• Microbiology and Biotechnology Letters
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• v.49 no.1
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• pp.45-56
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• 2021

Improved amylases were developed from protoplast fusants of two amylase-producing Aspergillus species. Twenty regenerated fusants were screened for amylase production using Remazol Brilliant Blue agar. Crude enzyme extracts produced by solid state fermentation of rice bran were assayed for activity. Three variable factors (temperature, pH and enzyme type) were optimized to increase the amylase activity of the parents and selected fusants using rice bran medium and solid state fermentation. Analysis of this optimization was completed using the Central Composite Design (CCD) of the Response Surface Methodology (RSM). Amylase activity assays conducted at room temperature and 80℃ demonstrated that Aspergillus designates, T5 (920.21 U/ml, 966.67 U/ml), T13 (430 U/ml, 1011.11 U/ml) and T14 (500.63 U/ml, 1012.00 U/ml) all exhibited improved function making them the preferred fusants. Amylases produced from these fusants were observed to be active over the entire pH range evaluated in this study. Fusants T5 and T14 demonstrated optimal activity under acidic and alkaline conditions, respectively. Fusants T13 and T14 produced the most amylase at 72 h while parents TA, TC and fusant T5 produced the most amylase after 96 h of incubation. Response surface methodology examinations revealed that the enzyme from fusant T5 was the optimal enzyme demonstrating the highest activity (1055.17 U/ml) at pH 4 and a temperature of 40℃. This enzyme lost activity with further increases in temperature. Starch hydrolysis using fusant T5 gave the highest yield of glucose (1.6158 g/100 ml). The significant activities of the selected fusants at 28 ± 2℃ and 80℃ and the higher sugar yields from cassava starch hydrolysis over their parental strains indicate that it is possible to improve amylase activity using the protoplast fusion technique.

• BMB Reports
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• v.40 no.4
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• pp.494-500
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• 2007

The endophytic bruchid pest Callosobruchus maculatus causes severe damage to storage cowpea seeds, leading to economical losses. For this reason the use of $\alpha$-amylase inhibitors to interfere with the pest digestion process has been an interesting alternative to control bruchids. With this aim, $\alpha$-amylase inhibitors from baru seeds (Dipteryx alata) were isolated by affinity chromatographic procedures, causing enhanced inhibition of C. maculatus and Anthonomus grandis $\alpha$-amylases. To attempt further purification, this fraction was applied onto a reversed-phase HPLC column, generating four peaks with remarkable inhibition toward C. maculatus $\alpha$-amylases. SDS-PAGE and MALDI-ToF analysis identified major proteins of approximately 5.0, 11.0, 20.0 and 55 kDa that showed $\alpha$-amylase inhibition. Results of in vivo bioassays using artificial seeds containing 1.0% (w/w) of baru crude extract revealed 40% cowpea weevil larvae mortality. These results provide evidence that several $\alpha$-amylase inhibitors classes, with biotechnological potential, can be isolated from a single plant species.

• Journal of Microbiology
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• v.43 no.6
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• pp.561-568
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• 2005

A newly-isolated thermophilic strain of the zygomycete fungus Rhizomucor pusillus 13.36 produced highly active dextrinogenic and saccharogenic enzymes. Cassava pulp was a good alternative substrate for amylase production. Dextrinogenic and saccharogenic amylases exhibited optimum activities at a pH of 4.0-4.5 and 5.0 respectively and at a temperature of $75^{\circ}C$. The enzymes were highly thermostable, with no detectable loss of saccharogenic or dextrinogenic activity after 1 hand 6 h at $60^{\circ}C$, respectively. The saccharogenic activity was inhibited by $Ca^{2+}$ while the dextrinogenic was indifferent to this ion. Both activities were inhibited by $Fe^{2+}\;and\;Cu^{2+}$ Hydrolysis of soluble starch by the crude enzyme yielded $66\%$ glucose, $19.5\%$ maltose, $7.7\%$ maltotriose and $6.6\%$ oligosaccharides.

