• Journal of Microbiology and Biotechnology
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• 제21권12호
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• pp.1250-1256
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• 2011

In this study, Bacillus licheniformis cells were immobilized by entrapment in calcium alginate beads and were used for production of alkaline protease by repeated batch process. In order to increase the stability of the beads, the immobilization procedure was optimized by statistical full factorial method, by which three factors including alginate type, calcium chloride concentration, and agitation speed were studied. Optimization of the enzyme production medium, by the Taguchi method, was also studied. The obtained results showed that optimization of the cell immobilization procedure and medium constituents significantly enhanced the production of alkaline protease. In comparison with the free-cell culture in pre-optimized medium, about 7.3-fold higher productivity was resulted after optimization of the overall procedure. Repeated batch mode of operation, using optimized conditions, resulted in continuous production of the alkaline protease for 13 batches in 19 days.

• Journal of Microbiology and Biotechnology
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• 제19권4호
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• pp.378-386
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• 2009

A sequential optimization strategy, based on statistical experimental designs, is employed to enhance the production of alkaline protease by a Bacillus pseudofirmus local isolate. To screen the bioprocess parameters significantly influencing the alkaline protease activity, a 2-level Plackett-Burman design was applied. Among 15 variables tested, the pH, peptone, and incubation time were selected based on their high positive significant effect on the protease activity. A near-optimum medium formulation was then obtained that increased the protease yield by more than 5-fold. Thereafter, the response surface methodology(RSM) was adopted to acquire the best process conditions among the selected variables, where a 3-level Box-Behnken design was utilized to create a polynomial quadratic model correlating the relationship between the three variables and the protease activity. The optimal combination of the major medium constituents for alkaline protease production, evaluated using the nonlinear optimization algorithm of EXCEL-Solver, was as follows: pH of 9.5, 2% peptone, and incubation time of 60 h. The predicted optimum alkaline protease activity was 3,213 U/ml/min, which was 6.4 times the activity with the basal medium.

• 한국미생물·생명공학회지
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• 제25권2호
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• pp.207-211
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• 1997

Media optimization for the production of thermostable protease specifically hydrolyzing defatted soybean meal (DSM) from Bacillus licheniformis NS70 was performed by two methods, one-at-a-time method and response surface methodology (RSM). The best carbon source and nitrogen source for the protease production were lactose and DSM, respectively. The maximum protease production estimated by RSM was 606 U/L at 1.11% lactose and 0.43% DSM, the value of which was nearly consistent to the experimental value of 599 U/L. Yeast extract suppressed the protease production. The medium pH was slightly increased at the beginning stage of fermentation, and it tended to decrease after 8 hours. The optimal pH for the protease production was 7.2 in the batch fermentation.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.282-282
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• 2003

• Journal of Microbiology and Biotechnology
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• 제22권1호
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• pp.58-68
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• 2012

An investigation was conducted on the enhancement of production and purification of an oxidant and SDS-stable alkaline protease (BHAP) secreted by an alkalophilic Bacillus horikoshii, which was screened from the body fluid of a unique Korean polychaeta (Periserrula leucophryna) living in the tidal mud flats of Kwangwha Island in the Korean West Sea. A prominent effect on BHAP production was obtained by adding 2% maltose, 1% sodium citrate, 0.8% NaCl, and 0.6% sodium carbonate to the culturing medium. The optimal medium for BHAP production contained (g/l) SBM, 15; casein, 10; $K_2HPO_4$, 2; $KH_2PO_4$, 2; maltose, 20; sodium citrate, 10; $MgSO_4$, 0.06; NaCl, 8; and $Na_2CO_3$, 6. A protease yield of approximately 56,000 U/ml was achieved using the optimized medium, which is an increase of approximately 5.5-fold compared with the previous optimization (10,050 U/ml). The BHAP was homogenously purified 34-fold with an overall recovery of 34% and a specific activity of 223,090 U/mg protein using adsorption with Diaion HPA75, hydrophobic interaction chromatography (HIC) on Phenyl-Sepharose, and ion-exchange chromatography on a DEAE- and CM-Sepharose column. The purified BHAP was determined a homogeneous by SDS-PAGE, with an apparent molecular mass of 28 kDa, and it showed extreme stability towards organic solvents, SDS, and oxidizing agents. The $K_m$ and $k_{cat}$ values were 78.7 ${\mu}M$ and $217.4s^{-1}$ for N-succinyl-Ala-Ala-Pro-Phe-pNA at $37^{\circ}C$ and pH 9, respectively. The inhibition profile exhibited by PMSF suggested that the protease from B. horikoshii belongs to the family of serine proteases. The BHAP, which showed high stability against SDS and $H_2O_2$, has significance for industrial application, such as additives in detergent and feed industries.

