• Journal of Microbiology
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• v.42 no.4
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• pp.340-345
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• 2004

To investigate the co-expression and crystallization of a fusion gene between the Bacillus thuringiensis crystal protein and a foreign protein in B. thuringiensis, the expression of the Cry1Ac fused with green fluorescent protein (GFP) genes in a B. thuringiensis $Cry^-B$ strain was examined. The cry1Ac gene was cloned in the B. thuringiensis-E. coli shuttle vector, pHT3101, under the control of the native cry1Ac gene promoter, while the GFP gene was inserted into the XhoI site upstream of the proteolytic cleavage site, in the middle region of the crylAc gene (pProAc-GFP). The B. thuringiensis $Cry^-B$ strain carrying pProAc-GFP (ProAc-GFP/CB) did not produce any inclusion bodies. However, the transformed strain expressed fusion protein forms although the expression level was relatively low. Furthermore, an immu­noblot analysis using GFP and Cry1Ac antibodies showed that the fusion protein was not a single spe­cies, but rather multiple forms. In addition, the N-terminal fragment of Cry1Ac and a non-fused GFP were also found in the B. thuringiensis $Cry^-B$ strain after autolysis. The sporulated cells before autolysis and the spore-crystal mixture after autolysis of ProAc-GFP/CB exhibited insecticidal activities against Plutella xylostella larvae. Accordingly, the current results suggest that a fusion crystal protein produced by the transfomant, ProAc-GFP/CB, can be functionally expressed but easily degraded in B. thuring­iensis.

• Korean Journal of Food Science and Technology
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• v.35 no.2
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• pp.201-205
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• 2003

The optimum autolysis conditions were investigated to prepare yeast autolyzate (extract) using yeast slurry collected from brewery plants. Brewers yeast slurry was washed with caustic soda to eliminate bitter hof substances attached to yeast cell walks. The pH of brewers yeast slurry was adjusted to 9.8 with caustic soda, and centrifuged at 3,000 rpm for 5 min. The supernatant was discarded, and the bottom cake was rehydrated and collected. Bitterness unit (BU) of washed yeast slurry was 24.1 BU, below the threshold value of 25.0. Yeast extract could be obtained from washed brewers yeast slurry at maximum yield up to 38% by autolyzing at pH 6.8 and $53^{\circ}C$ for 20 h.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.163-163
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• 2005

• Proceedings of the Zoological Society Korea Conference
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• 1999.10b
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• pp.224.2-224
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• 1999

No Abstract, See Full Text

• Journal of Microbiology
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• v.45 no.6
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• pp.593-596
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• 2007

In this study, we describe the development of a simple and efficient method for cell lysis via the insertion of a bacteriophage lambda lysis gene cluster into the pET22b expression vector in the following order; the T7 promoter, a gene for a target protein intended for production, Sam7 and R. This insertion of R and Sam7 into pET22b exerted no detrimental effects on cellular growth or the production of a target protein. The induction of the T7 promoter did not in itself result in the autolysis of cells in culture but the harvested cells were readily broken by freezing and thawing. We compared the efficiency of the cell lysis technique by freezing and thawing to that observed with sonication, and determined that both methods completely disintegrated the cells and released proteins into the solution. With our modification of pET22b, the lysis of cells became quite simple, efficient, and reliable. This strategy may prove useful for a broad variety of applications, particularly in experiments requiring extensive cell breakage, including library screening and culture condition exploration, in addition to protein purification.

• BMB Reports
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• v.44 no.10
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• pp.665-668
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• 2011

In order to clarify the impact of Ca-binding sites (Ca1 and 2) on the conformational stability of neutral proteases (NPs), we have analyzed the thermal, pH and organic solvent stability of a NP variant, V189P/A195E/G203D/A268E (Q-mutant), from Salinovibrio proteolyticus. This mutant has shown to bind calcium more tightly than the wild-type (WT) at Ca1 and to possess Ca2. Q-mutant was resisted against autolysis, thermoinactivation and pH denaturation in a Ca-dependent manner and exhibited better activity in organic solvents compared to the WT enzyme. These results imply that Ca1 and Ca2 are important for the conformational stability of NPs.

