the intact an particles on which the digestive enzymes could not easily act.
The thermal stability of sucrose phosphorylase (EC 188.8.131.52) from Streptococcus mutans was enhanced using random and site-directed mutageneses. Random mutagenesis studies revealed that eight single amino acid substitutions, T47S, S62P, Y77H, V128L, K140M, Q144R, N155S and D249G, contributed to the enhancement of thermal stability. These mutated enzymes retained their activity even after heat treatment at 55°C for 20 min, while the wild-type enzyme was drastically inactivated. The combinations of these eight mutations showed that the increase in the number of combined amino acid substitutions resulted in the higher thermal stability of the enzyme. The thermostable sucrose phosphorylase with all eight mutations retained more than 60% of initial activity after heating at 60°C for 20 min and exhibited the highest thermal stability among these mutated enzymes. The optimum temperature and pH of the thermostable sucrose phosphorylase were confirmed to be substantially similar to those of the wild-type enzyme. The thermostable sucrose phosphorylase was easily purified by heating at 65°C for 20 min with 20% sucrose, and the purified enzyme can be directly employed for the production of amylose without further purification.
The encapsulation of protein drugs in powdery forms is quite important in order to improve the stability, as well as to expand the application. When a protein solution is added to anhydrous sugars such as anhydrous maltose or anhydrous trehalose, water molecules are incorporated into sugars as water of crystallization, resulting in a protein encapsulated into sugar powders. This study investigated the enzyme encapsulation with crystal transformation of anhydrous maltose or anhydrous trehalose. The activity of the encapsulated enzyme depended on the crystal transformation rate of the anhydrous sugars. The remaining activity of the encapsulated alcohol dehydrogenase increased with the use of amorphous anhydrous trehalose and the addition of hydroxylpropyl-β-cyclodextrin into the enzyme solution.
In these studies, we found that the average starch particle size of Adzuki beans, and tebou var. and kintoki var. kidney beans was approximately 30-40 μm. In addition, these particles were globular with a C-type crystal structure. Heating enhanced the solubility and swelling power of the three kinds of isolated bean starch. No differences were noticed in the solubilities of the starches, and the swelling power of the Adzuki bean starch was higher than that of tebou var. and kintoki var. starch. The swelling power was also substantially altered by the various treatments. We found that the three different defatted starches had an A-type X-ray diffraction pattern and a higher solubility compared to the respective untreated starches. All three types of hot water-treated starch gave a similar X-ray diffraction pattern, but their solubility and swelling power were low compared to the untreated starch. Heat-moisture-treated starch gave an approximate A-type X-ray diffraction pattern, and the swelling power was even lower. In contrast, the starch that underwent the freeze-thawing treatment gave an amorphous X-ray diffraction pattern and had a much higher solubility at 50°C than any of the other samples. The resistant starch (RS) content for isolated starch from the three kinds of beans was approximately 1%. Although the RS content of defatted or hot water-treated starch did not differ from that of untreated starch, the RS content of the heat-moisture-treated starch increased to between 2.5 and 4.2%. Furthermore, the RS content of the starch exposed to the freeze-thawing treatment was the highest: approximately 4% for Adzuki bean, 9% for tebou var. and 8% for kintoki var. starch.
An acid-tolerant yeast strain MF-121 showing an excellent ability of ethanol fermentation in acidic media containing salt of pH 2.0-2.5 was isolated. Some brewing yeast, Saccharomyces cerevisiae, could scarcely ferment in the acidic media. The yeast strain MF-121 could ferment around 9% ethanol in acidic media of pH 2.5 containing 20% glucose and 1-5% sodium sulfate. Physiological properties and PCR-based 18S rRNA gene sequence showed that the yeast was closely related to Issatchenkia orientalis. The yeast could ferment ethanol in the acidic media prepared from complete hydrolysates of starch and bread.
