• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.1
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• pp.38-42
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• 2002

A chitinase gene (pCHi58) encoding a 58 kDa chitinase was isolated from the Serratia marcescens KCTC 2172 cosmid library. The chitinase gene consisted of a 1686 bp open reading frame that encoded 562 amino acids. Escherichia coil harboring the pChi58 gene secreted a 58 kDa chitinase into the culture supernatant. The 58 kDa chitinase was purified using a chitin affinity column and mono-S column. A nucleotide and N-terminal amino acid sequence analysis showed that the 58 kDa chitinase had a leader peptide consisting of 23 amino acids which was cleaved prior to the 24th alanine. The 58 KDa chitinase exhibited a $98\%$ similarity to that of S. marcescens OMB 1466 in its nuclotide sequence. The chitinolytic patterns of the 58 kDa chitinase released N,N'-diacetyl chitobiose (NAG2) as the major hydrolysis end-product with a trace amount of N-acetylglucosamine. When a 4-methylumbellyferyl-N-acetylglucosamin monomer, dimmer, and tetramer were used as substrates, the 58 kDa chitinase did not digest the 4-Mu-NAG monomer $(analogue\;of\;NAG_2)$, thereby indicating that the 58 kDa chitinase was likely an endochitinase. The optimum reaction temperature and pH of the enzyme were $50^{\circ}C$ and 5.0, respectively.

• The Plant Pathology Journal
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• v.25 no.4
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• pp.344-351
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• 2009

A biocontrol bacterium Bacillus licheniformis N1 grown in nutrient broth showed no chitinolytic activity, while its genome contains a gene which encodes a chitinase. The gene for chitinase from B. licheniformis N1 was amplified by PCR and the deduced amino acid sequence analysis revealed that the chitinase exhibited over 95% identity with chitinases from other B. licheniformis strains. Escherichia coli cells carrying the recombinant plasmid displayed chitinase activity as revealed by the formation of a clear zone on chitin containing media, indicating that the gene could be expressed in E. coli cells. Chitinase gene expression in B. licheniformis N1 was not detected by RT-PCR analysis. The protein was over-expressed in E. coli BL21 (DE3) as a glutathione S-transferase fusion protein. The protein could also be produced in B. subtilis 168 strain carrying the chitinase gene of N1 strain. The crude protein extract from E. coli BL21 carrying GST fusion protein or culture supernatant of B. subtilis carrying the chitinase gene exhibited enzyme activity by hydrolyzing chitin analogs, 4-methylumbelliferyl-$\beta$-D-N,N'-diacetylchitobioside and 4-methylumbelliferyl-$\beta$-D-N,N',N"-triacetylchitotrioside. These results indicated that even though the chitinase gene is not expressed in the N1 strain, the coding region is functional and encodes an active chitinase enzyme. Furthermore, B. subtilis 168 transformants expressing the chitinase gene exhibited antifungal activity against Fulvia fulva by suppressing spore germination. Our results suggest that the proper engineering of the expression of the indigenous chitinase gene, which will lead to its expression in the biocontrol strain B. licheniformis N1, may further enhance its biocontrol activity.

• 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.

• International Journal of Industrial Entomology and Biomaterials
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• v.19 no.2
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• pp.259-264
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• 2009

The AvChit gene encodes for a chitinase from the spider, Araneus ventricosus. This spider, A. ventricosus, is an abundant species in Korea. Arabidopsis thaliana plants were transformed with the AvChit gene using Agrobacterium tumefaciens. Thirteen transgenic lines expressing the AvChit gene were obtained. Functional expression of the AvChit gene in transgenic Arabidopsis was confirmed by Southern, northern and western blot analysis. The AvChit cDNA was expressed as a 61 kDa polypeptide in baculovirus-infected insect Sf9 cells. AvChit protein extracted from transgenic Arabidopsis exhibited high levels of chitinase activity. Phytopathological tests showed that two transgenic Arabidopsis lines expressing the AvChit gene displayed high levels of resistance to gray mold disease (Botrytis cinerea).

• Journal of Plant Biotechnology
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• v.1 no.2
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• pp.85-90
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• 1999

In order to protect fungal diseases, leaf disc explants of Solanum tuberosum cultivar, Belchip, was infected with an Agrobacterium MP90 strain containing chimeric gene construct, consisting of antibiotic resistance and chitinase gene driven by the CaMV 35S promoter, for transformation. Regenerated multiple shoots were selected on a medium containing kanamycin and carbenicillin after exposure to Agrobacterium. The presence and integration of the npt II and chitinase gene were confirmed by polymerase chain reaction(PCR). Northern blot analysis indicated that the genes coding for the enzyme could be expressed in potato plants. The chitinase activity of transgenic potato plants was higher than the control potato.

