• Journal of Microbiology and Biotechnology
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• v.23 no.4
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• pp.489-498
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• 2013

A new Bacillus strain degrading starch, named Bacillus sp. UEB-S, was isolated from a southern Tunisian area. Amylase production using solid-state fermentation on millet, an inexpensive and available agro-resource, was investigated. Response surface methodology was applied to establish the relationship between enzyme production and four variables: inoculum size, moisture-to-millet ratio, temperature, and fermentation duration. The maximum enzyme activity recovered was 680 U/g of dry substrate when using $1.38{\times}10^9$ CFU/g as inoculation level, 5.6:1 (ml/g) as moisture ratio (86%), for 4 days of cultivation at $37^{\circ}C$, which was in perfect agreement with the predicted model value. Amylase was purified by Q-Sepharose anion-exchange and Sephacryl S-200 gel filtration chromatography with a 14-fold increase in specific activity. Its molecular mass was estimated at 130 kDa. The enzyme showed maximal activity at pH 5 and $70^{\circ}C$, and efficiently hydrolyzed starch to yield glucose and maltose as end products. The enzyme proved its efficiency for digesting raw cereal below gelatinization temperature and, hence, its potentiality to be used in industrial processes.

• Preventive Nutrition and Food Science
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• v.14 no.1
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• pp.8-13
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• 2009

This study investigated the hypoglycemic effect of fermented soymilk extract (FSE) in STZ-induced diabetic mice. FSE was prepared via fermentation of soymilk with Bacillus subtilis followed by methanol extraction. The hypoglycemic effect was determined by inhibitory activities against ${\alpha}$-glucosidase and ${\alpha}$-amylase as well as the alleviation of postprandial glucose level. The non-fermented soymilk extract (SE) was used as control in this experiment. FSE showed higher (p<0.05) inhibitory activities than SE against ${\alpha}$-glucosidase and ${\alpha}$-amylase. The $IC_{50}$ values of FSE for ${\alpha}$-glucosidase and ${\alpha}$-amylase were 0.77 ancd 0.94 mg/mL, respectively, which were comparable or even superior to those of acarbose (0.79 and 0.68 mg/mL, respectively). In addition, a further suppression on the postprandial blood glucose levels were observed in the FSE than SE group for both STZ-induced diabetic mice and normal mice. Furthermore, FSE significantly lowered the incremental area under the curve (AUC) in the diabetic mice and the AUC in normal mice corroborated the hypoglycemic effect of FSE (p<0.05). Results from this study suggest that FSE may help decrease the postprandial blood glucose level via inhibiting ${\alpha}$-glucosidase and ${\alpha}$-amylase and the usefulness of FSE was proven to be better than SE.

• Journal of Microbiology and Biotechnology
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• v.9 no.2
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• pp.230-233
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• 1999

A plasmid vector was constructed for the expression and secretion of Bacillus macerans cyclodextrin glucanotransferase (CGTase) in Bacillus subtilis. The vector, pUBACGT, was composed of the ribosome-binding sequence, signal sequence, and cgt gene from B. macerans under the control of amyR2, the promoter of amyE gene coding for $\alpha$-amylase from B. subtilis var. natto. Bacillus subtilis LKS88, a mutant strain lacking genes for an amylase and two proteases, was used as a host for the transformation of the plasmid vector. The transformants were selected on kanamycin-containing Luria-Bertani plates. The starch hydrolyzing activity was observed on the starch-containing plates by the iodine method and cyclodextrin-forming activity was detected in the culture medium. A SDS-PAGE analysis showed that most of the expressed CGTase in the recombinant B. subtilis was secreted into the medium at a high expression level.