• Korean Journal of Food Science and Technology
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• v.38 no.1
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• pp.59-67
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• 2006

Effects of ${\alpha}-amylases$ and emulsifiers on characteristics of frozen bread dough were examined during 12 weeks of storage. Fungal or bacterial ${\alpha}-amylase$ and various emulsifiers, including monoglyceride (MG), sodium stearoyl-2-lactylate (SSL), and diacetyltartaric acid ester of mono- and diglycerides (DATEM), were added to frozen dough individually and as mixtures Height of frozen dough at maximum development time, total volume of $CO_2$ gas, and retention volume increased with increasing content of emulsifiers. indicating addition of enzymes and emulsifiers had significant effect on flexibility of starch-gluten complex in dough. Frozen dough made with bacterial ${\alpha}-amylase$ showed slightly higher pH during storage than that of frozen dough with fungal ${\alpha}-amylase$. Bread made from frozen dough prepared with both enzymes and emulsifiers showed lower specific loaf volume than that of control during storage, whereas highest specific loaf volume was obtained with addition of fungal ${\alpha}-amylase$ with SSL+MG and bacterial ${\alpha}-amylase$ with MG.

• Journal of Microbiology and Biotechnology
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• v.17 no.8
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• pp.1242-1248
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• 2007

Genomic analysis of a hyperthermophilic archaeon, Thermococcus onnurineus NA1 [1], revealed the presence of an open reading frame consisting of 1,377 bp similar to ${\alpha}$-amylases from Thermococcales, encoding a 458-residue polypeptide containing a putative 25-residue signal peptide. The mature form of the ${\alpha}$-amylase was cloned and the recombinant enzyme was characterized. The optimum activity of the enzyme occurred at $80^{\circ}C$ and pH 5.5. The enzyme showed a liquefying activity, hydrolyzing maltooligosaccharides, amylopectin, and starch to produce mainly maltose (G2) to maltoheptaose (G7), but not pullulan and cyclodextrin. Surprisingly, the enzyme was not highly thermostable, with half-life ($t_{1/2}$) values of 10 min at $90^{\circ}C$, despite the high similarity to ${\alpha}$-amylases from Pyrococcus. Factors affecting the thermostability were considered to enhance the thermo stability. The presence of $Ca^{2+}$ seemed to be critical, significantly changing $t_{1/2}$ at $90^{\circ}C$ to 153 min by the addition of 0.5 mM $Ca^{2+}$. On the other hand, the thermostability was not enhanced by the addition of $Zn^{2+}$ or other divalent metals, irrespective of the concentration. The mutagenetic study showed that the recovery of zinc-binding residues (His175 and Cys189) enhanced the thermo stability, indicating that the residues involved in metal binding is very critical for the thermostability.

• Journal of Sericultural and Entomological Science
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• v.27 no.1
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• pp.37-41
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• 1985

The haemolymph protein, digestive fluid proteins and digestive fluid amylase activity of wild silkworm, Theophila mandarina those of the were studied by polyacrylamide gel electrophoresis. In addition, they was also compared with silkworm. 1. 6 main protein bands in female and 7 main protein bands in male were detected in the larval haemolymph of T. mandarina where as 8 and 7 main protein bands in female and male of B. mori were observed. Some differences in the haemolymph protein ands of T. mandarina and B. mori were observed. 2. 15 protein bands and 12 protein bands were found in the larval digestive fluid of T. mandarina and B. mori respectively. Some differences in the mobility of digestive fluid proteins of T. mandarina and B. mori were noticed. 3. Larval digestive fluid amylases were anionic and moved near the tracking dye in both T. mandarina and B. mori. Mobility of the digestive fluid amylases relative to bromophenol blue were 0.019 and 0.020 in T. mandarina and B. mori respectively.

• Journal of Microbiology and Biotechnology
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• v.18 no.9
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• pp.1555-1563
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• 2008

