• Journal of Microbiology and Biotechnology
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• v.28 no.12
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• pp.2029-2035
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• 2018

Cycloisomaltooligosaccharide glucanotransferase (CITase) was isolated from alkaliphilic Paenibacillus daejeonensis via an amino acid homology search for the reported CITase. The recombinant alkaliphilic CITase (PDCITase) from P. daejeonensis was expressed in an Escherichia coli expression system and purified as a single protein band of 111 kDa. PDCITase showed optimum activity at pH 8.0 and retained 100% of activity within a broad pH range (7.0-11.5) after 18 h, indicating alkaliphilic or alkalistable CITase properties. In addition, PDCITase produced CI-7 to CI-17, CI-18, and CI-19, which are relatively large cycloisomaltooligosaccharides yet to be reported. Therefore, these large cycloisomaltooligosaccharides can be applied to the improvement of water solubility of pharmaceutical biomaterials.

• 한국당과학회:학술대회논문집
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• 2006.11a
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• pp.32-32
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• 2006

• Journal of Microbiology
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• v.35 no.3
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• pp.213-221
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• 1997

The secretion of the alkaliphilic Bacillus sp. S-1 extracellular pullulanase involves translocation across the cytoplasmic membrane of the Gram-positive bacterial cell envelope. Translocation of the intracellular pullulanase PUL-I, was traced to elucidate the mechanism and pathway of protein secretion from an alkaliphilic Bacillus sp. S-1. Pullulanase could be slowly bue quantitatively released into the medium during growth of the cells in medium contianing proteinase K. The released pullulanase lacked the N-terminal domain. The N-terminus is the sole membrane anchor in the pullulanase protein and was not affected by proteases, confirming that it is not exposed on the cell surface. Processing of a 180,000M$\_$r/ pullulanase to a 140,000M$\_$r/ polypeptide has been demonstrated in cell extracts using antibodies raised against 140,000M$\_$r/ extracellular form. Processing of the 180,000 M$\_$r/ protein occured during the preparation of extracts in an alkaline pH condition. A modified rapid extraction procedure suggested that the processing event also occured in vivo. Processing apparently increased the activity of pullulanase. The western blotting analysis with mouse anti-serum against 140-kDa extracellular pullulanase PUL-E showed that PUL-I is processed into PUL-X via intermediate form of PUL-E. Possible explanationa for the translocation are discussed.

• Journal of Microbiology and Biotechnology
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• v.5 no.1
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• pp.1-5
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• 1995

A genomic DNA of alkaliphilic bacterium, Micrococcus sp. Y-l, was analysed using the physical mapping method of pulsed-field gel electrophoresis (PFGE). Five restriction enzymes of Sspl, Hpal, Xbal, Ndel or EcoRI, which recognize the Adenine-Thymine-rich sequences of genomic DNA, were used for the generation of few (7 to 20) distinctly separate fragments, with average sizes in the range of 200～500 kb. However, the sites for Notl and SfiI, 8 base-recognizing enzymes, were highly frequent. The genome size of this strain was determined to be 4 mega base pairs (Mb) from restriction fragments separated by PFGE. This is the first case of restriction mapping in alkaliphilic bacterium.

• Journal of Microbiology and Biotechnology
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• v.23 no.6
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• pp.872-877
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• 2013

In a previous study, we isolated and reported a second species of the Saccharophagus genus, Saccharophagus sp. strain Myt-1. In the present study, an alginate lyase gene (algMytC) from the genomic DNA of Myt-1 was cloned and characterized. The DNA sequence fragment obtained contained an open reading frame of 1,032 bp that encoded a protein of 343 amino acids with an estimated molecular mass of 37.6 kDa and a pI of 6.60. The deduced protein, AlgMytC, had the conserved amino acid sequences (RTELREM, QIH, YFKAGVYNQ) of the polysaccharide lyase family 7. A BLAST homology search indicated that AlgMytC shared an amino acid sequence identity of 95.9% with alg7A of S. degradans 2-40. The cloned and purified AlgMytC protein showed optimal activity at $40^{\circ}C$, and retained more than 90% of its total activity even after treatment at $25^{\circ}C$ for 24 h. AlgMytC was very alkaliphilic with an optimal pH of 9.0, and more than 90% of its activity was retained in the pH range 8.5-10.0. Moreover, AlgMytC was stable over a wide pH range. The activity of AlgMytC was also stable in the presence of various detergents.

• Journal of Microbiology and Biotechnology
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• v.30 no.3
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• pp.404-416
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• 2020

Bacteria that are resistant to high temperatures and alkaline environments are essential for the biological repair of damaged concrete. Alkaliphilic and halotolerant Bacillus sp. AK13 was isolated from the rhizosphere of Miscanthus sacchariflorus. Unlike other tested Bacillus species, the AK13 strain grows at pH 13 and withstands 11% (w/v) NaCl. Growth of the AK13 strain at elevated pH without urea promoted calcium carbonate (CaCO₃) formation. Irregular vaterite-like CaCO₃ minerals that were tightly attached to cells were observed using field-emission scanning electron microscopy. Energy-dispersive X-ray spectrometry, confocal laser scanning microscopy, and X-ray diffraction analyses confirmed the presence of CaCO₃ around the cell. Isotope ration mass spectrometry analysis confirmed that the majority of CO₃^2- ions in the CaCO₃ were produced by cellular respiration rather than being derived from atmospheric carbon dioxide. The minerals produced from calcium acetate-added growth medium formed smaller crystals than those formed in calcium lactate-added medium. Strain AK13 appears to heal cracks on mortar specimens when applied as a pelletized spore powder. Alkaliphilic Bacillus sp. AK13 is a promising candidate for self-healing agents in concrete.

