Expression of serine proteinase P186 of Arthrobotrys oligospora and analysis of its nematode-degrading activity

Zhao, Hailong; Qiao, Jun; Meng, Qingling; Gong, Shasha; Chen, Cheng; Liu, Tianli; Tian, Lulu; Cai, Xuepeng; Luo, Jianxun; Chen, Chuangfu

doi:10.1007/s10482-015-0595-z

Cited by 7 publications

(3 citation statements)

References 33 publications

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“…According to the methods reported by Wang et al [13] and Zhao et al [21] the optimum temperature and optimum pH of reAO-379 chitinase were determined using a protein/nucleic acid analyzer (OD 585 nm ). Each experiment was repeated three times.…”

Section: Determination Of Optimum Temperature and Ph For A Oligospormentioning

confidence: 99%

Arthrobotrys oligospora Rekombinant Çitinaz AO-379’nın Enzim Özellikleri ve Nematod İndirgeyici Aktivitesi

Zhong¹,

Chen²,

Gong³

et al. 2019

Kafkas Univ Vet Fak Derg

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Chitinase is an important virulence factor produced by nematode trapping fungi in the process of infection, and plays an important role in the cleavage of nematodes and their eggshells. In this study, the cDNA sequence of Arthrobotrys oligospora chitinase AO-379 was amplified by RT-PCR and inserted into the vector pPIC9K to induce the expression of AO-379 in Pichia pastoris GS115. The recombinant AO-379 (reAO-379) was purified by nickel ion affinity chromatography, and enzymological properties and nematode-degrading activity of reAO-379 was analyzed. SDS-PAGE and Western blot analysis showed that the reAO-379 with molecular weight of about 44 kDa was successfully obtained. The reAO-379 showed strong chitinase activity at pH 5.5 and 30°C. Using reAO-379 to treat Strongylus equinus, Caenorhabditis elegans and Haemonchus contortus for 12, 24, and 36 h , the killing rates of reAO-379 in S. equinus were 42%, 89% and 100%; in C. elegans were 50%, 90% and 97%; in and H. contortus were 53%, 62% and 84%, respectively. Using reA-379 to treat Fasciola hepatica and Dicrocoelium chinensis eggs for 24, 48 and 72 h, the degradation rates of reAO-379 were 12%, 43% and 65% in F. hepatica eggs, and were 15%, 33% and 55% in D. chinensis eggs, respectively. Our study suggests that the reAO-379 is potentially valuable for development of biological control agent against digestive tract nematodes in livestocks.

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Section: Determination Of Optimum Temperature and Ph For A Oligospormentioning

confidence: 99%

Arthrobotrys oligospora Rekombinant Çitinaz AO-379’nın Enzim Özellikleri ve Nematod İndirgeyici Aktivitesi

Zhong¹,

Chen²,

Gong³

et al. 2019

Kafkas Univ Vet Fak Derg

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“…Z Hao et al amplified the gene sequence of the P. oligospora serine protease P186 and successfully expressed it in Pichia pastoris. The purified protease reP186 exhibited nematicidal activity [29]. In addition, subtilase genes obtained from the nematode-parasitic fungus Purpureocillium lilacinum formed a unique cluster, putatively indicating that the fungus might have developed distinctive mechanisms for nematode pathogenesis [30].…”

Section: Introductionmentioning

confidence: 99%

Comprehensive Analysis of Subtilase Gene Family and Their Responses to Nematode in Trichoderma Harzianum

Lu¹,

Liang²,

Deng³

et al. 2020

Preprint

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The subtilase family is the second largest family of serine proteases. Some fungi including Trichoderma species can capture and kill nematodes by secreting hydrolytic enzymes or toxins, among which serine proteases are important enzymes that allow fungi to infect nematodes. Subtilase can degrade nematode and insect body walls. In this study, subtilase family genes were identified from the Trichoderma harzianum genome database, and bioinformatics analysis of the characteristics and evolutionary status of these genes, along with structural and functional analyses of their proteins, was performed. Gene structure analysis revealed that all the 41 subtilase genes contained introns, while some did not have upstream or downstream regions. Chromosome localisation showed that subtilase family members were unevenly distributed in 22 Trichoderma chromosomes, and 3 clusters were present, indicating that they may be hot spots of subtilase genes. Conserved motif analyses showed these proteins contained a commonly conserved motif, and motifs belonging to the same subfamily remained highly similar. The upstream region of the subtilase genes were enriched with different type and numbers of cis-elements, indicating that subtilase genes are likely to play a role in the response to diverse stresses. Transcription of 31 genes was increased after 5 days of infection with nematodes, whereas that of 10 genes decreased. In these subtilase genes, ThSBT4, ThSBT5, ThSBT12, ThSBT27, ThSBT34, ThSBT35, ThSBT38, and ThSBT40 showed significantly upregulated expression with a log2 fold change value of more than 4, and ThSBT35 showed the highest peak. These results laid a theoretical foundation for further research on the function of the subtilase genes and the mechanism of the resistance response.

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“…A. oligospora-secreted extracellular hydrolases mainly include serine proteases and chitinases. The serine proteases PII, VCP1, Aoz1, P186, and Ver112 are known to degrade the nematode cuticle [6,9,10]. In addition, the secreted chitinases are involved in degrading the chitinous component of the nematode eggs and the cell wall of pathogenic fungi [5,8,[11][12][13].…”

Section: Introductionmentioning

confidence: 99%

Biological Characteristics of Recombinant Arthrobotrys oligospora Chitinase AO-801

Gong

Meng²,

Qiao³

et al. 2022

Korean J Parasitol

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Chitinase AO-801 is a hydrolase secreted by Arthrobotrys oligospora during nematode feeding, while its role remained elusive. This study analyzed the molecular characteristics of recombinant chitinase of Arthrobotrys oligospora (reAO-801). AO-801 belongs to the typical glycoside hydrolase 18 family with conserved chitinase sequence and tertiary structure of (α/β)8 triose-phosphate isomerase (TIM) barrel. The molecular weight of reAO-801 was 42 kDa. reAO-801 effectively degraded colloidal and powdered chitin, egg lysate, and stage I larval lysate of Caenorhabditis elegans. The activity of reAO-801 reached its peak at 40˚C and pH values between 4-7. Enzyme activity was inhibited by Zn2+, Ca2+, and Fe3+, whereas Mg2+ and K+ potentiated its activity. In addition, urea, sodium dodecyl sulfate, and 2-mercaptoethanol significantly inhibited enzyme activity. reAO-801 showed complete nematicidal activity against C. elegans stage I larvae. reAO-801 broke down the C. elegans egg shells, causing them to die or die prematurely by hatching the eggs. It also invoked degradation of Haemonchus contortus eggs, resulting in apparent changes in the morphological structure. This study demonstrated the cytotoxic effect of reAO-801, which laid the foundation for further dissecting the mechanism of nematode infestation by A. oligospora.

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Expression of serine proteinase P186 of Arthrobotrys oligospora and analysis of its nematode-degrading activity

Cited by 7 publications

References 33 publications

Arthrobotrys oligospora Rekombinant Çitinaz AO-379’nın Enzim Özellikleri ve Nematod İndirgeyici Aktivitesi

Arthrobotrys oligospora Rekombinant Çitinaz AO-379’nın Enzim Özellikleri ve Nematod İndirgeyici Aktivitesi

Comprehensive Analysis of Subtilase Gene Family and Their Responses to Nematode in Trichoderma Harzianum

Biological Characteristics of Recombinant Arthrobotrys oligospora Chitinase AO-801

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