• Journal of Microbiology and Biotechnology
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• v.7 no.4
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• pp.223-228
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• 1997

A DNA fragment containing the gene for cell wall hydrolase of alkalophilic Bacillus subtilis BL-29 was cloned into E. coli JM109 using pUC18 as a vector. A recombinant plasmid, designated pCWL45B, was contained in the fragment originating from the alkalophilic B. subtilis BL-29 chromosomal DNA by Southern hybridization analysis. The nucleotide sequence of a 1.6-kb HindIII fragment containing a cell wall hydrolase-encoding gene was determined. The nucleotide sequence revealed an open reading frame (ORF) of 900 bp with a concensus ribosome-binding site located 6 nucleotide upstream from the ATG start codon. The primary amino acid sequence deduced from the nucleotide sequence revealed a putative protein of 299 amino acid residues with an M.W. of 33, 206. Based on comparison of the amino acid sequence of the ORF with amino acid sequences in the GenBank data, it showed significant homology to the sequence of cell wall amidase of the PBSX bacteriophage of B. subtilis.

• BMB Reports
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• v.28 no.5
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• pp.443-450
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• 1995

Cloned staphylococcal peptidoglycan hydrolase was used in determining the physiological characteristics of peptidoglycan hydrolase. This enzyme hydrolyzed the bacterial cell walls and released the N-terminal alanine, but not the reducing groups. This cloned gene product was localized in the cytoplasm of transformed Escherichia coli. Activity gels indicated the enzyme had an Mr of about 54,000, which was consistent with the deduced Mr from sequencing of the cloned gene. The activity bound to CM-cellulose but not DEAE-cellulose resin, indicating it as a basic protein. Enhanced enzyme activity in a low concentration of cations, and inhibited enzyme activity in a solution with dissolved phospholipids, suggested that the activity and the availability of this basic protein may be regulated between negatively charged and positively charged cellular molecules. The activity against boiled crude cell wall was much greater than against purifed cell wall, suggesting protein associated with crude cell wall may aid in the binding of the peptidoglycan hydrolase The cloned peptidoglycan hydrolase showed positive activity on whole cells of some lysostaphin-resistant coagulase-negative staphylococci. The cloned enzyme may be an alternative for lysostaphin for lysis of staphylococci.

• Journal of Microbiology and Biotechnology
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• v.14 no.6
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• pp.1142-1149
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• 2004

Streptococcus mutans has the capacity of inducing dental caries. Thus, to develop a novel way of preventing dental caries, a cell wall hydrolase-producing strain was isolated and its characteristics were investigated. Among 200 alkalophilic strains isolated from soil, 8 strains exhibited lytic activities against Streptococcus mutans. However, strain YU5215 with the highest cell wall hydrolase activity was selected for further study. Strain YU5215 was identified as a novel strain of Bacillus based on analyzing its 16S rDNA sequence and Bergey's Manual of Systematic Bacteriology, and thus designated as Bacillus mutanolyticus YU5215. The optimal conditions for the production of the cell wall hydrolase from Bacillus mutanolyticus YU5215 consisted of glucose ($0.8\%$), yeast extract ($1.2\%$), polypeptone ($0.5\%$), $K_{2}HPO_{4}\;(0.1\%$), $MgSO_{4}{\cdot}7H_{2}O$ ($0.02\%$), and $Na_{2}CO_{3}\;(1.0\%$) at pH 10.0. Bacillus mutanolyticus YU5215 was cultured at 30^{circ}C for 72 h to produce the cell wall hydrolase, which was then purified by acetone precipitation and CM-agarose column chromatography. The molecular weight of the lytic enzyme was determined as 22,700 Da by SDS-PAGE. When the cell wall peptidoglycan of Streptococcus mutans was digested with the lytic enzyme, no increase in the reducing sugars was observed, while the free amino acids increased, indicating that the lytic enzyme had an endopeptidase-like property. The amino terminus of the cell wall peptidoglycan digested by the lytic enzyme was determined as a glutamic acid, while the lytic site of the lytic enzyme in the Streptococcus mutans peptidoglycan was identified as the peptide linkage of L-Ala and D-Glu.

