• Journal of Microbiology and Biotechnology
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• v.19 no.10
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• pp.1169-1175
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• 2009

An antifungal chitinase, ChiCW, produced by Bacillus cereus 28-9 is effective against conidial germination of Botrytis elliptica, the causal agent of lily leaf blight. ChiCW as a modular enzyme consists of a signal peptide, a catalytic domain, a fibronectin type-III-like domain, and a chitin-binding domain. When two C-terminal domains of ChiCW were truncated, $ChiCW{\Delta}FC$ (lacking the chitin-binding domain and fibronectin type III-like domain) lost its antifungal activity. Since $ChiCW{\Delta}C$ (lacking the chitin-binding domain) could not be expressed in Escherichia coli as $ChiCW{\Delta}FC$ did, a different strategy based on protein engineering technology was designed to investigate the involvement of the chitin-binding domain of ChiCW ($ChBD_{ChiCW}$) in antifungal activity in this study. Because ChiA1 of Bacillus circulans WL-12 is a modular enzyme with a higher hydrolytic activity than ChiCW but not inhibitory to conidial germination of Bo. elliptica and the similar domain composition of ChiA1 and ChiCW, the C-terminal truncated derivatives of ChiA1 were generated and used to construct chimeric chitinases with $ChBD_{ChiCW}$. When the chitin-binding domain of ChiA1 was replaced with $ChBD_{ChiCW}$, the chimeric chitinase named ChiAAAW exhibited both high enzyme activity and antifungal activity. The results indicate that $ChBD_{ChiCW}$ may play an important role in the antifungal activity of ChiCW.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2002.11a
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• pp.57.1-57
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• 2002

• BMB Reports
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• v.44 no.6
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• pp.375-380
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• 2011

Bacillus licheniformis SK-1 naturally produces chitinase 72 (CHI72) with two truncation derivatives at the C-terminus, one with deletion of the chitin binding domain (ChBD), and the other with deletions of both fibronectin type III domain (FnIIID) and ChBD. We constructed deletions mutants of CHI72 with deletion of ChBD (CHI72${\Delta}$ChBD) and deletions of both FnIIID and ChBD (CHI72${\Delta}$FnIIID${\Delta}$ChBD), and studied their activity on soluble, amorphous and crystalline substrates. Interestingly, when equivalent amount of specific activity of each enzyme on soluble substrate was used, the product yield from CHI72-${\Delta}$ChBD and CHI72${\Delta}$FnIIID${\Delta}$ChBD on colloidal chitin was 2.5 and 1.6 fold higher than CHI72, respectively. In contrast, the product yield from CHI72${\Delta}$ChBD and CHI72${\Delta}$FnIIID-${\Delta}$ChBD on ${\beta}$-chitin reduced to 0.7 and 0.5 fold of CHI72, respectively. These results suggest that CHI72 can modulate its substrate specificities through truncations of the functional domains at the C-terminus, producing a mixture of enzymes with elevated efficiency of hydrolysis.

• Journal of Microbiology and Biotechnology
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• v.16 no.12
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• pp.1897-1903
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• 2006

In order to investigate the functions of the C-terminal region of chitinase ChiCW of Bacillus cereus 28-9, a C-terminal truncated enzyme, ChiCW$\Delta$FC, was expressed in Escherichia coli and purified to homogeneity for biochemical characterization. Compared with ChiCW, ChiCW$\Delta$FC exhibited higher chitinase activity at high temperature and pH, but expressed lower hydrolytic and binding activities toward insoluble substrates. In addition, kinetic properties indicated that ChiCW$\Delta$MC hydrolyzed oligomeric and polymeric substrates less efficiently than ChiCW. These results suggest that the C-terminal region of ChiCW plays important roles in substrate binding and hydrolysis of chitin. In addition, the biological meaning of C-terminal proteolytic modification of ChiCW is discussed.

• Journal of Industrial Technology
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• v.36
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• pp.33-37
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• 2016

Cellulose binding domains (CBDs) of cellulases are thought to assist in the hydrolysis of insoluble crystalline cellulose. To gain sufficient amount of CBDs, the self-cleavable intein tag was used for expression and purification of Trichoderma reesei cellobiohydrolase I CBD in E. coli. Synthetic CBD genes, CBD or linker-CBD were cloned into expression vector pTYB11. Recombinant CBDs were successfully purified by intein mediated purification with an affinity chitin-binding domain. The final yields of recombinant CBD and linker-CBD were 3.2 mg/L and 1.4 mg/L, respectively. The functional bindings of recombinant CBDs were confirmed by Avicel binding experiments. The simple and easy purification method using self-cleavable intein tag can be further used in pretreatment of crystalline cellulose or characterization of engineered CBDs.

• Proceedings of the Microbiological Society of Korea Conference
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• 2006.05a
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• pp.97-99
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• 2006

A chitinase-producing bacterium strain KCTC10714 was isolated from sea sand around the King Sejong Station, King George Island in Antarctica. It was identified as Sanguibacter sp., based on the biochemical properties and 16S rRNA gene sequence. KCTC10714 chitinase showed enzyme activity in broad range of temperature from 0 to $70^{\circ}C$. At $0^{\circ}C$, it showed 70.9% of relative activity in comparison with 100%. The chitinase gene of KCTC10714 was cloned using inverse PCR cloning method. KCTC10714 chitinase gene was designated as chi21702. The ORF of chi21702 consisted of 1,449 bp (482 amino acid), and contained ChtBD3 (a chitin/cellulose binding domain) and an active site for chitinase family 18.

