Gene Cloning, Prokaryotic Expression, and Biochemical Characterization of a Soluble Trehalase in Helicoverpa armigera Hübner (Lepidoptera: Noctuidae)

Abstract

Trehalase is an indispensable component of insect hemolymph that plays important role in energy metabolism and stress resistance. In this study, we cloned and expressed the gene encoding soluble trehalase (HaTreh-1) of Helicoverpa armigera (cotton bollworm) and characterized the enzyme. HaTreh-1 had a full-length open reading frame encoding a protein of 571 amino acids. Sequence comparison indicated that HaTreh-1 was similar to some known insect trehalases. Two essential active sites (D321 and E519) and three essential residues (R168, R221, and R286) were conserved in HaTreh-1. The recombinant trehalase was expressed in Escherichia coli and purified by nickel exchange chromatography. Molecular weight of the recombinant protein was about 71 kDa, and the optimum HaTreh-1 enzyme activity is at 55°C with pH 6.0. Enzymatic assays showed a Km value of 72.8 mmol/liter and a Vmax value of 0.608 mmol/(liter·min). Inhibition assays in vitro indicated that castanospermine, a polyhydroxylated alkaloid, was an effective competitive inhibitor of trehalase with a Ki value of 6.7 μmol/liter. The inhibitor action of castanospermine was linked to its modification effect on trehalase structure. The circular dichroism spectrum showed that the percentage of α-helix increased under the presence of castanospermine. Results of our study will aid in developing effective trehalase inhibitors for controlling H. armigera in the future.

Purification and Characterization of Trehalase From Acyrthosiphon pisum, a Target for Pest Control

“…k cat is evaluated at 143 s −1 , and k cat /K m is 2 mM −1 .s −1 . This is in agreement with the K m values ranging from 0.5 to 73 mM that have been reported from other insects trehalases, such as Apis mellifera (0.52 mM) [52], Artemia Embryos (8.4 mM) [19], Bombyx mori (0.46 mM) [20], Aphis citricola (6.4 mM from alate -7.2 mM from apterous) [26], Chaetomium aureum (0.7 mM) [53] and Helicoverpa armigera (72.8 mM) [8].…”

“…The hydrolysis of trehalose into two glucose molecules takes place under the enzymatic control of trehalase (Treh) (EC 3.2.1.28). This enzyme was first discovered in Aspergillus niger in 1893 [5], and detected in various organisms [1][2][3][6][7][8]. According to the latest classification system of glycoside hydrolases based on their amino acid sequence similarity, Treh is an inverting glycosidase [9] belonging to either of the families 15, 65, and 37.…”

“…The reported extractions, purifications and characterizations of insect Trehs usually start from various crude enzyme solutions: blood for Phormia regina [6], embryos and larvae for Artemia [19], or whole midgut for the silkworm Bombyx mori [20] and Spodoptera frugiperda [21] and more recently on whole honeybees [22] and whole termites [23]. Gene cloning and prokaryotic expression were also used for Trehs from Chironomus ramosus [24], Drosophila melanogaster [25] and Helicoverpa armigera [8]. In the case of aphids, both soluble and membrane-bound trehalases in apterous and alate morphs of Aphis citricola have been observed [26].…”

Characterization, heterologous expression and engineering of trehalase for biotechnological applications

“…Trehalose and trehalase are found in several insects, such as Helicoverpa armigera [27], Spodoptera frugiperda [28], S. litura [29], Anisakis simplex [30], and the European honeybee [19]. Trehalose has important physiological effects on insect growth and development, energy for flight, chitin biosynthesis regulation [31], and restoration of abiotic stress.…”

The Molecular Characterization and Gene Expressions of Trehalase in Bumblebee, Bombus lantschouensis (Hymenoptera: Apidae)

“…Recent studies have indicated that only Tre-1 and Tre-2 are found in the majority of insects, such as Apolygus lucorum (Tan et al, 2014), Helicoverpa armigera (Ai et al, 2018), Spodoptera exigua (Tang et al, 2008), Omphisa fuscidentalis (Tatun et al, 2008), Drosophila melanogaster (Shukla et al, 2016), Cnaphalocrocis medinalis (Tian et al, 2016), and Bemisia tabaci (Wang et al, 2014). Multiple Tre-1 have been cloned in insects, including Harmonia axyridis (Tang et al, 2014;, Locusta migratoria , Nilaparvata lugens (Zhao et al, 2016), Tribolium castaneum (Tang et al, 2016), and Leptinotarsa decemlineata .…”