• KSBB Journal
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• v.26 no.3
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• pp.243-247
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• 2011

Using Bacillus subtilis spore display system, with cotG as an anchoring motif, levansucrase from Zymomonas mobilis, was displayed on the outer surface of Bacillus subtilis spore. Flow cytometry of DB104 (pSDJH-cotG-levU) spore, proved the surface localization of CotG-LevU fusion protein on the spore compared to that of DB104. Enzymatic activity of DB104 (pSDJH-cotG-levU) spore showed more than 1.5 times higher levansucrase specific activity compared to that of the host spore, which is a remarkable increase of enzymatic activity considering the existence of sacA (sucrase) and sacB (levansucrase) in the Bacillus subtilis chromosome. The spore integrity, revealed by sporulation frequency test after heat and lysozyme treatment of spore, did not changed at all in spite of the CotG-LevU fusion protein incorporation into the spore coat layer during spore formation process. These data prove again that Bacillus subtilis spore could be considered as good live immobilization vehicle for efficient bioconversion process.

• KSBB Journal
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• v.26 no.3
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• pp.199-205
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• 2011

Using tetrameric ${\beta}$-galactosidase as a model protein, anchoring motives were screened in Bacillus subtilis spore display system. Eleven spore coat proteins were selected considering their expression levels and the location in the spore coat layer. After chromosomal single-copy homologous integration in the amyE site of Bacillus subtilis chromosome, cotE and cotG were chosen as possible spore surface anchoring motives with their higher whole cell ${\beta}$-galactosidase activity. PAGE and Wester blot of extracted fraction of outer layer of purified spore, which express CotE-LacZ or CotG-LacZ fusion verified the existence of exact size of fusion protein and its location in outer coat layer of purified spore. ${\beta}$-galactosidase activity of spore with CotE-LacZ or CotG-LacZ fusion reached its highest value around 16~20 h of culture time in terms of whole cell and purified spore. After intensive spore purification with lysozyme treatment and renografin treatment, spore of BJH135, which expresses CotE-LacZ, retained only 1~2% of its whole cell ${\beta}$-galactosidase activity. Whereas spore of BJH136, which has cotG-lacZ cassette in the chromosome, retained 10~15% of its whole cell ${\beta}$-galactosidase activity, proving minor perturbation of CotG-LacZ, when incorporated in the spore coat layer of Bacillus subtilis compared to CotE-LacZ. Usage of Bacillus subtilis WB700, of which 7 proteases are knocked-out and thereby resulting in 99.7% decrease in protease activity of the host, did not prevent the proteolytic degradation of spore surface expressed CotG-LacZ fusion protein.

• Journal of Microbiology and Biotechnology
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• v.29 no.5
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• pp.749-757
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• 2019

Nitrilase is a valuable hydrolase that catalyzes nitriles into carboxylic acid and ammonia. Its applications, however, are severely restricted by the harsh conditions of industrial reaction processes. To solve this problem, a nitrilase from Acidovorax facilis 72W was inserted into an Escherichia coli-Bacillus subtilis shuttle vector for spore surface display. Western blot, enzyme activity measurements and flow cytometric analysis results all indicated a successful spore surface display of the CotB-nit fusion protein. In addition, the optimal catalytic pH value and temperature of the displayed nitrilase were determined to be 7.0 and $50^{\circ}C$, respectively. Moreover, results of reusability tests revealed that 64% of the initial activity of the displayed nitrilase was still retained at the $10^{th}$ cycle. Furthermore, hydrolysis efficiency of upscale production of cyanocarboxylic acid was significantly higher in the displayed nitrilase-treated group than in the free group expressed by E. coli (pET-28a-nit). Generally, the display of A. facilis 72W nitrilase on the spore surface of Bacillus subtilis may be a useful method for immobilization of enzyme and consequent biocatalytic stabilization.