• KSBB Journal
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• 제20권3호
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• pp.215-219
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• 2005

Bacillus clausii I-52에서 생산되는 염기성 단백질 분해효소를 통계학적 방법을 이용하여 최적화하였다. 7개의 배지성분 중 Plakett-Burman 방법을 이용하여 단백질 분해효소 활성에 영향을 주는 3개의 성분을 선택했고, 또 영향을 주는 농도 범위를 Box-behken 방법에 의해 결정했다. 세 성분을 포함하는 염기성 단백질 분해효소의 생산 함수식을 구했고 이로부터 최적조건을 표면반응분석법을 이용해서 계산했다. 이에 근거한 이론적 최대 생산 활성 (Wheat flour: 0 g/l, Sodium citrate: 5 g/l, Sodium carbonate: 10 g/1)일 때 74000 U/mL의 결과를 얻었다. 실제 최적 배지를 이용한 실험에서는 92000 U/mL의 염기성 단백질 분해효소를 보였다. 최적화 이전 경우 생산량은 49000 U/mL로서 $90\%$의 증가율을 보였다.

• Journal of Microbiology and Biotechnology
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• 제10권3호
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• pp.349-354
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• 2000

Abstract A facultative alkalophilic gram-negative Vibrio metschnikovii strain RH530, isolated from the wastewater, produced several alkaline proteases (VAP) including six alkaline serine proteases and a metalloprotease. From this strain, high yielding YAP mutants were isolated by NTG treatment. The isolated mutant KS1 showed nine times more activity than the wild-type after optimization of the culture media. The production was regulated by catabolite repression when glucose was added to the medium. The effects of several organic nitrogen sources on the production of the YAP were investigated to avoid catabolite repression. The combination of 4% wheat gluten meal (WGM), 1.5% cotton seed flour (eSF), and 5% soybean meal (SBM) resulted in the best production when supplemented with 1% NaCl. The YAP showed a resistance to surfactants such as $sodium-{\alpha}-olefin$ sulfonate (AOS), polyoxy ethylene oxide (POE), and sodium dodecyl sulfate (SDS), yet not to linear alkylbenzene sulfonate (LAS). However, the activity of the YAP was restored completely when incubated with LAS in the presence of POE or $Na_2SO_4$. The YAP was stable in a liquid laundry detergent containing 6.6% SLES (sodium lauryl ether sulfate), 6.6% LAS, 19.8% POE, and stabilizing agents for more than two weeks at $40^{\circ}C$, but the stability was sharply decreased even after 1 day when incubated at $60^{\circ}C$. A washing performance test with the YAP exhibited it to be a good washing power by showing 51 % and 60% activity at $25^{\circ}C{\;}and{\;}40^{\circ}C$, respectively, thereby indicating that the YAP also has a good detergency at a low temperature. All the results suggest that the YAP produced from the mutant strain KSI has suitable properties for use in laundry detergents.rgents.

• KSBB Journal
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• 제32권1호
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• pp.71-82
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• 2017

To verify anti-diabetic effect of oligopeptide with His-Pro repeats (mHP peptide), the oligopeptide was first secreted and optimized using the secretion vector, pRBAS with alkaline protease gene promoter and the signal sequence in Bacillus subtilis and directly the anti-diabetic effect of the mHP peptide was investigated in insulinoma cell, RINm5F cell line. The oligopeptide gene was obtained by annealing oligonucleotides with repeated His-Pro sequence and finally was constructed as 18 dipeptides (108 bp and 4.0 kDa) coding gene, named oligopeptide with His-Pro repeats (mHP peptide) to make cyclo(His-Pro) known to be anti-diabetic effects. The region encoding the oligopeptide gene was subcloned into the pRBAS secretion vector (E.coli-Bacillus shuttle vector) after PCR amplification using the designed primers including initiation and termination codons and His tag, named pRBAS-mHP (6.56 kb). To optimize secretion of the oligopeptide, various culture conditions were investigated in Bacillus subtilis LKS. As a result, the secreted oligopeptide was maximally measured (approximately $59.6{\mu}g/mL$) in 3 L batch culture and the highest secretion was achieved at $30^{\circ}C$, PY medium, and carbon sources (particularly barley and glycerol). In the RINm5F cells treated with 2 mM STZ, the oligopeptide treatment (0.1 mg/mL) restored the cell viability (10%) and reduced the nitric oxide (NO) generation (35%) and DNA fragmentation (90%). And also, insulin secretion level was increased to 17% higher than in STZ-treated RINm5F cells. These results suggest that the oligopeptide with His-Pro repeats could be a candidate material for anti-diabetic agent against STZ-induced diabetes.