Purification and characterization of trypsin produced by gut bacteria from Anticarsia gemmatalis

Abstract

Purification of active trypsin in the digestive process of insects is essential for the development of potent protease inhibitors (PIs) as an emerging pest control technology and research into insect adaptations to dietary PIs. An important aspect is the presence of proteolytic microorganisms, which contribute to host nutrition. Here, we purified trypsins produced by bacteria Bacillus cereus, Enterococcus mundtii, Enterococcus gallinarum, and Staphylococcus xylosus isolated from the midgut of Anticarsia gemmatalis. The trypsins had a molecular mass of approximately 25 kDa. The enzymes showed increased activity at 40°C, and they were active at pH values 7.5-10. Aprotinin, bis-benzamidine, and soybean Kunitz inhibitor (SKTI) significantly inhibited trypsin activity. The l-1-tosyl-amido-2-phenylethylchloromethyl ketone (TPCK), pepstatin A, E-64, ethylenediamine tetraacetic acid, and calcium ions did not affect the enzyme activity at the concentrations tested. We infer the purified trypsins do not require calcium ions, by which they differ from the trypsins of other microorganisms and the soluble and insoluble trypsins characterized from A. gemmatalis. These data suggest the existence of different isoforms of trypsin in the velvetbean caterpillar midguts.

Noncompetitive tight‐binding inhibition ofAnticarsia gemmatalistrypsins byAdenanthera pavoninaprotease inhibitor affects larvae survival

“…However, the mortality of the larvae was not 100%, which can be explained because the pests can produce serine proteases and/or alter expression of alternative gut proteases to circumvent the negative effects of PIs produced by those host plants during the evolutionary process (Meriño‐Cabrera et al, 2018; F. M. Pilon et al, 2017).…”

ppiGReMLIN: a graph mining based detection of conserved structural arrangements in protein-protein interfaces

“…The insect Anticarsia gemmatalis Hübner is a pest that attacks soybean. The plant, when injured by this insect, produces the Kunitz trypsin inhibitor (KTI), which impairs the process of proteases degradation in the caterpillar gut [27,28]. Inspired by this natural inhibitor produced by soybean, we are interested in proposing peptides or mimetic peptides to inhibit the proteases of the insect gut, that is, to carry out the ecological control of this pest insect.…”

Intestinal proteolytic profile changes during larval development of Anticarsia gemmatalis caterpillars

“…On the contrary, plants have developed multiple dynamic coevolutionary strategies of defense against phytophagous. In this interaction, plants can produce several protease inhibitors (PIs) and secondary metabolites (phytochemicals) via lipoxygenases, which affect the performance and resistance of herbivores (Feussner & Wasternack, ; Meriño‐Cabrera et al, ; Pilon et al, ).…”

“…PIs contribute up to 10% of the total protein content, generally present in a higher proportion in the reserve tissues and also detectable in leaves in response to insect attack (Meriño‐Cabrera et al, ; Pilon, Visôtto, Guedes, & Oliveira, ). In this sense, PIs can serve as pest control agents, either in the pulverized form or in transgenic plants that highly express these PIs (Meriño‐Cabrera et al, ; Pilon et al, ). Taking into account that the need for more appropriate formulations has raised the cost of agrochemicals over the years, interest in research into ecologically sound pesticides has increased (Krinski, Foerster, & Deschamps, ).…”

“…Taking into account that the need for more appropriate formulations has raised the cost of agrochemicals over the years, interest in research into ecologically sound pesticides has increased (Krinski, Foerster, & Deschamps, ). This alternative method is cheaper in application, more specific, less polluting, and has a long shelf life because it is more difficult for a pest to become resistant (Pilon et al, ). Currently, the control of agricultural pests tends towards reducing the use of agrochemicals due to their harmful effects on the environment and human health, being replaced by bioinsecticides that consider economic and ecological aspects (Chandler et al, ).…”