• BMB Reports
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• v.30 no.1
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• pp.46-54
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• 1997

Alkaliphilic Bacillus sp. S-1 secretes a large amount (approximately 80% of total pullulanase activity) of an extracellular pullulanase (PUL-E). The pullulanase exists in two forms: a precursor form (PUL-I: $M_r$ 180,000), and a processed form (PUL-E: $M_r$ 140,000). Two forms were purified to homogeneity and their properties were compared. PUL-I was different in molecular weight, isoelectric point, $NH_2$-terminal amino acid sequence, and stabilities over pH and temperature ranges. The catalytic activities of PUL-I were also distinguishable in the $K_m$ and $V_{max}$ values for various substrates, and in the specific activity for pullulan hydrolysis. PUL-E showed 10-fold higher specific activities than PUL-I. However. PUL-I is immunologically identical to PUL-E, suggesting that PUL-I is initially synthesized and proteolytically processed to the mature form of PUL-E. Processing was inhibited by PMSF, but not by pepstatin, suggesting that some intracellular serine proteases could be responsible for processing of the PUL-I. PUL-I has a different conformational structure for antibody recognition from that of PUL-E. It is also postulated that the translocation of alkaline pullulanase(AP) in the bacterium possibly requires processing of the $NH_2$-terminal region of the AP protein. Processing of the precursor involves a conformational shift. resulting in a mature form. Therefore. precursor processing not only cleaves the signal peptide, but also induces conformational shift. allowing development of active form of the enzyme.

• Journal of Microbiology and Biotechnology
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• v.16 no.4
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• pp.613-618
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• 2006

Two endoxylanases from an alkaliphilic bacterium, Bacillus halodurans C-1, were purified 3.8- and 7.9- fold with specific activities of 9.4 and 19.8U/mg protein, respectively. The molecular masses of both purified enzymes were 23 and 47 kDa, respectively, and 23 kDa xylanase I (Xyl I) exhibited an optimum pH at 7.0, whereas 47 kDa xylanase II (Xyl II) showed a broad pH range of 5.0 to 9.0. The temperature optima of both xylanases were $60^{\circ}C\;and\;70^{\circ}C$, respectively. Both were stable in the pH range of 6.0 to 9.0 and 5.0 to 10.0, respectively, and they were stable up to $60^{\circ}C\;and\;70^{\circ}C$, respectively. The $K_m\;and\;V_{max}$ of Xyl I were 4.33mg/ml and $63.5{\mu}mol/min/mg$, respectively, whereas Xyl II had a $K_m$ value of 0.30 mg/ml and $V_{max}$ of $210{\mu}mol/min/mg$. Both xylanases hydrolyzed xylans from birchwood, oat spelt, and larchwood. However, they showed different modes of action; a series of xylooligosaccharides larger than xylotriose were released as the major products by Xyl I, whereas xylobiose and xylotriose were the main products by Xyl II. The maximum synergistic action of the two enzymes on hydrolysis of xylan was 2.16 with the ratio of Xyl I to Xyl II at 1:9.

• Journal of Microbiology and Biotechnology
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• v.16 no.6
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• pp.921-926
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• 2006

The production and location of xylanolytic enzymes in alkaliphilic Bacillus sp. K-1, isolated from the wastewater treatment plant of the pulp and paper industry, was studied. When grown in alkaline xylan medium, the bacteria produced xylanolytic enzymes such as xylanase, $\beta$-xylosidase, arabinofuranosidase, and acetyl esterase. Two types of xylanases (23 and 45 kDa) were found to be extracellular, but another type of xylanase (35 and/or 40 kDa) was detected as pellet-bound that was eluted with 2% triethylamine from the residual xylan of the culture. The xylanases were different in their molecular weight and xylan-binding ability. Arabinofuranosidase and $\beta$-xylosidase were found to be intracellular and extracellular, respectively, and acetyl esterase was found to be extracellular. The extracellular xylanolytic enzymes effectively hydrolyzed insoluble xylan, lignocellulosic materials, and xylans in kraft pulps.

• ALGAE
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• v.35 no.1
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• pp.91-106
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• 2020

Incorporating renewable fuel into practice, especially from algae, is a promising approach in reducing fossil fuel dependency. Algae are an exceptional feedstock since they produce abundant biomass and oils in short timeframes. Algae also produce high-valued lipid products suitable for human nutrition and supplement. Achieving goals of producing algae fuels and high-valued lipids at competitive prices involves further improvement of technology, especially better control over cultivation. Manipulating microalgae cultivation conditions to prevent contamination is essential in addition to promoting optimal growth and lipid yields. Contamination of algal cultures is a major impediment to algae cultivation that can however be mitigated by choosing extremophile microalgae. This work describes the isolation of alkali-tolerant / alkaliphilic microalgae native to South Florida with ideal characteristics for cultivation. For that purpose, water samples from Lake Okeechobee were inoculated into Zarrouk's medium (pH 9-12) and incubated for 35 days. Selection resulted in isolation of three strains that were screened for biomass and lipid accumulation. Two alkali-tolerant algae Chloroidium sp. 154-1 and Chlorella sp. 154-2 were poor lipid accumulators. One of the isolates, the diatom Fistulifera sp. 154-3, was identified as a lipid accumulating, alkaliphilic organism capable of producing 0.233 g L^-1 d^-1 dry biomass and a lipid content of 20-30% dry weight. Lipid analysis indicated the most abundant fatty acid within Fistulifera sp. was palmitoleic acid (52%), or omega-7, followed by palmitic acid (17%), and then eicosapentanoic acid (15%). 18S rRNA phylogenetic analysis formed a well-supported clade with Fistulifera species.