• 한국식품조리과학회지
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• 제15권2호
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• pp.102-107
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• 1999

김치재료들의 chitinase 활성을 측정하고, 부추의 첨가량을 달리한 김치를 제조하여 15$^{\circ}C$에서 9일간의 숙성과정 중 김치의 chitinase 활성과 조직감의 변화를 측정한 결과는 다음과 같았다. 김치의 주요 재료들은 쪽파(특히 잎부분)＞마늘＞부추>대파(특히 잎부분) 순으로 chitinase 활성을 나타내었다. 김치숙성 중의 chitinase 활성은 김치액이 김치조직 보다 월등히 높게 나타났으며 각 김치시료간의 효소활성 차이도 김치액에서 더 뚜렷하게 나타났다. 김치액에서의 chitinase 활성은 숙성이 진행됨에 따라 점차적으로 감소한 반면, 김치조직에서의 chitinase 활성은 숙성 3일 또는 5일까지 증가한 후 감소하였다. 그러나 김치조직과 김치액 모두에서 대조구보다 부추첨가구가 더 높은 chitinase 활성을 나타내었으며 부추첨가구에서는 부추첨가량이 증가할수록 높은 chitinase 활성을 보였다. 숙성이 진행됨에 따라 모든 시료구에서는 김치조직의 침투관통력은 감소하였으며 부추첨가량에 따른 차이는 미미하였으나 대조구가 숙성 전 기간동안 가장 낮은 침투관통력을 보였다. 이상의 결과를 종합해보면 부추첨가는 김치 숙성 중 부추의 chitinase 활성에 의해 새우젓 등의 키틴질을 분해하여 chitosan을 형성하므로서 김치의 조직을 향상시키는 것으로 생각된다.

• 방사선산업학회지
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• 제4권1호
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• pp.65-71
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• 2010

Two chitinase producing strains CHI2 and CHI4 were isolated from soybean rhizosphere soil. Both the strains belonged to Lysobacter enzymogenes as indicated by 16S rDNA sequence analysis. Though strain CHI2 and CHI4 produced extracellular chitinase, they differ in their chitinolytic activity. CHI4 produced approximately three times the higher amounts of enzyme than that of CHI2 under specified conditions. CHI2 produced $535.67U\;l^{-1}$ of chitinase after 48 h incubation with a specific activity of $3.91U\;mg^{-1}$ of protein while strain CHI4 produced $1584.13U\;l^{-1}$ of chitinase with a specific activity of $10.88U\;mg^{-1}$ protein. SDS-PAGE analysis indicated that the molecular weight of chitinase enzyme was approximately 45 kDa. A faint band with a molecular weight of 55 kDa reveals the possibility for the presence of another kind of chitin binding protein. Mutant library was developed by exposing the isolates to gamma rays at their $LD_{99}$ value (0.23 kGy). Totally, 11 mutants of CHI2 and CHI4 are reported to have enhanced chitinase activity. Several leaky mutant clones with decreased enzyme activity and a defective mutant (CHI2-M16) with complete loss of chitinase activity were also identified. CHI4-M18, CHI4-M8 and CHI4-M29 showed 78.8, 41.5, and 31.9% increased chitinase activity over wild type CHI4.

• BMB Reports
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• 제36권2호
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• pp.185-189
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• 2003

Chitinase (EC 3.2.1.14) was isolated from the culture filtrate of Streptomyces sp. M-20 and purified by ammonium sulfate precipitation, DEAE-cellulose ion-exchange chromatography, and Sephadex G-100 gel filtration. No exochitinase activity was found in the culture filtrate. The molecular mass of the purified chitinase was 20 kDa, estimated by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was confirmed by activity staining with Calcofluor White M2R. Chitinase was optimally active at pH of 5.0 and at $30^{\circ}C$. The enzyme was stable from pH 4 to 8, and up to $40^{\circ}C$. Among the metals and inhibitors that were tested, the $Hg^+$, $Hg^{2+}$, and p-chloromercuribenzoic acid completely inhibited the enzyme activity. The chitinase activity was high on colloidal chitin, chitotriose, and chitooligosaccharide. The purified chitinase showed antifungal activity against Botrytis cinerea, and lysozyme activity against the cell wall of Botrytis cinerea.

• Journal of Microbiology and Biotechnology
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• 제21권6호
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• pp.604-612
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• 2011

Beauveria bassiana SFB-205 supernatant can effectively control cotton aphid populations, which is closely associated with its chitinase activity. The present work extends to optimizing a culture medium to produce more efficacious supernatant in flask conditions, followed by scale-up in 7 L, 300 L and 1.2 KL fermentors with the parameter of chitinase. In flask conditions, a combination of soluble starch and yeast extract produced the greatest amount of chitinase (5.1 units/ml) and its supernatant had the highest aphicidal activity. An optimal quantitative combination of the two substrates, estimated by a response surface method, enabled the supernatant to have 15.7 units/ml of chitinase activity and 3.7 ml/l of median lethal concentration ($LC_{50}$) of toxicity against cotton aphid adults in laboratory conditions. In the scale-up conditions, overall supernatant had 25-28 units/ml of chitinase activity. Decrease in pH and limitation of dissolved oxygen (DO) during cultures were significantly related to the yield of chitinase. These results suggest that the substrate-dependent chitinase production can be background information for optimizing a culture medium, and pH and DO are critical factors in maximizing the production in scale-up conditions.

