• Journal of Microbiology and Biotechnology
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• v.31 no.4
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• pp.570-583
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• 2021

Pyrococcus furiosus α-amylase can hydrolyze α-1,4 linkages in starch and related carbohydrates under hyperthermophilic condition (~ 100℃), showing great potential in a wide range of industrial applications, while its relatively low productivity from heterologous hosts has limited the industrial applications. Bacillus subtilis, a gram-positive bacterium, has been widely used in industrial production for its non-pathogenic and powerful secretory characteristics. This study was conducted to increase production of P. furiosus α-amylase in B. subtilis through three strategies. Initial experiments showed that co-expression of P. furiosus molecular chaperone peptidyl-prolyl cis-trans isomerase through genomic integration mode, using a CRISPR/Cas9 system, increased soluble amylase production. Therefore, considering that native P. furiosus α-amylase is produced within a hyperthermophilic environment and is highly thermostable, heat treatment of intact culture at 90℃ for 15 min was performed, thereby greatly increasing soluble amylase production. After optimization of the culture conditions (nitrogen source, carbon source, metal ion, temperature and pH), experiments in a 3-L fermenter yielded a soluble activity of 3,806.7 U/ml, which was 3.3- and 28.2-fold those of a control without heat treatment (1,155.1 U/ml) and an empty expression vector control (135.1 U/ml), respectively. This represents the highest P. furiosus α-amylase production reported to date and should promote innovation in the starch liquefaction process and related industrial productions. Meanwhile, heat treatment, which may promote folding of aggregated P. furiosus α-amylase into a soluble, active form through the transfer of kinetic energy, may be of general benefit when producing proteins from thermophilic archaea.

• Environmental Sciences Bulletin of The Korean Environmental Sciences Society
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• v.2 no.2
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• pp.159-168
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• 1998

Effect of plant growth regulators and end-product on the enzyme activities in cotyledons of Vigna angularis during germination was investigated by measuring the changes of $\alpha-amylase$ activities in attached and detached cotyledons applied growth regulators and sugars. The higher levels of $\alpha-amylase$ in detached cotyledons than those in cotyledons attached to the embryonic axis were due to both faster synthesis and slower degradation of the enzyme in the detached cotyledons than in the attached cotyledons. Levels of $\alpha-amylase$ activity were reduced by high concentrations of glucose and sucrose, and it is suggested that this effect was caused mostly by osmotic stress and partly by end-product repression. In detached cotyledons exogenously supplied $GA_3,$ IAA, kinetin, or their combinations has a small promotive effect on the developmental patterns of $\alpha-amylase$ activity ABA and uniconazole both prevented the synthesis of $\alpha-amylase$. Glucose inhibition of enzyme activity was partly reversed by the application of $GA_3,$ and CAMP. $GA_3,$ and cAMP seemed to act through a similar mechanism. The addition of inhibitors of protein and RNA synthesis largely prevented the increase of enzyme activity in the presence or absence of exogenous $GA_3,$. The pretreatment experiments with canavanine indicated that the earlier the time of addition was, the lower the amylase activity was.

• Journal of Microbiology and Biotechnology
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• v.21 no.5
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• pp.470-476
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• 2011

A potent fungus for amylase production, Chrysosporium asperatum, was isolated from among 30 different cultures obtained from wood samples collected in the Junagadh forest, India. All of the isolated cultures were screened for their ability to produce amylase by submerged fermentation. Among the selected cultures, C. asperatum (Class Euascomycetes; Onygenales; Onygenaceae) gave maximum amylase production. In all of the different media tested, potato starch was found to be a good substrate for production of amylase enzyme at $30^{\circ}C$ and pH 5.0. Production of enzyme reached the maximum when a combination of starch and 2% xylose, and organic nitrogen (1% yeast extract) and ammonium sulfate were used as carbon and nitrogen sources, respectively. There was no significant effect of metal ions on enzyme activity. The enzyme was relatively stable at $30^{\circ}C$ for 20 min, and no inhibitory effect of $Ca^{+2}$ ions on amylase production was observed.

