• 미생물학회지
• /
• 제42권3호
• /
• pp.235-237
• /
• 2006

먹물버섯은 버섯 시원체로부터 버섯이 성숙되는 과정에서 자가소화가 일어나 먹물이라 불리는 검은 액체를 생성한다. 이 과정에서 멜라닌을 생성하는 laccase, 균류 세포벽 성분의 하나인 키틴을 분해하는 chitinase의 관련을 분석하고자 Northern hybridization 방법을 이용하여 유전자의 발현을 분석하였다. 시원체가 생성되고 버섯이 성숙되어 먹물을 생성하는 시기에 따라 멜라닌색소 생성 효소인 laccase와 킨틴분해효소인 chitinase의 발현이 증가하는 것이 확인되었다.

• 한국미생물·생명공학회지
• /
• 제49권3호
• /
• pp.305-315
• /
• 2021

The cotton leafworm, Spodoptera littoralis, is a major pest in Egypt and many countries worldwide, and causes heavy economic losses. As a result, management measures to control the spread of the worm are required. S. littoralis nucleopolyhedrovirus (SpliNPV) is one of the most promising bioagents for the efficient control of insect pests. In this study, a chitinase gene (chitA) of a 1.8 kb DNA fragment was cloned and fully characterized from SpliNPV-EG1, an Egyptian isolate. A sequence of 601 amino acids was deduced when the gene was completely sequenced with a predicted molecular mass of 67 kDa for the preprotein. Transcriptional analyses using reverse transcription polymerase chain reaction (RT-PCR) revealed that chitA transcripts were detected first at 12 h post infection (hpi) and remained detectable until 168 hpi, suggesting their transcriptional regulation from a putative late promoter motif. In addition, quantitative analysis using quantitative RT-PCR showed a steady increase of 7.86-fold at 12 hpi in chitA transcription levels, which increased up to 71.4-fold at 120 hpi. An approximately 50 kDa protein fragment with chitinolytic activity was purified from ChitA-induced bacterial culture and detected by western blotting with an anti-recombinant SpliNPV chitinase antibody. Moreover, purification of the expressed ChitA recombinant protein showed in vitro growth inhibition of two different fungi species, Fusarium solani and F. oxysporum, confirming that the enzyme assembly and activity was correct. The results supported the potential role and application of the SpliNPV-ChitA protein as a synergistic agent in agricultural fungal and pest control programs.

• Mycobiology
• /
• 제39권3호
• /
• pp.182-186
• /
• 2011

Four Trichoderma strains (CH2, SH16, PQ34, and TN42) were isolated from soil samples collected from Quang Tri and Thua Thien Hue provinces in Vietnam. The strains exhibited high chitinolytic secretion. Strain PQ34 formed the largest zone of chitinase-mediated clearance (> 4 cm in diameter) in agar containing 1% (w/v) colloidal chitin. Analysis of the internal transcribed spacer regions of these strains indicated that they were Trichoderma asperellum. The molecular weights of the chitinases were approximately 42 kDa. Chitinase genes (chi42) of T. asperellum strains TN42, CH2, SH16, and PQ34 were 98~99% homologous to the ech42 gene of T. harzianum CB-Pin-01 (accession No. DQ166036). The deduced amino acid sequences of both T. asperellum strains SH16 and TN42 shared 100% similarity.

• KSBB Journal
• /
• 제29권3호
• /
• pp.131-138
• /
• 2014

In this study, we isolated two novel chitinase producing bacterial strains from yellow loess samples collected from Jullanamdo province. The chitinase producing bacteria were isolated based on the zone size of clearance in the chitin agar plates. Both of them were gram positive, rod ($2{\sim}3{\times}0.3{\sim}0.4{\mu}m$), spore-forming, and motility positive. They were facultative anaerobic, catalase positive and hydrolyzed starch, gelatin, and casein. From the 16s rRNA gene sequence analysis, the isolates were labeled as Bacillus licheniformis KYLS-CU01 and B. licheniformis KYLS-CU02. The isolates showed higher extracellular chitinase activities than B. licheniformis ATCC 14580 as a control. The optimum temperature and pH for chitinase production were $40^{\circ}C$ and pH 7.0, respectively. Response Surface Methodology (RSM) was used to optimize the culture medium for efficient production of the chitinase. Under this optimal condition, 1.5 times higher chitinase activity of B. licheniformis KYLS-CU02 was obtained. Extracellular chitinases of the two isolates were purified through ammonium sulfate precipitation and anion-exchange DEAE-cellulose column chromatography. The specific activities of purified chitinase from B. licheniformis KYLS-CU01 and B. licheniformis KYLS-CU02 were 7.65 and 5.21 U/mg protein, respectively. The molecular weights of the two purified chitinases were 59 kDa. Further, the purified chitinase of B. licheniformis KYLS-CU01 showed high antifungal activity against Fusarium sp.. In conclusion, these two bacterial isolates can be used as a biopesticide to control pathogenic fungi.