• 한국미생물·생명공학회지
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• 제9권4호
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• pp.179-183
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• 1981

진균류의 세포벽제거 및 이에 따른 protoplast생성 등에 많이 이용되어지고 있는 chitinase를 Streptomyces sp. 115-5 균주로부터 생산하여 순수 정제한 다음, 이 chitinase의 작용을 저해하는 포도당의 저해양상을 조사하였다. 포도당 외에 D-glucuronic acid, D-sorbitol및 D-xylose등도 chitinase의 활성을 저해하였다. 그런고로, 포도당 분자에 의한chitinase의 활성 저해 효과에는 위의 분자들의 공통부분인 2번, 3번 및 4번 탄소의 hydroxyl group들의 구조위치가 중요한 영향을 가진다는 것을 알 수 있다. 그리고 포도당에 의한 chitinase의 저해양상은 competitive inhibition 과 non-competitive inhibition 과의 혼합 저해형으로 나타났다.

• 한국식물병리학회지
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• 제10권4호
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• pp.277-283
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• 1994

An antimicrobial chitinase was purified from grapefruit extract and its properties were characterized. The chitinase was purified with a single step chromatography on regenerated chitin affinity gel column. The molecular weight of the purified chitinase was 29 kDa. The grapefruit extract contained the chitinase protein more than 50% of its total soluble proteins measured by coomassie stained protein bands. When the purified chitinase was incubated with polymers of N-acetylglucosamine (NAG), such as mycelia of Fusarium oxysproum and swollen chitin, they were degraded to oligosaccharides, and the oligosaccharides were then further hydrolyzed by the same enzyme to monomer and dimer of NAG. This result suggests that the chitinase contained both endo- and exo- chitinase activities. The chitinase was stable to heat and pH treatment; its activity was not diminished by the heat treatment upto 7$0^{\circ}C$ for 1 hr, and it showed a pH stability in the range of pH 4.0 to 12.0.

• Applied Biological Chemistry
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• 제35권3호
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• pp.191-195
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• 1992

강낭콩 잎에서 에틸랜에 의하여 유도되는 분자량 30KD인 염기성 키틴분해효소를 정제하고 그 항균활성을 연구하였다. 이 단백질은 chitinase 활성과 lysozyme 활성을 가졌으며, Aspergillus fumigatus, Botrytis cinerea, Fusarium oxysporum, Rhizoctonia solani의 균사 생장을 억제하였다. 그러나 함께 실험한 2종류의 미생물 chitinase, 달걀 lysozyme, 파파야 protease는 이들에 대한 항균작용을 갖지 않았다. 이상의 결과는 lysozyme 활성을 가진 식물 chitinase가 병원균의 균사생장을 억제하여 자신을 방어할 수 있음을 시사한다.

• Applied Biological Chemistry
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• 제36권1호
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• pp.1-6
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• 1993

Chitooligosaccharides 혼합물을 처리한 벼 세포 현탁배양액과 처리하지 않은 배양액의 단백질 및 효소활성을 비교하여 이 elicitor에 의해 유도되는 chitinase를 확인하였다. Chitooligosaccharide 혼합물 처리로 chitinase 활성은 비처리구에 비하여 3.5배 증가하였으며 증가율이 단백질 증가보다 10배 이상 높아 본 효소가 선택적으로 유도되는 것으로 나타났다. Polyacrylamide gel 전기영동상 나타나는 총 11종의 chitinase중 4종이 유도효소로 판명되었으며 DEAE-cellulose chromatography 결과 3개는 $26{\sim}58\;KD$의 분자량을 가진 acidic chitinase 분획에, 나머지 1개는 basic chitinase 분획에 속한 것으로 나타나서 주로 acidic chitinase가 유도되는 것으로 확인되었다.

• 한국미생물·생명공학회지
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• 제20권1호
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• pp.68-72
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• 1992

Aeromonas, salmonicida YA7-625가 생산하는 chitinase는 $Hg^{2+}$에 의해 저해를 받았으나, $\beta$-mercaptoethanol 처리에 의해서 활성이 보호되었으며 Na-thiosulfate, ascorbic acid등의 환원제들과 sodium azide, p-CMB 등의 -SH 저해제에대하여 전혀 작용을 받지 않았으므로 본 효소의 활성중심에는 -SH가 없는 것으로 생각되었다. 또한 histidine, cysteine, tyrosine 및 trytophan에 작용하는 iodine에 의하여 저해를 받았으며 tryptophan에 대해 특이적으로 작용하는 N-bromosuccinimide에 의해 실활되는 것으로 보아 활성중심에 tryptophan, histidine 및 tyrosine 가 존재하는 것으로 생각되었다.

• The Plant Pathology Journal
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• 제25권4호
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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.