• Journal of Microbiology and Biotechnology
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• v.19 no.5
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• pp.495-501
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• 2009

A new spore display method is presented that enables recombinant proteins to be displayed on the surface of Bacillus spores via fusion with InhA, an exosporium component of Bacillus thuringiensis. The green fluorescent protein and $\beta$-galactosidase as model proteins were fused to the C-terminal region of InhA, respectively. The surface expression of the proteins on the spores was confirmed by flow cytometry, confocal laser scanning microscopy, measurement of the enzyme activity, and an immunogold electron microscopy analysis. InhA-mediated anchoring of foreign proteins in the exosporium of Bacillus spores can provide a new method of microbial display, thereby broadening the potential for novel applications of microbial display.

• Journal of Microbiology and Biotechnology
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• v.17 no.4
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• pp.677-680
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• 2007

To analyze a cotG-based Bacillus subtilis spore display system directly, $GFP_{UV}$ was expressed on the surface of Bacillus subtilis spores. When $GFP_{UV}$ was fused to the C-terminal of the cotG structural gene and expressed, the existence of a $CotG-GFP_{UV}$ fusion protein on the B. subtilis spore was confirmed by flow cytometry confocal microscopic analysis. When the cotG anchoring motif was deleted, no fluorescence emission was observed under flow cytometry and confocal microscopic analysis from the purified spore, confirming the essential role of CotG as an anchoring motif. This $GFP_{UV}$ displaying spore might be used for another signaling application triggered by intracellular or extracellular stimuli.

• Journal of Microbiology and Biotechnology
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• v.29 no.2
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• pp.179-190
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• 2019

Bacillus subtilis spore surface display (BSSD) technology is considered to be one of the most promising approaches for expressing heterologous proteins with high activity and stability. Currently, this technology is used for various purposes, such as the production of enzymes, oral vaccines, drugs and multimeric proteins, and the control of environmental pollution. This paper presents an overview of the latest developments in BSSD technology and its application in protein engineering. Finally, the major limitations of this technology and future directions for its research are discussed.

• Journal of Microbiology and Biotechnology
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• v.29 no.9
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• pp.1383-1390
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• 2019

In this study, we expressed cotA laccase from Bacillus subtilis on the surface of B. subtilis spores for efficient decolorization of synthetic dyes. The cotE, cotG, and cotY genes were used as anchoring motifs for efficient spore surface display of cotA laccase. Moreover, a $His_6$ tag was inserted at the C-terminal end of cotA for the immunological detection of the expressed fusion protein. Appropriate expression of the CotE-CotA (74 kDa), CotG-CotA (76 kDa), and CotY-CotA (73 kDa) fusion proteins was confirmed by western blot. We verified the surface expression of each fusion protein on B. subtilis spore by flow cytometry. The decoloration rates of Acid Green 25 (anthraquinone dye) for the recombinant DB104 (pSDJH-EA), DB104 (pSDJH-GA), DB104 (pSDJH-YA), and the control DB104 spores were 48.75%, 16.12%, 21.10%, and 9.96%, respectively. DB104 (pSDJH-EA) showed the highest decolorization of Acid Green 25 and was subsequently tested on other synthetic dyes with different structures. The decolorization rates of the DB104 (pSDJH-EA) spore for Acid Red 18 (azo dye) and indigo carmine (indigo dye) were 18.58% and 43.20%, respectively. The optimum temperature for the decolorization of Acid Green 25 by the DB104 (pSDJH-EA) spore was found to be $50^{\circ}C$. Upon treatment with known laccase inhibitors, including EDTA, SDS, and $NaN_3$, the decolorization rate of Acid Green 25 by the DB104 (pSDJH-EA) spore decreased by 23%, 80%, and 36%, respectively.