• Journal of Microbiology and Biotechnology
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• v.20 no.3
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• pp.563-568
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• 2010

The Val289 residue in the $\alpha$-amylase of Bacillus amyloliquefaciens, which is equivalent to the Ala289 and Val286 residues in the $\alpha$-amylases of B. stearothermophilus and B. licheniformis, respectively, was studied by site-directed mutagenesis. This residue was substituted with 10 different amino acids by random substitution of the Val codon. In these mutant $\alpha$-amylases, Val289 was substituted with Ile, Tyr, Phe, Leu, Gly, Pro, Ser, Arg, Glu, and Asp. Compared with the wild-type $\alpha$-amylase, the mutant $\alpha$-amylase Val289Ile showed 20% more hydrolytic activity, whereas Val289Phe and Val289Leu showed 50% lesser activity. On the other hand, the mutant $\alpha$-amylases Val289Gly, Val289Tyr, Val289Ser, and Val289Pro showed less than 15% activity. The substitution of Val289 with Arg, Asp, or Glu resulted in complete loss of the $\alpha$-amylase activity. Interestingly, the mutant $\alpha$-amylase Val289Tyr had acquired a transglycosylation activity, which resulted in the change of product profile of the reaction, giving a longer oligosaccharide.

• Journal of Microbiology and Biotechnology
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• v.2 no.3
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• pp.183-188
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• 1992

A soil bacterium which produces raw starch-digesting $\beta$-amylase in culture medium, has been screened from soils. One strain, isolated and identified as Bacillus polymyxa No. 26, was selected as a $\beta$-amylase producing bacterium. Morphological and biological characteristics of the strain were found to be similar to those of a strain belonging to B. polymyxa. The electron microscopic observations of the bacterial vegetative cells and sporulated cells were extensively done to know the corelation between the enzyme synthesis and sporulation. When the bacterium was cultured on the appropriate media (3% dextrin, 0.3% beef extract, 0.5% polypeptone, 1% yeast extract and 0.3% NaCl at pH 7.0 for 4 days) raw starch-digestible $\beta$-amylase was produced extracellularly. This strain produced 130 units of $\beta$-amylase per ml in a culture medium containing 3% dextrin at $30^\circ{C}$. This value is compared to those of other $\beta$-amylase-producing strains. The optimum pH and temperature for crude enzymes were pH 6.5 to 7.0 and $50^\circ{C}$, respectively. The enzymes were stable between pH 5.5 and 9.0 for 30 min at $45^\circ{C}$.

• Microbiology and Biotechnology Letters
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• v.27 no.2
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• pp.129-135
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• 1999

EH-1 was highest at 9 days of incubation. This regrowth and enzymatic activity of Haloarcular sp. EH-1 was highest at 9 days of incubation. This amylase was purified by acetone fractionation, DEAG-Cellulose column chromatography, 1st Sephadex G-75 gel filtration, CM-Cellulose column chromatography and 2nd Sephadex G-75 gel filtration. The amylase was purified about 98.64 fold with a yield of 11.75%. The molecular weight of amylase was estimated to be about 43,000and 40,000 by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively, suggesting that the enzyme was a monomer. Amylase had an optimal temperature of 4$0^{\circ}C$, and an optimum pH of 7.0, and the thermal stability was observed the above 50% at 10$0^{\circ}C$ after 1 hour, and the stable range of pH was 6.0 to 8.0. The enzymatic activity was increased in the presence of 10 mM 2-mercaptoethanol, slightly by 10 mM SnCl2.2H2O.FeCl2.4H2O.CuCl2.2H2O.HgCl2.6H2O and SDS. End products from soluble starch were glucose, maltose and maltotriose, and Km value for soluble starch was 2.5mg/ml.