• Journal of Microbiology and Biotechnology
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• v.11 no.3
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• pp.508-514
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• 2001

A stuructural gene (ycl) encoding novel yeast cell wall hydrolase, YCL, was cloned from alkalophilic Bacillus alcalophilus subsp. YB380 by PCR, and transformed into E. coli JM83. Based on the N-terminal and internal amino acid sequences of the enzyme, primers were designed for PCr. The positive clone that harbors 1.8 kb of the yeast cell wall hydrolase gene was selected by the colony hybridization method with a PCR fragment as a probe. According to the computer analysis, this gene contained a 400-base-paired N-terminal domain of the enzyme. Based on nucletide homology of the cloned gene, a 850 bp fragment was amplified and the C-terminal domain of the enzyme was sequenced. With a combination of the two sequences, a full nucleotide sequence for YCL was obtained. This gene, ycl, consisted of 1,297 nucleotides with 27 nucleotides with 27 amino acids of signal sequence, 83 redundant amino acids of prosequence, and 265 amino acids of the mature protein. This gene was then cloned into the pJH27 shuttle vector and transformed into the Bacillus subtilis DB104 to express the enzyme. It was confirmed that the expressed cell wall hydrolase that was produced by Bacillus subtilis DB104 was the same as that of the donor strain, by Western blot using polyclonal antibody (IgY) prepared from White Leghorn hen. Purified yeast cell wall hydrolase and expressed recombinant protein showed a single band at the same position in the Western blot analysis.

• Journal of Microbiology and Biotechnology
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• v.8 no.5
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• pp.501-508
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• 1998

An alkalophilic mi.croorganism (strain YB380), which produces yeast cell wall hydrolase extracellulary, was isolated from Korean soil. The rod-shaped cells were 0.3~0.4 by 2~4${\mu}{\textrm}{m}$ long, motile, aerobic, gram-positive, and spore-forming. The color of the colony was light yellow. The temperature range for growth at pH 9.0 was 25 to $45{\circ}C, with optimum growth at $35{\circ}C. The pH range for growth at $35{\circ}C was 8 to 11 with an optimum pH of 9.0. Therefore, the strain YB380 is an obligate alkalophile. The 16S rRNA of strain YB380 has a 99% sequence similarity with that of Bacillus alcalophilus. On the basis of physiological properties, cell wall fatty acid composition, and phylogenetic analysis, we propose that the isolated strain is Bacillus alcalophilus. The yeast cell wall hydrolase from Bacillus alcalophilus subsp. YB380 has been purified and partially characterized. The molecular weight was estimated to be 27,000 daltons with an optimum temperature and pH of $60{\circ}C and 9.0, respectively. The N-terminal amino acid sequence of the enzyme was analyzed as Gln- Thr- Val- Pro- Trp- Gly- Ile- Asn- Arg- Val.

• Proceedings of the Korean Society of Crop Science Conference
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• 2007.04a
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• pp.65-73
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• 2007

The objective of this study is to understand how regulatory mechanisms respond to sugar status for more efficient carbon utilization and source-sink regulation in plants. So, we need to identify and characterize many components of sugar-response pathways for a better understanding of sugar responses. For this end, genes responding change of sugar status were screened using Arabidpsis cDNA arrays, and confirmed thirty-six genes to be regulated by sucrose supply in detached leaves by RNA blot analysis. Eleven of them encoding proteins for amino acid metabolism and carbohydrate metabolism were repressed by sugars. The remaining genes induced by sugar supply were for protein synthesis including ribosomal proteins and elongation factors. Among them, I focused on three hydrolase genes encoding putative $\beta$-galactosidase, $\beta$-xylosidase, and $\beta$-glucosidase that were transcriptionally induced in sugar starvation. Homology search indicated that these enzymes were involved in hydrolysis of cell wall polysaccharides. In addition to my results, recent transcriptome analysis suggested multiple genes for cell wall degradation were induced by sugar starvation. Thus, I hypothesized that enzyme for cell wall degradation were synthesized and secreted to hydrolyze cell wall polysaccharides producing carbon source under sugar-starved conditions. In fact, the enzymatic activities of these three enzymes increased in culture medium of Arabidopsis suspension cells under sugar starvation. The $\beta$-galactosidase encoded by At5g56870 was identified as a secretory protein in culture medium of suspension cells by mass spectrometry analysis. This protein was specifically detected under sugar-starved condition with a specific antibody. Induction of these genes was repressed in suspension cells grown with galactose, xylose and glucose as well as with sucrose. In planta, expression of the genes and protein accumulation were detected when photosynthesis was inhibited. Glycosyl hydrolase activity against galactan also increased during sugar starvation. Further, contents of cell wall polysaccharides especially pectin and hemicellulose were markedly decreased associating with sugar starvation in detached leaves. The amount of monosaccharide in pectin and hemicellulose in detached leaves decreased in response to sugar starvation. These results supported my idea that cell wall has one of function to supply carbon source in addition to determination of cell shape and physical support of plant bodies.