• Journal of Microbiology and Biotechnology
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• v.27 no.3
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• pp.507-513
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• 2017

Bacillus subtilis spores can be used for protein display to engineer protein properties. This method overcomes viability and protein-folding concerns associated with traditional protein display methods. Spores remain viable under extreme conditions and the genotype/phenotype connection remains intact. In addition, the natural sporulation process eliminates protein-folding concerns that are coupled to the target protein traveling through cell membranes. Furthermore, ATP-dependent chaperones are present to assist in protein folding. CotA was optimized as a whole-cell biocatalyst immobilized in an inert matrix of the spore. In general, proteins that are immobilized have advantages in biocatalysis. For example, the protein can be easily removed from the reaction and it is more stable. The aim is to improve the pH stability using spore display. The maximum activity of CotA is between pH 4 and 5 for the substrate ABTS (ABTS = diammonium 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate). However, the activity dramatically decreases at pH 4. The activity is not significantly altered at pH 5. A library of approximately 3,000 clones was screened. A E498G variant was identified to have a half-life of inactivation ($t_{1/2}$) at pH 4 that was 24.8 times greater compared with wt-CotA. In a previous investigation, a CotA library was screened for organic solvent resistance and a T480A mutant was found. Consequently, T480A/E498G-CotA was constructed and the $t_{1/2}$ was 62.1 times greater than wt-CotA. Finally, E498G-CotA and T480A/E498G-CotA yielded 3.7- and 5.3-fold more product than did wt-CotA after recycling the biocatalyst seven times over 42 h.

• Journal of Microbiology and Biotechnology
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• v.26 no.7
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• pp.1267-1277
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• 2016

Currently, enzymatic synthesis of lactulose, a synthetic prebiotic disaccharide, is commonly performed with glycosyl hydrolases. In this work, a new type of lactulose-producing biocatalyst was developed by displaying β-galactosidase from Bacillus stearothermophilus IAM11001 (Bs-β-Gal) on the surface of Bacillus subtilis 168 spores. Localization of β-Gal on the spore surface as a fusion to CotX was verified by western blot analysis, immunofluorescence microscopy, and flow cytometry. The optimum pH and temperature for the resulting spore-displayed β-Gal was 6.0 and 75℃, respectively. Under optimal conditions, it showed maximum activity of 0.42 U/mg spores (dry weight). Moreover, the spore-displayed CotX-β-Gal was employed as a whole cell biocatalyst to produce lactulose, yielding 8.8 g/l from 200 g/l lactose and 100 g/l fructose. Reusability tests showed that the spore-displayed CotX-β-Gal retained around 30.3% of its initial activity after eight successive conversion cycles. These results suggest that the CotX-mediated spore-displayed β-Gal may provide a promising strategy for lactulose production.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.46 no.3
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• pp.296-302
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• 2013

The effects of temperature on spore release, growth and photosynthetic efficiency of Lithophyllum yessoense and Hildenbrandia rubra were examined. L. yessoense was collected at Galnam and H. rubra was collected at Gyeokpo, Korea. The experimental temperatures were different for spore release (10, 15, $20^{\circ}C$), sporeling growth (10, 15, 20, 25, $30^{\circ}C$) and photosynthetic efficiency (10, 15, 20, $25^{\circ}C$). All other culture conditions were the same: 34 psu, 12:12 LD and $50{\mu}mol$ photon $m^{-2}s^{-1}$. Spore liberation was maximal at $10^{\circ}C$ for L. yessoense and at $20^{\circ}C$ for H. rubra. After 14 days, the surface area of L. yessoense was 0.031 $mm^2$ at $25^{\circ}C$ and for H. rubra was 0.032 $mm^2$ at $20^{\circ}C$. Sporelings of L. yessoense were a dark-red color and grew in a round shape. In contrast, H. rubra was bright pink and changed from a round shape in the early growth stage to later become flabelliform. Photosynthetic efficiency was highest between $20-25^{\circ}C$ in both species. In conclusion, L. yessoense and H. rubra display different physiological features based on the optimal temperatures for spore release and sporling growth.