A rapid and sensitive microassay for determination of chitinolytic activity

McCreath, Kenneth J.; Gooday, Graham W.

doi:10.1016/0167-7012(92)90055-9

Cited by 127 publications

(100 citation statements)

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“…The different activities toward these substrates indicated the presence of Nacetylglucosaminidase, exo-chitinase, and endo-chitinase, respectively, in the preparation (McCreath and Gooday, 1992). Therefore Trichoderma viride secreted multiple chitinolytic enzymes in the culture filtrate during the preparation of Usukizyme.…”

Section: Discussionmentioning

confidence: 99%

“…4-Methylumbelliferyl N-acetylglucosamine (4-MU-GlcNAc), 4-methylumbelliferyl N,NЈ-diacetylchitobiose [4-MU-(GlcNAc) 2 ], and 4-methylumbelliferyl N,NЈ,NЉ-triacetylchitotriose [4-MU-(GlcNAc) 3 ] were used to assay the different chitin-degrading activities of the crude enzyme according to the method of McCreath and Gooday (1992). To 1 ml of the appropriately diluted enzyme solution incubated at 40°C for 5 min, 0.5 ml of substrate was added to a final concentration of 3.8 mM.…”

Section: Methodsmentioning

confidence: 99%

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Purification and characterization of a chitinase from Trichoderma viride.

Omumasaba

Yoshida

Ogawa

2001

J. Gen. Appl. Microbiol.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Purification and characterization of a chitinase from Trichoderma viride.

Omumasaba

Yoshida

Ogawa

2001

J. Gen. Appl. Microbiol.

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“…for the detection of endo-chitinolytic activity, as reported elsewhere (McCreath and Gooday, 1992;Rao et al, 2004). Briefly, for the ChiA protein purification procedure, leaves were homogenized in 1x PBS, in presence of EDTA 5 mM, PMSF 1 mM and PVP-40 1.5%, by 20-s bursts at full power using a Waring Blender (Waring Products, CT, USA).…”

Section: Chia Purification From Tobacco Transgenic Plantsmentioning

confidence: 99%

A viral chitinase enhances oral activity of TMOF

Fiandra

Terracciano

Fanti

et al. 2010

Insect Biochemistry and Molecular Biology

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In this study we investigate the combined effect on Heliothis virescens (Lepidoptera, Noctuidae) larvae of Aedes aegypti-Trypsin Modulating Oostatic Factor (Aea-TMOF), a peptide that inhibits trypsin synthesis by the gut, impairing insect digestive function, and Autographa californica nucleopolyhedrovirus Chitinase A (AcMNPV ChiA), an enzyme that is able to alter the permeability of the peritrophic membrane (PM). Aea-TMOF and AcMNPV ChiA were provided to the larvae by administering transgenic tobacco plants, co-expressing both molecules. Experimental larvae feeding on these plants, compared to those alimented on plants expressing only one of the two molecules considered, showed significantly stronger negative effects on growth rate, developmental time and mortality. The impact of AcMNPV ChiA on the PM of H. virescens larvae, measured as increased permeability to molecules, was evident after five days of feeding on transgenic plants expressing ChiA. This result was confirmed by in vitro treatment of PM with recombinant ChiA, extracted from the transgenic plants used for the feeding experiments. Collectively, these data indicate the occurrence of a positive interaction between the two transgenes concurrently expressed in the same plant. The hydrolytic activity of ChiA on the PM of tobacco budworm larvae enhances the permeation of TMOF molecules to the ectoperitrophic space, and its subsequent absorption. The permeation through the paracellular route of Aea-TMOF resulted in a spotted accumulation on the basolateral domain of enterocytes, which suggests the occurrence of a receptor on the gut side facing the haemocoel. The binding of the peptide, permeating at increased rates due to the ChiA activity, is considered responsible for the enhanced insecticide activity of the transgenic plants expressing both molecules. These data corroborate the idea that ChiA can be effectively used as gut permeation enhancer in oral delivery strategies of bioinsecticides targeting haemocoelic receptors. Dear Rene,We have revised the manuscript as requested. Below are the reviewers comments and for each issue raised our remedial action or an explanation to address their concerns is reported in bold.I think that this version is certainly improved, and we will be glad to consider any further suggestion you may have. All the best, FrancoReviewer #1: Fiandra et al.In this manuscript Fiandra et al present some interesting data on apparent enhancement of TMOF action by chitinase. Overall, this is a sound study that will be improved with the following modifications:1. What is the mechanism of action of TMOF? This should be introduced upfront. The action of TMOF in inhibiting trypsin synthesis and impeding larval digestive processes is key to the logic behind these experiments. Not until page 16 is this information provided for the reader! This information should actually be included in the abstract. Clearly introduce TMOF.The information requested has been added in the manuscript (Abstract and Introduction). Page 4What is the size o...

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“…Direct plate screening method using fluorescent chitin oligomer analogues. A direct plate screening method was modified from that described previously (McCreath & Gooday, 1992;Robbins et af., 1988) to screen colonies for chitinase activity. The method involved growing either bacterial colonies or a lawn of plaques on appropriate agar plates.…”

Section: S T E C H K a R N J A N A R U K A N D A E G O O D M A N Wmentioning

confidence: 99%

Multiple genes involved in chitin degradation from the marine bacterium Pseudoalteromonas sp. strain S91

Techkarnjanaruk¹,

Goodman²

1999

Microbiology

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A cluster of three closely linked chitinase genes organized in the order chiA, chiB and chic, with the same transcriptional direction, and two unlinked genes, chip and chiQ, involved in chitin degradation in Pseudoalteromnas sp. strain S91 were cloned, sequenced and characterized. The deduced amino acid sequences revealed that ChiA, Chis and Chic exhibited similarities to chitinases belonging to family 18 of the glycosyl hydrolases while Chip and ChiQ belonged to family 20. Chip and ChiQ showed different enzymic activities against fluorescent chitin analogues, but neither was able to degrade colloidal chitin. ChiA possessed chitinase activity but did not bind chitin; Chis bound chitin but had no chitinase activity; Chic possessed strong chitinase activity and also bound chitin. Production of Chic in 591 appeared to be controlled by chiA expression, since insertion of a transposon into the ORF of chiA resulted in the loss of chitinase activity as well as loss of Chic proteins in a chitinasenegative mutant. In Escherichia coli, Chic appeared to be expressed from its own promoter.

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A rapid and sensitive microassay for determination of chitinolytic activity

Cited by 127 publications

References 15 publications

Purification and characterization of a chitinase from Trichoderma viride.

Purification and characterization of a chitinase from Trichoderma viride.

A viral chitinase enhances oral activity of TMOF

Multiple genes involved in chitin degradation from the marine bacterium Pseudoalteromonas sp. strain S91

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