• Journal of Microbiology and Biotechnology
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• v.19 no.6
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• pp.573-581
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• 2009

The complex enzyme pool secreted by the phytopathogenic fungus Fusarium graminearum in response to glucose or hop cell wall material as sole carbon sources was analyzed. The biochemical characterization of the enzymes present in the supernatant of fungal cultures in the glucose medium revealed only 5 different glycosyl hydrolase activities; by contrast, when analyzing cultures in the cell wall medium, 17 different activities were detected. This dramatic increase reflects the adaptation of the fungus by the synthesis of enzymes targeting all layers of the cell wall. When the enzymes secreted in the presence of plant cell wall were used to hydrolyze pretreated crude plant material, high levels of monosaccharides were measured with yields approaching 50% of total sugars released by an acid hydrolysis process. This report is the first biochemical characterization of numerous cellulases, hemicellulases, and pectinases secreted by F. graminearum and demonstrates the usefulness of the described protein cocktail for efficient enzymatic degradation of plant cell wall.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2005.11a
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• pp.371-374
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• 2005

In our previous work in transcriptional regulation of sugar, expression of genes encoding putative glycosyl hydrolases in Arabidopsis was induced by sugar starvation. They were annotated as b-galactosidase (At5g56870), ${\beta}-xylosidase$ (At5g49360) and ${\beta}-glucosidase$ (At3g60140), which belong to glycosyl hydrolase family that has a catalytic domain of polysaccharides. From the primary structure of deduced amino acid sequence, they were predicted to localize to cell wall. Further investigation of these cell wall hydrolases implicated that cell wall polysaccharides provide metabolizable sugars to nutrient allocation under sugar starvation.

• Korean Journal of Microbiology
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• v.37 no.1
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• pp.15-20
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• 2001

In this study, we have isolated bacterial cell wall lytic enzyme in the culture supernatant of Aspergillus sp. HCLF-4. This hydrolase showed cell wall lytic activity against Anabaena cylindrica. The extracellular enzyme was produced by Aspergillus sp. HCLF-4 when it was grown in a PDB media containing 0.05% heat killed Micrococcus luteus cells. The molecular weight of lytic enzyme was about 14.3 kDa. The optimal pH and temperature for the activity of this enzyme were 3.0~4.0 and $30^{\circ}C$, respectively. This hydrolase activity was reduced by $Na^{+}$, $Li^{+}$, $Ca^{2+}$, $Cu^{2+}$, $Fe^{3+}$, EDTA, and PMSF, whereas it was increased by $Mg^{2+}$, $Mn^{2+}$>. The enzyme has N-acetylmuramyl-L-amidase or endopeptidase activity.

• Journal of Microbiology and Biotechnology
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• v.18 no.5
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• pp.946-951
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• 2008

The opd gene, encoding organophosphorus hydrolase (OPH) from Flavobacterium sp. capable of degrading a wide range of organophosphate pesticides, was surface- and intracellular-expressed in Synechococcus PCC7942, a prime example of photoautotrophic cyanobacteria. OPH was displayed on the cyanobacterial cell surface using the truncated ice nucleation protein as an anchoring motif. A minor fraction of OPH was displayed onto the outermost surface of cyanobacterial cells, as verified by immunostaining visualized under confocal laser scanning microscopy and OPH activity analysis; however, a substantial fraction of OPH was buried in the cell wall, as demonstrated by proteinase K and lysozyme treatments. The cyanobacterial outer membrane acts as a substrate (paraoxon) diffusion barrier affecting whole-cell biodegradation efficiency. After freeze-thaw treatment, permeabilized whole cells with intracellular-expressed OPH exhibited 14-fold higher bioconversion efficiency ($V_{max}/K_m$) than that of cells with surface-expressed OPH. As cyanobacteria have simple growth requirements and are inexpensive to maintain, expression of OPH in cyanobacteria may lead to the development of a low-cost and low-maintenance biocatalyst that is useful for detoxification of organophosphate pesticides.