• Preventive Nutrition and Food Science
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• v.6 no.4
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• pp.206-210
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• 2001

Starch is an abundant resource in plant biomass, and it should be hydrolyzed enzymatically into fermentable sugars for ethanol fermentation. A genetic recombinant yeast, Saccharomyces cerevisiae GA-7458, was constructed by integrating the structural gene of both $\alpha$-amylase from Bacillus stearothermophilus and the gene (STA1) encoding glucoamylase from S. diastaticus into the chromosome of S. cerevisiae SH7458. The recombinant yeast showed active enzymatic activities of $\alpha$-amylase and glucoamylase. The productivity of ethanol fermentation from the pH-controlled batch culture (pH 5.5) was 2.6 times greater than that of the pH-uncontrolled batch culture. Moreover, in a fed-batch culture, more ethanol was produced (13.2 g/L), and the production yield was 0.38 with 2% of corn starch. Importantly, the integrated plasmids were fully maintained during ethanol fermentation.

• Journal of Microbiology and Biotechnology
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• v.22 no.8
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• pp.1077-1083
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• 2012

Previously, an extracellular ${\alpha}$-amylase (BKA) had been purified from the culture of Bacillus sp. KR8104. Subsequently, the crystal structure of the active enzyme revealed a 422 amino acids polypeptide. In this study, the bka was cloned into E. coli, which encoded a polypeptide of 659 amino acids including two additional fragments: one 44 residues N-terminal fragment and another 193 residues C-terminal fragment. In order to investigate the role of the C-terminal fragment, two constructs with and without this region [$BKA{\Delta}$(N44) and $BKA{\Delta}$(N44C193)] were designed and expressed in E. coli BL21. The optimum pH, thermal stability, and the end-products of starch hydrolysis were found to be similar in both constructs. The $K_m$ and $V_{max}$ values for $BKA{\Delta}$(N44) were lower than $BKA{\Delta}$(N44C193), using either starch or ethylidene-blocked 4-nitrophenylmaltoheptaoside as a substrate.

• Journal of Microbiology and Biotechnology
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• v.24 no.7
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• pp.898-904
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• 2014

The stability of Bacillus licheniformis alpha-amylase (BLA) under acid condition was enhanced through direct evolution using the error-prone polymerase chain reaction. One beneficial mutation site, H281I, was obtained in BLA. The specific activity of H281I was 161/352 U/mg, which was 62.6/27.5% higher than that of the wild-type (WT) (99/276 U/mg) at pH 4.5/6.5 and $95^{\circ}C$. The pH optimum for H281I was decreased about 1 unit, whereas no significant changes of optimum temperature and thermostability were observed compared with the wild type (WT). The $k_{cat}/K_m$ value of H281I was 1.7-/1.4-fold higher at pH 4.5/6.5, respectively, than that of WT. The structure model analysis indicated that the H281I mutation altered the predicted interaction between the amino acid residues at 281 and 273, thus creating a conducive local environment for substrate binding, as reflected by its decreased $K_m$, and consequently increased the specific activity.

• Microbiology and Biotechnology Letters
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• v.47 no.1
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• pp.125-131
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• 2019

We have isolated and identified 72 bacterial strains from four bentonite samples collected at the mining areas located in Gyeongsangbuk-do, Republic of Korea, and measured their hydrolytic enzyme (${\alpha}$-amylase, protease, and cellulase) activities to identify the isolates with industrial-use potential. Most of the isolates belonged to the Bacillaceae, with minor portions being from the Paenibacillaceae, Micrococcaceae, and Bacillales Family XII at the family level. Of the strains isolated, 33 had extracellular ${\alpha}$-amylase activity, 30 strains produced cellulase, and 35 strains produced protease. Strain MBLB1268, having the highest ${\alpha}$-amylase activity, was identified as Bacillus siamensis ($0.38{\pm}0.06U/ml$). Bacillus tequilensis MBLB1223, isolated from Byi33-b, showed the highest cellulase activity ($0.26{\pm} 0.04U/ml$), whereas Bacillus wiedmannii MBLB1197, isolated from Zdb130-b, exhibited the highest protease activity ($54.99{\pm}0.78U/ml$). These findings show that diverse bacteria of the Bacillaceae family adhere to and exist in bentonite and are potential sources of industrially useful hydrolytic enzymes.