A novel $\alpha$-amylase ($\alpha$-1,4-$\alpha$-D-glucan glucanohydrolase, E.C. 3.2.1.1), ScAmy43, was found in the culture medium of the phytopathogenic fungus Sclerotinia sclerotiorum grown on oats flour. Purified to homogeneity, ScAmy43 appeared as a 43 kDa monomeric enzyme, as estimated by SDS-PAGE and Superdex 75 gel filtration. The MALDI peptide mass fingerprint of ScAmy43 tryptic digest as well as internal sequence analyses indicate that the enzyme has an original primary structure when compared with other fungal a-amylases. However, the sequence of the 12 N-terminal residues is homologous with those of Aspergillus awamori and Aspergillus kawachii amylases, suggesting that the new enzyme belongs to the same GH13 glycosyl hydrolase family. Assayed with soluble starch as substrate, this enzyme displayed optimal activity at pH 4 and $55^{\circ}C$ with an apparent $K_m$ value of 1.66 mg/ml and $V_{max}$ of 0.1${\mu}mol$glucose $min^{-1}$ $ml^{-1}$. ScAmy43 activity was strongly inhibited by $Cu^{2+}$, $Mn^{2+}$, and $Ba^{2+}$, moderately by $Fe^{2+}$, and was only weakly affected by $Ca^{2+}$ addition. However, since EDTA and EGTA did not inhibit ScAmy43 activity, this enzyme is probably not a metalloprotein. DTT and $\beta$-mercaptoethanol strongly increased the enzyme activity. Starting with soluble starch as substrate, the end products were mainly maltotriose, suggesting for this enzyme an endo action.

• Korean journal of food and cookery science
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• v.9 no.4
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• pp.266-271
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• 1993

In order to investigate the traditional value of Ewhaju (traditional wine) and to establish the brewing condition, studies on of traditional background and field inquiry were carried out. Scientific evaluation and possibility of revealation of Ewhaju were searched by the experiments of microbial and enzymatic properties of brewed Ewhaju and Nuruk by traditional method. In flora of microorganisms in Nuruk of Ewhaju, Aspergillus oryzae and Hansenula sP. were isolated, and, showed a level of 1.2$\times$$10^6$ CHU/g, respectiveln but other microorganisms were not grown in diluted cultivation test. The a-and f-amylase activity of Nuruk were 30.74 and 34.4, respectively and their activities of two amylases were 19.28 and 18.8 at first stage of brewing, 21.21 and 19.80 at 100 day after brewing, and 20.25 and 19.90 at one year aged Ewhaju, respectively. The brewed Ewhaju could be remained with high quality long period without teat treatment or addition of preservatives, also, stored Ewhaju contains remarka-bly high activity of amylases which might contribute to digestion.

• Microbiology and Biotechnology Letters
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• v.8 no.4
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• pp.247-253
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• 1980

This experiment was carried out to improve the method of koji preparation by using stainless steel frays which were specially designed and covered with lids. To elucidate changes in chemical composition and formation of enzymes during the preparation of koji with glutinous rice for red pepper paste. professes, amylases, reducing sugars, nitrogens, and microbial contaminations were determined and compared with the case of using trays without lids. The results obtained were as follows. 1. The activities of protease and amylases (both liquefying and saccharifying activities) during the koji preparation were found to be higher incase of using the trays with lids than that without lids. 2. The contents of moisture, soluble nitrogen, amino nitrogen, and reducing sugar were also higher in case of using the trays with lids. 3. Contamination by yeasts and bacteria were markedly low in the trays with lids. 4. Temperature of koji was higher in the trays with lids, however the level of moisture loss was 1ower.

• Microbiology and Biotechnology Letters
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• v.40 no.4
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• pp.296-302
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• 2012

A bacterial strain was isolated from soybean paste fermented in a Korean Buddhist temple as a producer of the extracellular thermostable ${\alpha}$-amylase. The isolate YB-1234 has been identified as Bacillus licheniformis on the basis of its 16S rDNA sequence, morphology and biochemical properties. A gene encoding the thermostable ${\alpha}$-amylase of B. licheniformis YB-1234 was cloned into Escherichia coli and its nucleotide sequence was determined. The deduced amino acid sequence of ${\alpha}$-amylase was very highly homologous to those of the thermostable ${\alpha}$-amylases of B. licheniformis belonging to the glycosyl hydrolase family 13. The ${\alpha}$-amylase produced by recombinant E. coli carrying the ${\alpha}$-amylase gene exhibited maximal activity at pH 6.0, identical to ${\alpha}$-amylase in the culture filtrate of B. licheniformis, while the temperature profile was somewhat different between the two. Particularly, ${\alpha}$-amylase produced from B. lcheniformis is much more thermostable than that from recombinant E. coli. The predominant products resulting from the ${\alpha}$-amylase hydrolysis were glucose, maltose and maltotriose for maltotetraose and maltohexaose.