• BMB Reports
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• v.37 no.4
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• pp.422-428
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• 2004

Raw-starch-digesting $\alpha$-amylase (Amyl III) was purified to an electrophoretically pure state from the extract of a koji culture of Aspergillus awamori KT-11 using wheat bran in the medium. The purified Amyl III digested not only soluble starch but also raw corn starch. The major products from the raw starch using Amyl III were maltotriose and maltose, although a small amount of glucose was produced. Amyl III acted on all raw starch granules that it has been tested on. However, it was considered that the action mode of the Amyl III on starch granules was different from that of glucoamylase judging from the observation of granules under a scanning electron microscope before and after enzyme reaction, and also from the reaction products. Glucoamylase (GA I) was also isolated and it was purified to an electrophoretically pure state from the extract. It was found that the electron micrographic features of the granules after treatment with the enzymes were quite different. A synergistic effect of Amyl III and GA I was observed for the digestion of raw starch granules.

• Food Science and Biotechnology
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• v.17 no.1
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• pp.180-183
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• 2008

Digestion properties of 3 types of cereals, white rice, brown rice, and barley, were measured after cooking or grinding. Regardless of the processing methods, white rice showed the highest rate and the greatest extent of digestion, whereas barley showed the lowest values. During the early digestion period, cooked white rice kernels had a larger k (kinetic constant) value than uncooked white rice flour, indicating that cooking induced faster digestion than grinding. In the case of brown rice and barley, the cell wall in cooked kernels remained intact and resulted in a lower k values than those of uncooked flour. However, after 3 hr of digestion, the total digestion extent was greater for the cooked brown rice and barley than that for uncooked flours. The high content of slowly digestible starch (SDS) in cooked brown rice and barley might be due to the starch fraction which was protected by the cell wall. The resistant starch (RS) content, however, was greater for the uncooked flours than that for cooked kernels. The cooked kernels of 3 cereal samples tested showed higher glycemic index (GI) values than the uncooked flours.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2001.06a
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• pp.40-45
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• 2001

Raw starch-digesting amylase (BF-2A, M.W. 93, 000 Da) from Bacillus circulans F-2 was converted to two components during digestion with subtilisin. Two components were separated and designated as BF-2A' (63, 000 Da) and BF-2B (30, 000 Da), respectively. BF-2A' exhibited the same hydrolysis curve for soluble starch as the original amylase (BF-2A). Moreover, the catalytic activities of original and modified enzymes were indistinguishable in $K_{m}$, Vmax for, and in their specific activity for soluble starch hydrolysis. However, its adsorbability and digestibility on raw starch was greatly decreased. Furthermore, the enzymatic action pattern on soluble starch was greatly different from that of the BF-2A. A smaller peptide (BF-2B) showed adsorb ability onto raw starch. By these results, it is suggested that the larger peptide (BF-2A') has a region responsible for the expression of the enzyme activity to hydrolyze soluble substrate, and the smaller peptide (BF-2B) plays a role on raw starch adsorption. A similar phenomenon is observed during limited proteinase K, thermolysin, and endopeptidase Glu-C proteolysis of the enzyme. Fragments resulting from proteolysis were characterized by immunoblotting with anti-RSDA. The proteolytic patterns resulting from proteinase K and subtilisin were the same, producing 63- and 30-kDa fragments. Similar patterns were obtained with endopeptidase Glu-C or thermolysin. All proteolytic digests contained a common, major 63-kDa fragment. Inactivation of RSDA activity results from splitting off the C-terminal domain. Hence, it seems probable that the protease sensitive locus is in a hinge region susceptible to cleavage. Extracellular enzymes immunoreactive toward anti-RSDA were detected through whole bacterial cultivation. Proteins of sizes 93-, 75-, 63-, 55-, 38-, and 31-kDa were immunologically identical to RSDA. Of these, the 75-kDa and 63-kDa proteins correspond to the major products of proteolysis with Glu-C and thermolysin. These results postulated that enzyme heterogeneity of the raw starch-hydrolysis system might arise from the endogeneous proteolytic activity of the bacterium. Truncated forms of rsda, in which the gene sequence encoding the conserved domain had been deleted, directed the synthesis of a functional amylase that did not bind to raw starch. This indicates that the conserved region of RSDA constitutes a raw starch-binding domain, which is distinct from the active centre. The possible role of this substrate-binding region is discussed.d.