• Journal of Microbiology and Biotechnology
• /
• 제17권3호
• /
• pp.474-480
• /
• 2007

Fusarium wilt of cucumbers was effectively controlled by Escherichia coli expressing an endochitinase gene (chiA), and the rate was as effective (60.0%) as the wild-type strain S. proteamaculans 3095 (55.0%) where the gene was cloned. However, live cells of soil inoculated E. coli host harboring the chiA gene did not proliferate but declined 100-fold from $10^8$ CFU during the first week and showed less than 10 cells after day 14, suggesting that E. coli was able to express and produce the chitinase enzyme to the soil even as the population was gradually decreasing. Because the majority of the strains was alive for only a short period of time and the Fusarium-affected seedlings showed symptoms of wilting within 7-10 days, it seems that the pathogen control was decided early after the introduction of the biocontrol agent, eliminating the survival of the antagonist. These results indicated that soil inoculated E. coli could sufficiently express and produce the recombinant protein to control the pathogen, and root or soil colonization of the antagonist might not be a significant factor in determining the efficacy of biological control.

• 한국미생물·생명공학회지
• /
• 제20권5호
• /
• pp.511-518
• /
• 1992

Serratia marcescens ATCC 27117에서 부터 클로닝한 58KD 키티나아제 유전자를 subcloning 하여 2.6Kb DNA 삽입단편을 가진 플라스미드 pCHI26을 제조하고 대장균에서 발현과 분비를 살펴보았다. 키티니아제 유전자는 대장균에서 자신의 prmoter를 이용하여 매우 낮은 수준(<5mU/m$\ell$)으로 발현되었으며 lac promoter를 이용하는 경우 키티니아제 발현이 증가되어 약 80mU/m$\ell$가 되었다. 발현된 키티니아제는 거의 전적으로 대장균의 periplasm에 위치(약 87.8)하고 있었다. 배양시간에 따라서 세포내 키티니아제 활성을 측정해본 결과 초기정지기까지는 균체 성장과 비례해서 세포내 효소활성이 증가되었으나 정지기부터 세포내 효소활성이 급격히 줄어드는 양상을 보였다. 그러나 이 기간 동안 세포의 효소활성의 변화는 거의 없었다. 이러한 결과로 보아 periplasm에 위치한 키티나아제가 대장균의 단백분해효소에 의해서 분해되는 것으로 추정되었다.

• 농약과학회지
• /
• 제18권3호
• /
• pp.202-209
• /
• 2014

RNA interference(RNAi)는 살아있는 세포 내에서 유전자의 표현 형을 억제하는 작용을 하고 Chitinase는 곤충이 탈피를 하는 동안 오래된 큐티클의 분해와 재흡수를 도와주는 효소로 알려져 있다. 이러한 작용기작을 이용하는 연구를 수행하기 위하여 담배거세미나방의 chitinase와 관련하여 탈피저해 효과를 조사하였다. 담배거세미나방 5령 유충으로부터 RNA를 추출하고 이용하여 cDNA를 합성하고 약 700 bp의 chitinase를 증폭 하였다. 증폭한 PCR product를 pGEM T-easy vector에 cloning하여 competent cell (E.coli)에 형질전환 시키고 mixture를 배양 후 colony를 선발하고 plasmid DNA를 추출하였다. 그 결과 약 3 kb size의 vector band와 약 700 bp의 insert band를 확인 할 수 있었다. dsRNA를 합성하기 위해 각각의 DNA를 Spe I과 Nco I의 제한 효소 처리를 하여 linear form의 DNA로 만들었다. dsRNA 합성 후 약 $10{\mu}g/{\mu}l$의 농도로 $5{\mu}l$씩 담배거세미나방 4령 유충에 주입하였다. 그 결과 유충-유충간의 탈피에서는 기형발육, 탈피저해, 표피의 색소 변이가 나타났다. 번데기-성충 간의 탈피에서는 탈피저해, 날개변이, 기형발육 현상을 볼 수 있었다. 용화율의 경우 무처리구 83.3%, DW 처리구 78.3%, dsRNA 처리구 66.7%로 나타났다. 우화율의 경우 무처리구 90.0%, DW 처리구 72.3%, dsRNA 처리구 65.0%로 나타나 dsRNA를 처리한 그룹에서 상대적인 탈피 저해 효과를 확인할 수 있었다. 그러나 변이율의 경우 무처리구 8.9%, DW 처리구 2.9%, dsRNA 처리구 19.2%로 dsRNA를 주입한 처리구에서 변이율이 가장 높게 나타난 것을 확인할 수 있었다. 표현형적 변이는 dsRNA 주입 후 약 18 시간 이후부터 뚜렷하게 나타나는 것을 볼 수 있었다.