• Applied Biological Chemistry
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• v.39 no.4
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• pp.293-296
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• 1996

The present study was carried out to investigate the effect of Ni on germination, cell elongation, ${\alpha}-amylase$ activity, contents of chlorophyll and protein in radish were determined in the water culture. As the concentration of Ni was increased in the water culture, germination of radish was 55% by Ni 10 mg/kg and 30% by Ni 20 mg/kg. The ratio of cell elongation injury was 50%, by two days after Ni 20 mg/kg treatment. The injury ratio of ${\alpha}-amylase$ activity was 45% in the same condition and as the time goes on, inhibition of ${\alpha}-amylase$ activity were slightly decreased. Contents of chlorophyll a and b were decreased two days after treatment and chlorophyll a was more inhibited than chlorophyll b. Also changes of the protein contents was slightly decreased. Activity of ${\alpha}-amylase$ was decreased at germination stage, contents of chlorophyll a and b were decreased at growing stage.

• Food Science and Biotechnology
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• v.14 no.1
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• pp.32-38
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• 2005

Effects of acid and ${\alpha}$-amylase on resistant starches including retrograded RS3 and cross-linked RS4 prepared from normal maize starch were investigated. Acid and ${\alpha}$-amylase hydrolytic patterns of RS3 were similar, while those of native starch and RS4 differed. Acid hydrolysis rate of RS3 was markedly higher at initial stage, then slowly decreased up to 20 days, whereas that of RS4 increased continuously. The sizes of acid- and ${\alpha}$-amylase-treated RS3 residues decreased, but those of RS4 remained unchanged. X-ray patterns of all treated residues did not change; however, the peak intensities increased. Swelling power of RS3 increased to 150% at $95^{\circ}C$, whereas that of RS4 differed depending on the treatment condition. Swelling power of acid-treated RS4 residue increased markedly, but that of ${\alpha}$-amylase-treated one remained constant. Gel filtration chromatography profiles of untreated RS3 and RS4 residues were similar, whereas that of acid-treated RS4 residue was different from them. RS showed different hydrolytic behavior by acid and ${\alpha}$-amylase depending on the type, and susceptibility of RS3 was higher than that of RS4.

• Korean Journal of Food Science and Technology
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• v.26 no.4
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• pp.398-402
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• 1994

The participation of thermostable amylase in the decrease of viscosity of pine nut's porridge was investigated using the crude enzyme obtained from ammonium sulfate fractionation of pine nut extracts. The fraction precipitated at $35{\sim}55%$ saturation of ammonium sulfate had the highest specific activity of the enzyme. ${\alpha}-amylase$ activity was maximal at $75^{\circ}C$, pH 5.4. Amylograph data showed that addition of the enzyme to rice flour resulted in the significant decrease of its viscosity, suggesting the existence of thermostable ${\alpha}-amylase$ in pine nut.

• Microbiology and Biotechnology Letters
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• v.3 no.2
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• pp.73-82
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• 1975

In the course of studies on the production of thermostable amylases by thermophilic actinomycetes isolated from soils the investigation was carried out on the production of $\alpha$-amylase by G-1011 strain which had presented the most remarkable $\alpha$-amylase formation ability among 128 amylolytic isolates. The results were as follows ： 1. Characteristics of G-1011 strain were compared with those descriptions of thermophilic actinomycetes given in Bergey's Manual. The strain was identical to these species of actinomycetes. The details of physiological properties of the strain luould he published in near future. 2. The optimum temperature for incubation of the cell growth of G-1011 strain and $\alpha$-amylase production by the strain was revealed to 5$0^{\circ}C$. 3. The effective medium for $\alpha$-amylase formation by the strain was consisted of 3.0%, soluble starch, 1.0%, peptone, 0.5%, yeast extract, 0.5%, NaCl, 0.1%, MgSO$_4$ㆍ7$H_2O$ 0.02%, $K_2$MPO$_4$and 0.002% FeSO$_4$ㆍ7$H_2O$. The pH of the medium was ajusted to 7.0 with phosphate buffer solution. 4. The maximum production of $\alpha$-amylase (3420 D. U/ml) by G-1011 strain resulted when it was grown for 16 hours with the culture of reciprocal shaking.