Abstract: This article deals with characterizations of two Aspergillus niger exo-polygalacturonases (exo-PGs; EC 184.108.40.206) and a Penicillium chrysogenum exo-arabinanase (no EC number). Two exo-PGs, termed exo-PG1 and -PG2, purified from a commercial A. niger enzyme preparation (Pectinex AR) had molar masses of 82 and 56 kDa, respectively. Exo-PG1 was stable over wider pH and temperature ranges than exo-PG2. Exo-PG1 had a broad specificity towards oligogalacturonates with different DPs, while digalacturonate was the most favorable substrate for exo-PG2. Both enzymes degraded xylogalacturonan from pea hull in an exo manner to produce galacturonic acid (GalA) and Xyl-GalA disaccharide, as identified by electrospray ion trap mass spectrometry (ESI-ITMS). Moreover, exo-PGs split acetylated homogalacturonan in an exo manner, producing GalA and acetylated GalA, as shown by ESI-ITMS. An exo-arabinanase, termed Abnx, was purified from a culture filtrate of P. chrysogenum 31B. The enzyme released only arabinobiose from the non-reducing terminus of α-1,5-L-arabinan and showed no activity towards p-nitrophenyl α-L-arabinofuranoside or α-1,5-L-arabinofuranobiose. The nucleotide sequence of the abnx cDNA gene, which encodes Abnx, was determined. Abnx was found to be structurally distinct from known arabinan-degrading enzymes based on its amino acid sequence and a hydrophobic cluster analysis (HCA). The abnx cDNA gene product expressed in Escherichia coli catalyzed the release of arabinobiose from α-1,5-L-arabinan. The activity of the recombinant Abnx towards a series of arabino-oligosaccharides, as expressed by kcat/Km value, was greatest with arabinohexaose. The recombinant enzyme was found to possess trans-arabinobiosylation activity on various acceptors, such as aliphatic alcohols, sugars and sugar alcohols. The transfer product of glycerol was identified as O-α-L-arabinosyl-(1→5)-O-α-L-arabinosyl-(1→1)-glycerol on the basis of the spectral data, fast atom bombardment-mass (FAB-MS) and 1H- and 13C-NMR. Unlike endo-arabinanases, Abnx catalyzed the hydrolysis of linear arabinan without inverting the anomeric configuration.
Random mutagenesis by error-prone PCR was introduced to kojibiose phosphorylase (KP; EC 220.127.116.11) from Thermoanaerobacter brockii ATCC35047. One thermostable mutant and two DP-mutants that were defined as the mutant producing kojioligosaccharides with a degree of polymerization (DP) different from that of wild-type KP were isolated. The half-lives of a thermostable mutant, D513N were estimated to be 135 h at 60°C, 110 min at 70°C and 6 min at 75°C, respectively. They were about 1.6-fold, 7-fold and 6-fold longer than those of the wild-type enzyme, respectively. Two DP-mutants, S676N and N687I showed higher productivity of kojioligosaccharides of DP ≥ 4 than the wild-type KP under the same condition of reaction. In the case of S676N, the amount of kojipentaose increased more than 3 fold that for the wild type. Furthermore, kojioligosaccharides with a DP of ten or greater was detected by using S676N, but not by the wild type. Chimeric phosphorylases were constructed from the KP gene and trehalose phosphorylase (TP; EC 18.104.22.168) gene from T. brockii. One unique enzyme, chimera V-III was isolated by chimerization from KP and TP. Although only 125 amino acid residues in 785 residues of chimera V-III were from that of wild-type KP, chimera V-III showed not TP-type but KP-type enzyme. Since both KP and TP catalyze transglucosylation using β-G1P as the glucosyl donor, it is strongly suggested that 125 amino acid residues exchanged from KP play an important role in the binding of glucosyl acceptors. The Km value of chimera V-III for kojibiose was remarkable increased, and this chimera accepted little or no monosaccharide other than glucose; furthermore, the enzyme was able to act on laminaribiose in the absence of inorganic phosphate, and produced trisaccharides, 6-O-β-D-glucosyl-laminaribiose and laminaritriose from laminaribiose. From these results, it is assumed that chimerization causes a conformational change of the catalytic site where the reducing-end of molecules of substrates would bind, and the marked decrease of the substrate-binding activity in this region.