• 한국어병학회지
• /
• 제6권2호
• /
• pp.103-110
• /
• 1993

Sau3AI으로 부분절단한 Serratia marcescens genomic DNA(5Kb 이상)을 pUC19의 BamHI site에 삽입하여 total genomic library를 준비하였다. Swollen colloidal chitin media에서 halo를 형성하는 2개의 E.coli 형질전환주를 선별하였다. 이들 colony가 chitinase 유전자를 갖음을 재확인하기 위하여 4-methylumbelliferyl N-acetyl-$\beta$-D-glucosaminide(4-MuFGlcNAc)를 이용하였다. 4-MuFGlcNAc는 chitinase에 대한 기질특이성을 나타내며 형광을 나타내는 기질로서 positive clone들은 360nm의 자외선을 조사하였을 경우 밝은 형광을 나타낸다. pUC19으로 부터 유래된 2 종류의 다른 chitinase clone, pCH1(11.0Kb) 및 pCH2(7.5Kb)를 genomic DNA library로 부터 분리하였으며, 이들의 제한효소지도를 작성한 결과 서로 다른 제한효소지도를 나타내었다. pCH1EA 및 pCH2로 부터 각각의 EcoRI-Xbal fragment를 subcloning함으로써 두개의 다른 chitinase 유전자의 위치를 결정하였다. pCH1EA 및 pCH2를 cross hybridization 한 결과 hybridization signal을 나타내지 않아 서로 유사성이 없는 것으로 사료된다.

• Journal of the Korean Wood Science and Technology
• /
• 제47권3호
• /
• pp.371-380
• /
• 2019

Termites are pests that cause serious economic and cultural damage by digesting wood cellulose. Termites are arthropods and have an epidermis surrounded by a chitin layer. To maintain a healthy epidermis, termites have chitinase (${\beta}$-1,4-poly-N-acetyl glucosamidinase, EC 3.2.1.14), an enzyme that hydrolyzes the ${\beta}$-1,4 bond of chitin. In this study, the amino acid sequence of the gene, which is presumed to be termite chitinolytic enzyme (NCBI accession no. KC477099), was obtained from a transcriptomic analysis of Reticulitermes speratus KMT001 in Bukhan Mountain, Korea. An NCBI protein BLAST search confirmed that the protein is a glycoside hydrolase family 18 (GH18). The highest homology value found was 47%, with a chitinase from Araneus ventricosus. Phylogenetic analysis indicated that the KC477099 protein has the same origins as those of arthropods but has a very low similarity with other arthropod chitinases, resulting in separation at an early stage of evolution. The KC477099 protein contains two conserved motifs, which encode the general enzymatic characteristics of the GH18 group. The amino acid sequences $Asp^{156}-Trp^{157}-Glu^{158}$, which play an important role in the enzymatic activity of the GH18 group, were also present. This study suggests that the termite KC477099 protein is a new type of chitinase, which is evolutionarily distant from other insect chitinases.

• 식물병연구
• /
• 제20권3호
• /
• pp.216-218
• /
• 2014

Lysobacter antibiocus HS124 is a chitinase-producing rhizobacterium with proven capacities to suppress plant diseases. Bacterial cultures of L. antibioticus HS124 showed strong biocontrol efficacies against various plant diseases compared to those of bacterial cultures of Bacillus subtilis QST713 which is an active ingredient of a commercial biopesticide, Serenade. Here, we report the draft genome sequence and automated annotation of strain HS124. This draft genome sequence indicates the novelty of L. antibiocus HS124 and a subset of gene functions that may be related to its biocontrol activities.