• The Korean Journal of Food And Nutrition
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• v.36 no.5
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• pp.368-378
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• 2023

Changes in contents of free sugars, amino acids, and fatty acids of legumes were analyzed for each phase of in vitro digestion. In addition, contents of resistant starch in raw and digested pulses were compared. Soybeans, kidney beans, cowpeas, and chickpeas were analyzed. An in vitro digestion model was used to analyze contents of nutrients using LC-MS and GC-MS. Stachyose in kidneybean, cowpea, and chickpea increased as the digestion phase progressed. In four types of legumes, raffinose slightly decreased or showed no significant difference between the Oral phase and the BBMV phase. Content of glucose, a monosaccharide, increased during the BBMV phase. During the digestion phase, levels of free amino acids and free fatty acids also increased. Content of resistant starch was reduced compared to that in the raw material. It was 0.01g/100 g food in soybean, 1.06 g/100 g food in red kidney bean, 0.77g/ 100g food in cowpea, and 0.76 g/100 g food in chickpea. It was confirmed that nutrients in the in vitro digestion model were liberated at each digestion phase with changes in the content of resistant starch. These results are expected to be used as fundamental data for obtaining bioavailability of nutrients.

• Microbiology and Biotechnology Letters
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• v.16 no.3
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• pp.231-237
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• 1988

Several methods to produce ethanol from tapioca starch were examined. Among four methods tested, alcohol fermentation with extruded tapioca starch was the most effective, which alcohol yield was 460.5 f/ton. After 69hours reaction with Rhizopus sp. glucoamylase, 108.7mg/$m\ell$ of reducing sugar were produced from extruded tapioca and 43.8mg/$m\ell$ from raw tapioca starch. In alcohol fermentation with extruded tapioca, the high concentration of alcohol at early stage prevented bacterial contamination and the fermentation rate was increased due to the high saccharifying power of glucoamylase on the extruded starch, but extrusion temperature had no influence on the fermentability, Scanning electron microscopy showed that the extrusion process changed the structure of tapioca starch granule to more susceptible form to glucoamylase attack than the raw starch. And glucoamylase of Rhizopus sp. had stronger digestion activity on both extruded tapioca and raw tapioca starch than that of Aspergillus usamii.

• BMB Reports
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• v.37 no.4
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• pp.429-438
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• 2004

Complementary DNAs encoding $\alpha$-amylases (Amyl I, Amyl III) and glucoamylase (GA I) were cloned from Aspergillus awamori KT-11 and their nucleotide sequences were determined. The sequence of Amyl III that was a raw starch digesting $\alpha$-amylase was found to consist of a 1,902 bp open reading frame encoding 634 amino acids. The signal peptide of the enzyme was composed of 21 amino acids. On the other hand, the sequence of Amyl I, which cannot act on raw starch, consisted of a 1,500 bp ORF encoding 499 amino acids. The signal peptide of the enzyme was composed of 21 amino acids. The sequence of GA I consisted of a 1,920 bp ORF that encoded 639 amino acids. The signal peptide was composed of 24 amino acids. The amino acid sequence of Amyl III from the N-terminus to the amino acid number 499 showed 63.3% homology with Amyl I. However, the amino acid sequence from the amino acid number 501 to C-terminus, including the raw-starch-affinity site and the TS region rich in threonine and serine, showed 66.9% homology with GA I.

• Journal of Microbiology and Biotechnology
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• v.2 no.3
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• pp.189-196
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• 1992

A $\alpha$-l, 4-D-glucan maltohydrolase $(\beta$-amylase), secreted by the mesophilic aerobic bacterium Bacillus polymyxa No.26, was purified and characterized. The enzyme production was increased after a logarithmic phase of bacterial growth and paralleled with the onset of bacterial sporulation. By applying anion exchange chromatography and gel filtration the enzyme was purified 16.7-fold and had a specific activity of 285.7 units/mg. Two enzyme activities were eluted on a column of DEAE-Sephadex chromatography, and they were designated as E-I for a major enzyme peak and E-II for a minor peak. Of them, E-I enzyme peak was further purified by using gel chromatography. The molecular mass of this enzyme was determined to be 64, 000 daltons and consisted of a single subunit, showing an isoelectric point of 8.9. The enzyme was able to attack specifically the $\alpha$-l, 4-glycosidic linkages in soluble starch and caused its complete hydrolysis to maltose and $\beta$-limited dextrin. This amylolytic enzyme displayed a temperature optimum at $45^\circ{C}$ and a pH optimum at 7.0. The amino acid composition of the purified enzyme was quite similar to the other bacterial $\beta$-amylases reported. Surprisingly, the purified enzyme from this aerobe only exhibited hydrolytic activity on soluble starch, not on starch granules. The degradation of from starch by $\beta$-amylase was greatly stimulated by pullulanase addition. These results differentiated from other $\beta$-amylases reported. Based on a previous result that showed the enzyme system involves in effective degradation of raw starch granules, this result strongly suggested that the purified enzyme (E-I) can be a synergistic part of starch granule-digestion and E-II plays a crucial role in digestion of starch granules.