Alkaliphilic Bacillus sp. strain 41M-1 secretes a xylanase (termed xylanase J) that has an alkaline pH optimum. Xylanase J is a multidomain enzyme and consists of two functional domains: a glycoside hydrolase family 11 catalytic domain and an additional domain of unknown function. Protein engineering study of xylanase J indicated that the functionally unknown domain should be a xylan-binding domain (XBD) belonging to carbohydrate binding module family 36. The XBD bound to insoluble xylan and enhanced hydrolyzing activity of the adjacent catalytic domain. The XBD was successfully displayed on the surface of filamentous phage. Random mutations were introduced into the XBD gene and the repertoire was cloned for display on phage. Sequencing analysis of the xylan-binding activity-deficient mutants revealed that Phe284, Asp286, Asp313, Trp317 and Asp318 might contribute to the xylan-binding activity of XBD. The mutant XBD with amino acid substitution T316I (Thr317 was replaced by Ile) showed higher xylan-binding activity compared to the wild-type XBD. Furthermore, hydrolyzing activity of xylanase J toward insoluble xylan was improved by introducing mutation T316I.
In germination of plant seeds, storage starch is principally degraded by the combination of amylolytic enzymes. As starch is an insoluble granule, a conventional view of the degradation pathway is that the initial attack is performed by α-amylase having the starch granule-binding ability. Plant α-glucosidase was also capable of adsorbing and hydrolyzing starch granules directly, indicating a possible second pathway: the direct liberation of glucose from starch granules by plant α-glucosidase rather than the α-amylase-mediated system. We found that the starch-binding site of plant α-glucosidase was situated in its C-terminal region, of which function was independent of the catalytic domain. Site-directed mutagenesis analysis on the aromatic amino acid residues conserved in this region revealed that Trp803 and Phe895 of rice α-glucosidase were responsible for binding to starch granules. Mold α-glucosidases were devoid of the ability to attack starch granules. In plant seeds, multiple α-glucosidases have been observed. Two types of α-glucosidases, insoluble and soluble enzymes, were found in the germinating stage of rice. Expression patterns of their activities classified 14 rice varieties into two groups (Groups 1 and 2). In Group 1 varieties, insoluble enzyme decreased immediately after germination. The soluble enzyme increased by de novo synthesis. Group 2 maintained a constant activity level of insoluble and soluble α-glucosidases in germination. From Groups 1 and 2, we selected varieties of Akamai and Nipponbare, respectively, of which analysis elucidated interesting molecular mechanisms of insoluble and soluble enzymes: i) isoform and isozyme formations by post-translational proteolysis as well as by chromosomal gene expression; ii) characterization of purified enzymes exhibiting different activities to starch granules.
As the first known structures of a glycoside hydrolase family 54 enzyme, we determined the crystal structures of free and arabinose-complex forms of α-L-arabinofuranosidase from Aspergillus kawachii IFO4308 (AkAbf54). AkAbf54 comprises two domains: a catalytic domain and an arabinose-binding domain. The catalytic domain has a β-sandwich fold slightly similar to those of clan-B glycoside hydrolases. The arabinose-binding domain has a β-trefoil fold similar to that of carbohydrate-binding module (CBM) family 13. However, the arabinose-binding domain shows a number of distinctive characteristics from those of CBM family 13. Therefore, it was classified into a new CBM family, CBM42, and was referred to as AkCBM42. In the arabinose-complex structure, one of three arabinofuranose molecules bound to the catalytic domain through many interactions. Interestingly, a disulfide bond formed between two adjacent cysteine residues recognized the arabinofuranose molecule. From the location of this arabinofuranose and the results of a mutation study, the nucleophile and acid/base residues were determined to be Glu221 and Asp297, respectively. The other two arabinofuranose molecules bound to AkCBM42. The O1 atoms of both these arabinofuranose molecules are exposed to the solvent, indicating that AkCBM42 binds arabinofuranose residues linked to the xylan backbones of arabinoxylans. Binding assay and affinity gel electrophoresis analysis with insoluble polysaccharides, and ITC analysis with mono- or oligosaccharides revealed a unique function of AkCBM42. This is the first example of a CBM that can specifically recognize the side-chain moieties of branched polysaccharides.
