• Journal of Microbiology and Biotechnology
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• v.12 no.5
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• pp.766-772
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• 2002

The plasmid, pJHKJ4, containing the endoxylanase gene, was introduced into Bacillus subtilis DB 104. The recombinant cells produced 587 unit/ml of endoxylanase at 33 h. The endoxylanase was immobilized covalently on the surface of silica fur effective xylan hydrolysis. The activities of the immobilized and free endoxylanases were optimal at pH 6.5 and 10 mM $MnSO_4$. The optimal temperature of the immobilized endoxylanase was $60^{\circ}C$, whereas that of the free endoxylanase was $65^{\circ}C$. Under these optimal conditions, the activity of the immobilized endoxylanase was 1.7 times higher than that of the fee endoxylanase. From microscope photographs, the immobilized endoxylanase was found to be bounded and evenly distributed on the surface of silica, a nonporous solid support. The enzyme kinetics between the immobilized and free endoxylanases was estimated to be uncompetitive, when plotting double-reciprocal plots against xylan concentrations and endoxylanase activities. These results suggest that the higher activity of the immobilized endoxylanase may be due to increased formation of enzyme-substrate complex, because of the easy accessibility of the immobilized enzyme to the polysaccharide-xylan as a high molecular weight substrate.

• Journal of Life Science
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• v.10 no.2
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• pp.125-130
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• 2000

To overproduce endoxylanase from a recombinant Bacillus subtilis harboring the pJHKJ4 plasmid, the effects of carbon and nitrogen sources on the cell growth and expression level of endoxylanase were investigated in the flask cultures. Among the various carbon and nitrogen sources tested, glucose and maltose as carbon source and yeast extract as nitrogen source were found to be the most effective for the cell growth and the endoxylanase expression. When the concentration of glucose was increased from 0.5% to 5%, the highest activity of extracellular endoxylanse, 166 unit/$m\ell$, was observed at 2% glucose. In case of maltose, the endoxylanase was stably produced at the level of 180 unit/$m\ell$, regardless of the concentration of maltose. The higher the concentration of yeast extract, the greater cell growth and endoxylanase expression were obtained. However, the highest endoxylanase activity per unit cell mass was observed with 1% yeast extract. With the optimized medium (2% glucose, 1% yeast extract, etc), about 630 unit/$m\ell$ of endoxylanse was expressed through the batch fermentation in a fermentor, which expression level corresponded to about 0.7 g-endoxylanase protein /$\ell$. It was also found that the plasmid was stably maintained above 70% level, and more than 90% of endoxylanase activity was detected in the extracellular medium.

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

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

• Microbiology and Biotechnology Letters
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• v.36 no.4
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• pp.307-313
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• 2008

The yeast surface expression system, pCTXYN (6.8 kb), of Bacillus endoxylanase gene (xynB, 642 bp) was constructed and introduced into Saccharomyces cerevisiae EBY100 cell. The transformed yeast cell showing the highest endoxylanase activity was selected through the active staining of colonies grown on YPDG medium containing xylan. With the yeast transformant, EBY100/pCTXYN, grown on galactose containing medium, it was found that the endoxylanase was successfully displayed on the yeast cell surface and the xylooligosaccharides were efficiently produced from xylan. The most of endoxylanase activity was detected in the cell fraction and reached about 1.9 unit/mL after 48 h cultivation. The optimized conditions for xylooligosaccharides production from xylan were determined as follows: substrate and its concentration, oat spelt xylan 6%; concentration of yeast whole-cell, 5 unit/mL; temperature, $50^{\circ}C$, and reaction time $2{\sim}4\;h$. When the oat spelts xylan and corncob xylan were hydrolyzed by treatment with cell surface-displayed endoxylanase, xylotriose was formed as a main product.

• Microbiology and Biotechnology Letters
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• v.46 no.4
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• pp.390-394
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• 2018

For the coexpression of endoxylanase and endoglucanase genes in yeast Saccharomyces cerevisiae, the genes were separately inserted downstream of the yeast ADH1 promoters, resulting the plasmid pAGX3 (9.83 kb). In the batch culture on YPD medium of the yeast transformant, S. cerevisiae SEY2102/pAGX3, the total activities of the enzymes reached about 7.91 units/ml for endoxylanase and 0.43 units/ml for endoglucanase. In the fed-batch culture with intermittent feeding of yeast extract and glucose, the total activities of 24.9 units/ml for endoxylanase and 0.84 units/ml for endoglucanase were produced which were about 3.1-fold and 2.0-fold increased levels, respectively, compared to those of the batch culture. Most of endoxylanase and endoglucanase activities were found in the extracellular media. This recombinant yeast could be useful for the development of simultaneous saccharification bioprocess of the cellulose and xylan mixture.