• The Plant Pathology Journal
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• v.21 no.3
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• pp.195-206
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• 2005

Spatial and temporal expression of pathogenesis-related (PR) gene and proteins has been recognized as inducible defense response in pepper plants. Gene expression and/or protein accumulation of PR-1, $\beta-1,3-glucanase$ and chitinase was predominantly found in pepper plants during the inoculations by Xanthomonas campestris pv. vesicatoria, Phytophthora capsici and Colletotrichum coccodes. PR-1 and chitinase genes were also induced in pepper plants in response to environmental stresses, such as high salinity and drought. PR-1 and chitinase gene expressions by biotic and abiotic stresses were regulated by their own promoter regions containing several stress-related cis-acting elements. Overexpression of pepper PR-1 or chitinase genes in heterogeneous transgenic plants showed enhanced disease resistance as well as environmental stress tolerances. In this review, we focused on the putative function of pepper PR-1, $\beta-1,3-glucanase$ and chitinase proteins and/or genes at the biochemical, molecular and cytological aspects.

• The Plant Pathology Journal
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• v.31 no.3
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• pp.252-258
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• 2015

This is the first study reporting the evaluation of transgenic lines of tomato harboring rice chitinase (RCG3) gene for resistance to two important fungal pathogens Fusarium oxysporum f. sp. lycopersici (Fol) causing fusarium wilt and Alternaria solani causing early blight (EB). In this study, three transgenic lines TL1, TL2 and TL3 of tomato Solanum lycopersicum Mill. cv. Riogrande genetically engineered with rice chitinase (RCG 3) gene and their R1 progeny was tested for resistance to Fol by root dip method and A. solani by detached leaf assay. All the R0 transgenic lines were highly resistant to these fungal pathogens compared to nontransgenic control plants. The pattern of segregation of three independent transformant for Fol and A. solani was also studied. Mendelian segregation was observed in transgenic lines 2 and 3 while it was not observed in transgenic line 1. It was concluded that introduction of chitinase gene in susceptible cultivar of tomato not only enhanced the resistance but was stably inherited in transgenic lines 2 and 3.

• Journal of Ginseng Research
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• v.27 no.1
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• pp.37-42
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• 2003

For study about introduce of gene connected with disease and transformation system of gingseng, chitinase gene cloned from soybene and disease resistant gene were carried out for expression and transformation of plant using Agrobacterium. The disease resistance gene(DR-49), 35S-35S-AMV, has been constructed. The disease resistance gene and chitinase gene were introduced into the binary vector pRD 400, which were mobilized into Agrobacterium tumefaciens faciens strain MP 90 and LBA 4404 harboring disarmed Ti-plasmid. As a result of induce transformants using ginseng embryo and petiole, multi shoots were formed on MS medium supplemented 1 mg/ι 2,4-D and 0.5 mg/ι kinetin. Also transformation by cotyledonwas effective on MS medium supplemented 1 mg/ι 2,4-D and 0.5 mg/ι kinetin, transformation percent of disease resistant gene and chitinase gene were showed 18%, 14% respectively. As transformed tissue is under pre-embryoid condition, normal shoot is required through the process of matured embryo.

• Journal of Microbiology and Biotechnology
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• v.5 no.2
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• pp.87-91
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• 1995

The chitinase gene was cloned from Acinetobacter sp. WC-17 for investigating the genetic control and enzymatic properties of bacterial chitinase. A genomic library of Acinetobacter sp. WC-17 was prepared in E.coli JM109 by using pUC18 as a vector. The chitinase-positive clone containing 3.2kb insert fragment was obtained from 5, 000 insert-bearing transformants. The optimum pH and temperature of cloned enzyme were 6.0 and $55^{\circ}C$, respectively. Almost all the chitinase activity of E.coli recombinant was localized in the periplasmic fraction, while most of the enzyme activity of Acinetobacter sp. WC-17 was found in the extracellular fraction.

• Applied Biological Chemistry
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• v.41 no.2
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• pp.125-129
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• 1998

Xanthomonas sp. isolated from soil exhibited cell wall lytic activity of Candida albicans and secreted chitinase in chitin media. Especially, the chitinase activity was induced by chitin and reached a maximum level at 3 days culture in chitin media. We constructed genomic library of Xanthomonas sp. using cosmid vector in E. coli. Oligonucleotide probe was synthesized from the consensus sequence corresponding to chitinase active site, which was derived from the comparison of amino acid sequences of bacterial chitinase genes. Using this oligonucleotide probe, we screened the genomic library. By restriction enzyme mapping of the positive clones, we identified 4 independent clones which may contain the chitinase gene. One of the clones, named pXCH1 (1.2 kb insert), was further analyzed. Northern blot analysis indicated that is transcripts, 1 kb and 0.8 kb, were induced by chitin. When the cloned gene was induced by IPTG in E.coli cell, chitinase activity which was secreted onto culture media was not observed. However, when the cell was disrupted by using sonicator and then centrifuged, the supernatant exhibited chitinase activity. SDS-PAGE of the supernatant indicated that about 35 kDa protein was induced by IPTG. From these results, it was concluded that the cloned DNA was one of the chitinase genes of Xanthomonas sp.