• Nutrition Research and Practice
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• v.3 no.2
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• pp.149-155
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• 2009

In this study, we investigated the heat effect of digestion-resistant fraction (RF) from soybean on retarding bile acid transport in vitro. The RFs from soybean retarded bile acid transport. A raw, unheated RF of soybean (RRF-SOY) was significantly more effective than the heated RF of soybean (HRF-SOY). The RS1 which physically trapped in milled grains and inaccessible to digestive enzyme after 18 hrs incubation level of content in RRF-SOY was found to be as high as 24.1% and after heating the RS1 of HRF-SOY was significantly reduced to 16.8%. The X-ray diffraction pattern of RF from soybean was altered after heat treatment. The RFs from soybean were characterized by peak at diffraction angles of $12.0^{\circ}$ and $20.0^{\circ}$ corresponding to RS content. Cellulose contents of RRF-SOY was 5% higher than that of HRF-SOY and pentosan contents of RRF-SOY was 5% higher than that of HRF-SOY, too. Whereas the hemicellulose content of RRF-SOY was 13% lower than HRF-SOY.

• Microbiology and Biotechnology Letters
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• v.13 no.4
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• pp.329-337
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• 1985

A potent mold strain was selected to digest raw starch, which was classified as a strain of Rhizopus of zoe. Its amylase production was maximized when grown on wheatbran media for 3 days at 3$0^{\circ}C$ and initial pH 4. The crude enzyme was tested for ethanol fermentation by yeast, Saccharomyces cerevisiae IFO 7026, on various starchymaterials and the ethanol production after 4 days was: 9.4％ from rice powder, 9％ from corn powder, 8.1％ from sweet potato powder, and 5.4％ from potato powder, respectively.

• Microbiology and Biotechnology Letters
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• v.10 no.2
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• pp.123-132
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• 1982

These experiments were conducted to investigate the enzymatic characteristics of the purified $\alpha$-amylase (F-2A) of Bacillus circulans F-2 and the digestion rate of various starches. 1. The molecular weight was estimated to be 93000 by SDS-polyacrylamide disc gel electrophoresis. The isoelectric point was about pH 5.0. The optimum pH for the enzyme action was 6.0-6.5 and the stable pH ranged pH 5.5-12.0. The optimum temperature was 6$0^{\circ}C$, and the purified $\alpha$-amylase was stable below 4$0^{\circ}C$. 2. The purified $\alpha$-amylase was activated by Mn$^{＋＋}$ and Co$^{＋＋}$, whereas it was inhibited by Ag$^{＋}$, HT$^{＋＋}$, Cu$^{＋＋}$ and Pb$^{＋＋}$. 3. The purified $\alpha$-amylase is considered to have no sulfhydryl residue essential for its catalytic activity. 4. Michaelis constant (Km) was 1.704 mg/$m\ell$. Activation energy between 25-4$0^{\circ}C$ was 12.297 Kcal/mole, and between 40-6$0^{\circ}C$, it was 7.831 Kcal/mole. 5. The hydrolysis product from soluble starch, amylose and amylopectin in the early stage of hydrolysis was G$_{6}$, and as hydrolysis proceeds, G$_4$and G$_2$appeared. 6. Products from each oligosaccarides are as follows: G$_4$longrightarrow G$_2$＋ G$_2$,G$_3$ ＋G$_1$,G$_{5}$longrightarrow G$_4$＋G$_1$,G$_{6}$longrightarrowG$_4$＋ G$_2$,G$_{7}$ G$_4$,G$_{8}$longrightarrow G$_4$＋G$_4$, 7. On raw potato starch, raw sago starch and raw yam starch, the purified enzyme exhibited a remarkably high digestion rate than Porcine pancreatic amylase and Streptococcus bovis amylase.