• Journal of Microbiology and Biotechnology
• /
• v.10 no.4
• /
• pp.551-553
• /
• 2000

A strong constitutive $P_{JH}$ promoter from Bacillus was applied to overexpress the endoxylanase gene in B. subtilis. The expression plasmid, pHJKJ4, was designed to contain the $P_{JH}$ promoter and endoxylanase promoter ($P_B$), and introduced into B. subtilis DB104. Through batch fermentation of the trasformant cell on a maltose medium, endoxylanase was produced in a growth-associated manner as the predominant protein. The total activity reached about 600 unit/ml at the end of the cultivation, which corresponded to 698 mg endoxylanase protein/l with a specific activity of 860 unit/mg protein. It was also found that the segregational plasmid instability was less than 30% and most of the endoxylanase activity was detected in the culture medium. This result suggests that the secretory production of endoxylanase can be significantly enhanced with the use of the $P_{JH}$ promoter and high-cell density culture techniques, quantitatively as well as qualitatively.

• Journal of Microbiology and Biotechnology
• /
• v.28 no.2
• /
• pp.314-322
• /
• 2018

Bacillus subtilis 8 is highly efficient at degrading feather keratin. We observed integrated feather degradation over the course of 48 h in basic culture medium while studying the entire process with scanning electron microscopy. Large amounts of ammonia, sulfite, and $\text\tiny{L}$-cysteic acid were detected in the fermented liquid. In addition, four enzymes (gamma-glutamyltranspeptidase, peptidase T, serine protease, and cystathionine gamma-synthase) were identified that play an important role in this degradation pathway, all of which were verified with molecular cloning and prokaryotic expression. To the best of our knowledge, this report is the first to demonstrate that cystathionine gamma-synthase secreted by B. subtilis 8 is involved in the decomposition of feather keratin. This study provides new data characterizing the molecular mechanism of feather degradation by bacteria, as well as potential guidance for future industrial utilization of waste keratin.

• Journal of Microbiology and Biotechnology
• /
• v.16 no.6
• /
• pp.855-862
• /
• 2006

Hexavalent chromium ($Cr^{6+}$) is a highly harmful pollutant, which can be detoxified and precipitated through reduction to $Cr^{3+}$. Bacillus megaterium TKW3 previously isolated from chromium-contaminated marine sediments was capable of reducing $Cr^{6+}$ in concomitance with metalloids ($Se^{4+}$, $Se^{6+}$, and $As^{5+}$). Notwithstanding approximately 50% inhibition, it was the first report of simultaneous bacterial reduction of $Cr^{6+}$ and $Se^{4+}$ (to elemental Se). No significant difference was observed among electron donors (glucose, maltose, and mannitol) on $Cr^{6+}$ reduction by B. megaterium TKW3. The reduction was constitutive and determined to be non-plasmid mediated. Peptide mass fingerprints (PMF) revealed a novel aerobic membrane-associated reductase with $Cr^{6+}$-induced expression and specific reductive activity (in nmol $Cr^{6+}$/mg protein/min) of 0.220 as compared with 0.087 of the soluble protein fraction. Respiratory inhibitor $NaN_3$ did not interfere with the reductase activity. Transmission electron microscopy with energy dispersive X-ray (TEM-EDX) analysis confirmed the aggregation of reduced chromium along the intracellular membrane region. Future identification of the N-terminal amino acid sequence of this reductase will facilitate purification and understanding of its enzymatic action.

• Journal of Microbiology and Biotechnology
• /
• v.30 no.7
• /
• pp.1082-1091
• /
• 2020

Microbial transglutaminases (MTGs) are widely used in the food industry. In this study, the MTG gene of Streptomyces sp. TYQ1024 was cloned and expressed in a food-grade bacterial strain, Bacillus subtilis SCK6. Extracellular activity of the MTG after codon and signal peptide (SP Ync M) optimization was 20 times that of the pre-optimized enzyme. After purification, the molecular weight of the MTG was 38 kDa and the specific activity was 63.75 U/mg. The optimal temperature and pH for the recombinant MTG activity were 50℃ and 8.0, respectively. MTG activity increased 1.42-fold in the presence of β-ME and 1.6-fold in the presence of DTT. Moreover, 18% sodium chloride still resulted in 83% enzyme activity, which showed good salt tolerance. Cross-linking gelatin with the MTG increased the strength of gelatin 1.67 times and increased the thermal denaturation temperature from 61.8 to 75.8℃. The MTG also significantly increased the strength and thermal stability of gelatin. These characteristics demonstrated the huge commercial potential of MTG, such as for applications in salted protein foods.

• Journal of Microbiology and Biotechnology
• /
• v.1 no.4
• /
• pp.240-245
• /
• 1991

We have constructed a promoter-probe vector pKU20 using pKT230, a derivative of broad-host-range plsmid RSF1010, as a base. The pKU20 contains structural gene for aminoglycoside phos-photransferase (aph), without promoter, and a multiple cloning site upstream the aph. Using this vector, a 412base pairs (bp) PstI fragment showing strong promoter activity both in Escherichia coli LE392 and Pseudomonas putida KCTC1644 has been cloned from Pseudomonas fluorescens chromosomal DNA on the basis of streptomycin resistance. The nucleotide sequence of the 412 bp fragment has been determined and the putative - 35 and -10 region was observed. Insecticidal protein gene of Bacillus thuringiensis subsp. kurstaki HD-73 inserted on downstream of the promoterlike DNA fragment was efficiently expressed in E. coli and P. putida. The toxin protein was efficiently synthesized in an insoluble form in both strains.