• Journal of Life Science
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• v.18 no.1
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• pp.52-57
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• 2008

Xylan is a major hemicellulose component of the cell walls of monocots and hardwood, representing up to 30% of the dry weight of these plants. To efficiently hydrolyze xylan, the endoxylanase gene from Bacillus sp. was expressed in B. subtilis DB431 by introducing the plasmid pJHKJ4. The total activity of the recombinant endoxylanase reached about 857 unit/ml by batch fermentation of B. subtilis DB431/pJHKJ4 in LB maltose medium. The majority (>92%) of endoxylanase was efficiently secreted into the culture medium. The recombinant endoxylanase hydrolyzed more the birchwood xylan efficiently than the other xylans. When 4 % concentration of xylan was used, the highest production of xylooligosaccharide was observed, and xylobiose and xylotriose were the major products. Optimal amount of enzyme and reaction time for producing xylooligosaccharide were found to be 10 unit and 1 hr, respectively. In addition, the temperature of $40^{\circ}C{\sim}50^{\circ}C$ gave the highest production of xylooligosaccharide. Consequently, the optimized conditions for the production of xylooligosaccharide through the hydrolysis of xylan were determined as follows: 10 unit endoxylanase, $50^{\circ}C$, 4% birchwood xylan, 1 hr reaction.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.171-177
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• 2004

The endoxylanase (642 bp; 213 amino acids) and $\beta$-xylosidase (1,602 bp; 533 amino acids) genes from Bacillus sp. were amplified by PCR and separately inserted into the downstream of the yeast ADH1 promoters, resulting in the pAEDX-1 (7.63 kb) and pAEX (8.47 kb) plasmids, respectively. When the yeast transformants, S. cerevisiae SEY2102 harboring pAEDX-1 or pAEX, were grown on YPD medium, the total activities of the enzymes were approximately 9.8 unit/ml for endoxylanase and 2.9 unit/m1 for $\beta$-xylosidase. When the three kinds of xylan from oat spelts, birch wood, and corncob were hydrolyzed by treating with recombinant endoxylanase and $\beta$-xylosidase, it was found that xylose, xylobiose, and xylotriose were produced. To efficiently hydrolyze xylan, various reaction conditions such as amount of enzymes, substrate type, substrate concentration, temperature, and reaction time were examined. The optimized conditions for the hydrolysis of xylan were as follows: amount of endoxylanase, 10 units; amount of $\beta$-xylosidase, 10 units; temperature, $50^\circ{C}$; substrate type, oat spelts xylan; substrate concentration, 6%; reaction time, 1 h. Under the optimal condition, xylose was mainly produced from oat spelts xylan by cooperative action of endoxylanase and $\beta$-xylosidase.

• Journal of Microbiology and Biotechnology
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• v.17 no.12
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• pp.2076-2080
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• 2007

The endoxylanase (GenBank Access No. U51675) of Bacillus spp. and endoglucanase (GenBank Access No. AY466436) of Trichoderma spp. were separately inserted downstream of the yeast constitutive ADHI promoter, resulting in three different plasmids (pAGX1, pAGX2, and pAGX3) according to the transcription direction of two genes. When the yeast transformants, S. cerevisiae SEY2102 harboring each expression plasmid, were grown on YPD medium, the total activities of the enzymes were approximately 3.01 unit/ml, 3.24 unit/ml, and 7.56 unit/ml for endoxylanase and 0.60 unit/ml, 0.54 unit/ml, and 0.39 unit/ ml for endoglucanase, in the following order: the pAGX1, pAGX2, and pAGX3. More than 70% of the endoxylanase and endoglucanase activities was found in the extracellular media.

• Proceedings of the Microbiological Society of Korea Conference
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• 2003.05a
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• pp.122-124
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• 2003

Cyclodextrin glucanotransferase (CGTase) and endoxylanase genes of Bacillus sp. were subcloned down-stream of yeast ADH1 promoter and expressed in S. cerevisiae. Most of the CGTase and endoxylanase expressed were detected in the extracellular medium with activity of 0.6 and 7-8 unit/ml, respectively. The recombinant enzymes were secreted as N-linked-glycosylated forms, resulting an enhanced thermal stability. CGTase predominantly produced $\alpha-cyclodextrin$ from starch and endoxylanase produced xylobiose and xylotriose from xylan.