• Journal of Life Science
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• v.24 no.12
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• pp.1316-1324
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• 2014

Most beta-glucans obtained from various fruit bodies of mushrooms and mushroom mycelial cultures have high-molecular weight glycoproteins, conjugated with beta-glucans. We report that isoflavone-conjugated glycolproteins (designated as gluvone) were isolated and exhibited stronger anticarcinogenic activities. Agaricus blazei mycelia (ABM) was cultured in a liquid medium containing soybean flakes for 14 days. The liquid culture was autolyzed by incubating at $53^{\circ}C$ (pH 5.5) for 3 h. A crude glycoprotein (CGP) fraction with a cytotoxic effect on a mouse ascite cancer cell line (S-180) and a human breast cancer cell line (MCF-7) was isolated from the autolyzed ABM cultures by 80% ethanol treatment. Gluvone was isolated from the CGP with Sephadex G-75 column chromatography. It exhibited a stronger anticancer effect than CGP against the S-180 cell-induced female ICR mouse ascites carcinogenesis. Gluvone with 9,400 daltons was identified as a glycoprotein conjugated with isoflavone. According to HPLC and GC analysis, in conjunction with $^1H$-NMR spectral analysis, it contained 60% carbohydrates (glucose, fructose, and ribose), 31% protein, and 2% isoflavone (daidzein and genistein), which is a novel material. These results indicate that a strong anticarcinogenic gluvone was isolated from the autolyzed product of a submerged liquid culture of ABM, suggesting that autolysis could be a useful tool to produce antitumor agents.

• The Korean Journal of Food And Nutrition
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• v.14 no.2
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• pp.161-166
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• 2001

The optimum condition for production of yeast extract by autolysis and enzymatic method were studied with waste brewers\` yeast. The major components of brewer\`s yeast in proximate compositions were carbohydrate(46.0%) and protein(41.7%). There was a little appreciable difference in the promaximate composition of waste brewers\` and bakery yeast. The major minerals were identified as K, P, Mg and Ca, and their contents were about 1,659.7%, 1,197.4mg%, 210.4mg% and 105.6mg%, respectively. The major vitamins were identified as vitamin C and vitamin B group, and their contents were about 7.9IU/100g and 4.2IU/100g, respectively. The optimum yeast concentration in the reaction suspension was 10% (w/v) waste brewer\`s yeast slurry. The protein content in brewer\`s yeast extract was increased with addition of NaCl as plasmolyzers. However, the plasmolyzer effect of ethanol a little showed as the concentration increasd from 3% ∼7%. The 5\`-nucleotides(5\`-GMP and 5\`-IMP) content in brewer\`s yeast extract was increased with addition of glucanase. The total content of 5\`-nucleotides (5\`-GMP and 5\`-IMP) increased by 160% than those by the autolysis of waste brewer\`s and bakery yeast.

• Proceedings of the Korean Biophysical Society Conference
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• 1997.07a
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• pp.44-44
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• 1997

Ca$^{2＋}$ is one of the most common metal ions associated with proteins, playing more or less well-defined functional roles in biological activities. In protease, the presence of $Ca^{2＋}$ slows down autolysis and enhances thermal stability. Subtilisin, one of the best studied proteases, is known to have two $Ca^{2＋}$ -binding sites.(omitted)

• Journal of Microbiology and Biotechnology
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• v.8 no.6
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• pp.639-644
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• 1998

Extracellular serine proteases were isolated from a soil bacterium, alkalophilic coryneform bacterium TU-19, which have been grown in a liquid medium optimized at 3$0^{\circ}C$ and pH 10.0. Three different sizes, 120 kDa (protease I), 80 kDa (protease II), and 45 kDa (protease III), of serine pro teases were purified using Sephadex G-150 and QAE-Sephadex chromatography (Kang et al. 1995. Agric. Chem Biotech. 38: 534-540). SDS-PAGE showed that the 120 kDa protease was degraded into the 80 kDa protease in 20 mM Tris-HCI (pH 8.0) buffer solution. This degradation was enhanced in the presence of 0.5 M NaCl and 5 mM EDTA, but was inhibited in the presence of 5 mM $CaCl_2$. These results indicated that the $Ca^{2＋}$ ion seems to stabilize the 120 kDa protease like other proteases derived from Bacillus species. The $NH_2$-terminal amino acid sequences of the 10 residues of both proteases were completely identical: Met-Asn-Thr-Gln-Asn-Ser-Phe-Leu-Ile-Lys. In contrast to this, the 80 kDa protease has 1.5 times higher specific activity than the 120 kDa protease does (Kang et al. 1995. Agric. Chern. Biotech. 38: 534-540). Therefore the C-terminal of the 120 kDa protease seems to be autolyzed to the 80 kDa protease but this autolysis did not decrease the protease activity. Optimum pH and temperature of both 80 kDa and 120 kDa proteases were pH 10.5 and $45^{\circ}C$, respectively, and pH and thermal stability were almost identical. Several divalent ions except the $Fe^{2＋}$ ion showed similar effects on activities of both proteases, which are similarly resistant to three different detergents.