• Journal of Microbiology and Biotechnology
• /
• v.23 no.6
• /
• pp.750-758
• /
• 2013

Anthrax is a bacterial disease caused by the aerobic spore-forming bacterium Bacillus anthracis, which is an important pathogen owing to its ability to be used as a terror agent. B. anthracis spores can escape phagocytosis and initiate the germination process even in antimicrobial conditions, such as oxidative stress. To analyze the oxidative stress response in B. anthracis and thereby learn how to prevent antimicrobial resistance, we performed protein expression profiling of B. anthracis strain HY1 treated with 0.3 mM hydrogen peroxide using a comparative proteomics-based approach. The results showed a total of 60 differentially expressed proteins; among them, 17 showed differences in expression over time. We observed time-dependent changes in the production of metabolic and repair/protection signaling proteins. These results will be useful for uncovering the metabolic pathways and protection mechanisms of the oxidative response in B. anthracis.

• International Journal of Industrial Entomology and Biomaterials
• /
• v.2 no.2
• /
• pp.185-189
• /
• 2001

Wild type Bacilus thuringiensis subsp. israelensis and B. sphaericus toxins have been used separately as active in ingredients for bacterial insecticides to control mosquito larvae due to their comparable toxicity to chemical insecticides. Cry11B, recently cloned from B. thuringiensis subsp. jegathesan, shows higher toxicity against three major species of mosquito larvae than Cry11A, one of the major component of B. thuringiensis subsp. israelensis inclusion body. To determine whether the combination of cry11B and B. sphaericus binary toxins is as toxic as B. thuringiensis subsp. israelensis parental strain, cry11B and B. sphaericus binary toxins genes were co-expressed as an operon using cytlA promoters/STAB-SD hybrid expression system in B. thuringiensis subsp. israelensis acrystalliferous strain 4Q7. However, unexpectedly, B. sphaericus binary toxins were barely produced, whereas relatively large amount of Cry11B was produced. When this strain was grown in four different media, NB+G and Peptonized Milk produced more toxin proteins and spores per unit of media than GYS and G-Tris. Toxicity of this strain against fourth instar Culex quinquefasciatus was ranged from of 8.3 to 45.7 ng/ml, with NB+G culture being the highest, and GYS culture was the lowest.

• Applied Biological Chemistry
• /
• v.41 no.2
• /
• pp.137-140
• /
• 1998

The transgenic tomato plants showing the insecticidal activity against the coleopteran insect larvae have been bred to the 4th generation $(R_4)$. The Bacillus thuringiensis subsp. tenebrionis (B.t.t.)-toxin gene and the expression were detected in the $R_4$ transgenic plants. The expression of the toxin gene conferred a coleopteran insect larvae tolerance to the transgenic tomato plants. The ploidy levels of the $R_4$ transgenic plants were diploid. The results indicated that the toxin gene was inherrited to the next generation and expressed. Such a molecular breeding can provide a method for a permanent control of insects a agronomic relevance.

• Journal of Life Science
• /
• v.23 no.7
• /
• pp.863-868
• /
• 2013

The XylP gene, which encodes endoxylanase in Bacillus sp. HY-20, was subcloned, and two expression plasmids, pG-xylP and pGMF-xylP were constructed. These plasmids, which contain different signal sequences, XylP s.s and $MF{\alpha}_{opt}$ s.s, respectively, for the secretory expression of endoxylanase, were transformed into Saccharomyces cerevisiae SEY2102 and FY833, respectively. The recombinant endoxylanases were successfully expressed, with a total activity range of 23.7-70.1 unit/ml according to the expression system and host strain. The endoxylanase activity in SEY2102/pGMF-xylP reached a maximum of 88.1 unit/ml in baffled flask culture. Most of the recombinant endoxylanase was efficiently secreted in the extracellular fraction, and the $MF{\alpha}_{opt}$ s.s was more efficient for secreting endoxylanase in yeast than the XylP s.s. Therefore, the expression system developed in this study produces large extracellular amounts of endoxylanase using S. cerevisiae as the host strain, and it could be used in bioethanol production and industrial applications.

• Environmental Engineering Research
• /
• v.15 no.3
• /
• pp.141-147
• /
• 2010

The objective of this study was to assess the biofouling characteristics of the Bacillus biofilm formed on reverse osmosis (RO) membranes. For the study, a sporogenic Bacillus sp. was isolated from the seawater intake to a RO process, with two distinct sets of experiments performed to grow the Bacillus biofilm on the RO membrane using a lab-scale crossflow membrane test unit. Two operational feds were used, 9 L sterile-filtered seawater and 109 Bacillus cells, with flow rates of 1 L/min, and a constant 800 psi-pressure and pH 7.6. From the results, the membrane with more fouling, in which the observed permeate flux decreased to 33% of its initial value, showed about 10 and 100 times greater extracellular polymeric substances and spoOA genes expressions, respectively, than the those of the less fouled membrane (flux declined to 20% of its initial value). Interestingly; however, the number of culturable Bacillus sp. in the more fouled membrane was about 10 times less than that of the less fouled membrane. This indicated that while the number of Bacillus had less relevance with respect to the extent of biofouling, the activation of the genes of interest, which is initiative of biofilm development, had a more positive effect on biofouling than the mass of an individual Bacillus bacterium.