• Food Science and Preservation
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• v.22 no.4
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• pp.545-552
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• 2015

This study investigated the quality characteristics of popped rice Doenjang prepared with different Bacillus strains (Bacillus subtilis KACC 15935, and Bacillus subtilis HJ18-9). The changes in the enzyme activity (protease, cellulase, and ${\alpha}$-amylase), amino-type nitrogen and ammonia-type nitrogen contents, and the reducing sugar were investigated during the fermentation period. Enzymes such as protease, cellulase, and a-amylase plays an important role in the changes in composition of nutrients, and in flavor and taste of popped rice Doenjang. Protease activities of the popped rice deonjang fermented with different Bacillus strains (control, B. subtilis KACC 15935, and B. subtilis HJ18-9) was in the range of 171.77-185.97 unit/g at the beginning of fermentation, and there were no significant differences among the samples. On the other hand, the protease activity in popped rice Doenjang fermented with B. subtilis HJ18-9 increased significantly up to $248.77{\pm}4.53unit/g$ at the end of fermentation (p<0.05). Cellulase activity and a-amylase activity of popped rice Doenjang in HJ18-9 was higher than these of other samples. After 56 days of fermentation, amino-type nitrogen in popped rice deonjang fermented with control, B. subtilis KACC 15935, and B. subtilis HJ18-9 increased significantly up to $174.99{\pm}3.70$, $166.59{\pm}1.40$, $225.39{\pm}3.70mg%$, respectively (p<0.05). These results suggested that B. subtilis HJ18-9 was a suitable starter for the preparation of soybean paste.

• Korean Journal of Food Science and Technology
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• v.41 no.4
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• pp.369-373
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• 2009

${\alpha}$-Waxy corn starch was used as a feed for twin-screw extrusion in order to enhance starch liquefaction with added thermostable ${\alpha}$-amylase (derived from Bacillus licheniformis). The residence time distribution and starch liquefaction were investigated. The starch liquefaction was analyzed in terms of reducing sugar contents, molecular size from gel permeation chromatography (GPC), and microstructure from scanning electron microscopy (SEM). The use of ${\alpha}$-starch contributed to the production of more reducing sugar than the use of raw starch use alone. From GPC, the effect of ${\alpha}$- starch on the molecular size reduction was shown to be small. From SEM, irregular and damaged surface were observed on the extrudate from ${\alpha}$-starch, as compared to those from raw starch. The spread of residence time distribution curves was greater with feed of ${\alpha}$-starch than raw starch, indicating that ${\alpha}$-starch was hard to flow forward during extrusion. This could be improved by increasing the feed moisture content and barrel temperature of extruder.

• Applied Biological Chemistry
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• v.47 no.2
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• pp.176-181
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• 2004

This study was carried out to investigate the effect of yucca extract on the quality characteristics of Chungkookjang using Bacillus subtilis p01. The changes in the contents of amino-type N, ammonia type N, volatile compounds and organic acids, and those in the activities of ${\alpha}-amylase$ and protease were also determined during aging of Chungkookjang. The amount of amino-type N increased gradually with time for aging. The content of amino-type N was slightly higher in Chungkookjang fermented by adding yucca extract than in the control without yucca extract. The content of ammonia-type N was slightly lower in Chungkookjang with yucca extract than in the control without yucca extract. The activities of amylase and protease were higher in Chungkookjang with yucca extract than in the control and the highest in Chungkookjang containing 0.5 mg/g of yucca extract. Organic acid contents in Chungkookjang was the highest at the initial period of fermentation. The contents of organic acids in Chungkookjang with yucca extract was higher than that in control for 48 hr of aging. The amounts of 2,5-dimethylpyrazine were increased by addition of yucca extract, while those of cis-3-hexanol were decreased.