• Journal of Life Science
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• v.32 no.2
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• pp.135-141
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• 2022

Thymosin β-4 (TB4) is a small peptide composed of 43 amino acids. To obtain sufficient biologically active mouse TB4 economically, we cloned and overexpressed this gene in an Escherichia coli system. With the isopropyl β-D-1-thiogalactopyranoside induction of the E. coli transformant, TB4 fusion protein with intein- and chitin-binding domain was successfully expressed in the soluble fraction within the E. coli cell. The TB4-intein - chitin-binding domain fusion protein was purified from the soluble fraction of E. coli cell lysate. The affinity chromatography with chitin beads and dithiothreitol-mediated intein self-cleavage reaction releases the TB4 peptide into the stripping solution. Sodium dodecyl sulphate - polyacrylamide gel electrophoresis and Western blot analyses were used to confirm that the recombinant TB4 peptide was produced with the expected size of 5 kDa. We found that the recombinant TB4 stimulated cell migration in the transwell plate chamber assay. After 18 hr of the treatment of the recombinant TB4 with 1 ng/ml concentration, the migration of the HT1080 cell was increased by 20% compared with that of the chemically synthesized TB4. The recombinant TB4 was also observed to promote the healing of a wound area in C57BL/6 mice by as high as 35% compared with that of the chemically synthesized TB4. These results suggest that the recombinant TB4 has better biological activity for cell migration and wound healing than that of the chemically synthesized TB4 peptide.

• Journal of Microbiology and Biotechnology
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• v.19 no.3
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• pp.277-285
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• 2009

The nucleotide sequence of the Paenibacillus curdlanolyticus B-6 xyn10A gene, encoding a xylanase Xyn10A, consists of 3,828 nucleotides encoding a protein of 1,276 amino acids with a predicted molecular mass of 142,726 Da. Sequence analysis indicated that Xyn10A is a multidomain enzyme comprising nine domains in the following order: three family 22 carbohydrate-binding modules (CBMs), a family 10 catalytic domain of glycosyl hydrolases (xylanase), a family 9 CBM, a glycine-rich region, and three surface layer homology (SLH) domains. Xyn10A was purified from a recombinant Escherichia coli by a single step of affinity purification on cellulose. It could effectively hydrolyze agricultural wastes and pure insoluble xylans, especially low substituted insoluble xylan. The hydrolysis products were a series of short-chain xylooligosaccharides, indicating that the purified enzyme was an endo-$\beta$-1,4-xylanase. Xyn10A bound to various insoluble polysaccharides including Avicel, $\alpha$-cellulose, insoluble birchwood and oat spelt xylans, chitin, and starches, and the cell wall fragments of P. curdlanolyticus B-6, indicating that both the CBM and the SLH domains are fully functioning in the Xyn10A. Removal of the CBMs from Xyn10A strongly reduced the ability of plant cell wall hydrolysis. These results suggested that the CBMs of Xyn10A play an important role in the hydrolysis of plant cell walls.

• The Plant Pathology Journal
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• v.33 no.3
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• pp.318-328
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• 2017

Chitinase-producing Paenibacillus elgii strain HOA73 has been used to control plant diseases. However, the antimicrobial activity of its extracellular chitinase has not been fully elucidated. The major extracellular chitinase gene (PeChi68) from strain HOA73 was cloned and expressed in Escherichia coli in this study. This gene had an open reading frame of 2,028 bp, encoding a protein of 675 amino acid residues containing a secretion signal peptide, a chitin-binding domain, two fibronectin type III domains, and a catalytic hydrolase domain. The chitinase (PeChi68) purified from recombinant E. coli exhibited a molecular mass of approximately 68 kDa on SDS-PAGE. Biochemical analysis indicated that optimum temperature for the actitvity of purified chitinase was $50^{\circ}C$. However, it was inactivated with time when it was incubated at $40^{\circ}C$ and $50^{\circ}C$. Its optimum activity was found at pH 7, although its activity was stable when incubated between pH 3 and pH 11. Heavy metals inhibited this chitinase. This purified chitinase completely inhibited spore germination of two Cladosporium isolates and partially inhibited germination of Botrytis cinerea spores. However, it had no effect on the spores of a Colletotricum isolate. These results indicate that the extracellular chitinase produced by P. elgii HOA73 might have function in limiting spore germination of certain fungal pathogens.

• Journal of the Korea Organic Resources Recycling Association
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• v.18 no.3
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• pp.31-37
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• 2010

Chitinases (EC 3.2.1.14) hydrolyze the ${\beta}$-1,4-linkages in chitin, the second most abundant polymer of N-acetyl-${\beta}$-D-glucosamine which is a structural component of protective biological matrices such as fungal cell walls and insect exoskeletons. The glycosyl hydrolases 18 family including chitinases is an ancient gene family widely expressed in archea, prokaryotes and eukaryotes. Since earthworms live in the soil with a lot of microbial activities and fungi are supposed to be a major component of the diet of earthworm, it has been reported that there would be appropriate immune system to protect themselves from microorganisms attacks. In this study, the novel chitinase, EaChi, from the midgut of earthworm, Eisenia andrei, were identified and characterized. To obtain full-length cDNA sequence of chitinase, RT-PCR and RACE-PCR analyses were carried out by using the previously identified EST sequence amongst cDNA library established from the midgut of E. andrei. EaChi, a partial chitinase gene, was composed of 927 nucleotides encoding 309 amino acids. By the multiple sequence alignments of amino acids with other different species, it was revealed that EaCHI is a member of glycosyl hydrolases 18 family, which has two highly conserved domains, substrate binding and catalytic domain.