N-Linked oligosaccharide processing enzymes are key enzymes in the biosynthesis of N-linked oligosaccharides. These enzymes are a molecular target for inhibition by anti-viral agents that interfere with the formation of essential glycoproteins required in viral assembly, secretion and infectivity. We think that the molecular recognition of three kinds of glucosidases (family 13 and family 31 α-glucosidases and endoplasmic reticulum glucosidases) are different. Therefore, glycon and aglycon specificity profiling of glucosidases was an important approach for the research of glucosidase inhibitors. We carried out the profiling of glucosidases using small molecules as a probe. Moreover, we designed and synthesized three types of glucosidase inhibitors. These compounds were evaluated with regard to their ability to inhibit glucosidases in vitro, and were also tested in a cell culture system. We found some compounds having glucosidase inhibitory activity and anti-viral activity.
We found a new enzyme, neopullulanase (EC 22.214.171.124), and showed that it catalyzes the hydrolysis of α-1,4- and α-1,6-glucosidic linkages, as well as transglycosylation to form α-1,4- and α-1,6-glucosidic linkages. Based on the series of experimental results using neopullulanase, we pointed out the same catalytic machinery and the common catalytic mechanism of the enzymes that catalyze these four reactions, and thus, proposed and defined the concept of the α-amylase family. Mutational analyses provided the evidence that one active center of neopullulanase participates in all four reactions; the hydrolysis of α-1,4- and α-1,6-glucosidic linkages and transglycosylation to form α-1,4- and α-1,6-glucosidic linkages. Structural analyses provided the conclusive proof that one active center of neopullulanase participates in all four reactions. We have been trying to interconvert glucanohydrolases/glucanotransferases, and their specificities and create tailor-made industrially useful enzymes based on the concept of the α-amylase family. Based on the concept, we engineered Thermus amylomaltase to essentially erase hydrolytic activity and created perfect 4-α-glucanotransferase for the industrial production of cycloamylose. The concept of the α-amylase family is demonstrated here again as a rational tool for interconverting glucanohydrolases/glucanotransferases, and their specificities.
Barley α-amylase binds sugars at two sites on the enzyme surface in addition to the active site. Crystallography and site-directed mutagenesis highlight the importance of aromatic residues at these surface sites as demonstrated by Kd values determined for β-cyclodextrin by surface plasmon resonance and for starch granules by adsorption analysis. Activity towards amylopectin and amylose follows two different kinetic models, degradation of amylopectin being composed of a fast and a slow component, perhaps reflecting attack on A and B chains, respectively, whereas amylose hydrolysis follows a simple Michaelian kinetics. β-cyclodextrin binding at surface sites inhibits only the fast reaction in amylopectin degradation. Site-directed mutagenesis and activity analysis, furthermore show that one of the surface binding sites as well as individual subsites in the active site cleft have distinct roles in the multiple attack on amylose. Although the two isozymes AMY1 and AMY2 share ligands for three structural calcium ions, they differ importantly in the effect of calcium on activity and stability, AMY1 having the higher affinity and the lower stability. The role of the individual calcium ions is studied by mutagenesis, crystallography and microcalorimetry. Further improvement of recombinant AMY2 production allows future direct mutational analysis in this isozyme. Specific proteinaceous inhibitors act on α-amylases of different origin. In the complex of barley α-amylase/subtilisin inhibitor (BASI) with AMY2, a fully hydrated calcium ion at the protein interface mediates contact between inhibitor residues and the enzyme catalytic groups in a manner that depends on calcium and which can be suppressed by site-directed mutagenesis of Glu168 in BASI. Finally certain inhibitors and enzymes are targets of the disulphide reductase thioredoxin h that attacks a specific disulphide bond in BASI and, remarkably, reduces two different disulphide bonds in the barley monomeric and dimeric amylase inhibitors that both belong to the CM-proteins and inhibit animal α-amylase.