A novel beta-fructofuranosidase in Coleoptera: Characterization of a beta-fructofuranosidase from the sugarcane weevil, Sphenophorus levis

Autor(es)

Pedezzi, Rafael ^[1] ; Fonseca, Fernando P. P. ^[1] ; Santos Junior, Celio Dias ^[1] ; Kishi, Luciano T. ^[1] ; Terra, Walter R. ^[2] ; Henrique-Silva, Flavio ^[1]

Afiliação do(s) autor(es)

^[1] Univ Fed Sao Carlos, Dept Genet & Evolut, Mol Biol Lab, BR-13565905 Sao Carlos, SP - Brazil

^[2] Univ Sao Paulo, Inst Chem, Dept Biochem, BR-05513970 Sao Paulo - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Insect Biochemistry and Molecular Biology; v. 55, p. 31-38, DEC 2014.
• Citações Web of Science: 8

Resumo

beta-fructofuranosidases or invertases (EC 3.2.1.26) catalyze the hydrolysis of sucrose into fructose and glucose. beta-fructofuranosidases have been widely described in microorganisms, but were not known in the animal kingdom until very recently. There are studies reporting lepidopteran beta-fructofuranosidases, but no beta-fructofuranosidase gene sequence or encoding transcript has previously been identified in beetles. Considering the scarcity of functional studies on insect beta-fructofuranosidases and their apparent non-occurrence among coleopterans, the aim of the present study was to investigate the occurrence and characterize a beta-fructofuranosidase transcript identified in a cDNA library from the sugarcane weevil, Sphenophorus levis (Curculionidae). To validate that the beta-fructofuranosidase sequence (herein denominated Sl-beta-fruct) is indeed encoded by the S. levis genome, PCRs were performed using genomic DNA extracted from the larval fat body as well as DNA from the midgut with microbial content. Amplification of Sl-beta-fruct gene using larval fat body DNA indicated its presence in the insect's genomic DNA. The Sl-beta-fruct gene was cloned in Pichia pastoris to produce the recombinant enzyme (rSl-beta-fruct). Molecular weight of the recombinant protein was about 64 kDa, indicating possible glycosylation, since the theoretical weight was 54.8 kDa. The substrate specificity test revealed that rSl-beta-fruct hydrolyzes sucrose and raffinose, but not melibiose or maltose, thereby confirming invertase activity. The pH curve revealed greatest activity at pH 5.0, demonstrating rSl-beta-fruct to be an acidic beta-fructofuranosidase. Quantitative PCR (qRT-PCR) analyses indicated that the production of mRNA only occurs in the midgut and reaches the greatest expression level in 30-day-old larvae, which is the expected pattern for digestive enzymes. Chromatography of glycosidases from S. levis midguts showed two enzymes acting as beta-fructofuranosidase, indicating the presence of a Sl-beta-fruct isoform or a beta-fructofuranosidase from insect intestinal microbiota. Moreover, it was found that alpha-glucosidases do not act on sucrose hydrolysis. Phylogenetic analyses indicated this enzyme to be similar to enzymes found in other coleopteran and lepidopteran beta-fructofuranosidases, but also closely similar to bacterial enzymes, suggesting potential horizontal gene transfer. Despite this, the enzyme seems to be restricted to different groups of bacteria, which suggests distinct origin events. The present study expands the concept of the occurrence of beta-fructofuranosidase in insects. Despite the few descriptions of this gene in the animal kingdom, it is possible to state that beta-fructofuranosidase is crucial to the establishment of some insects throughout their evolutionary history, especially members of the Lepidoptera and Coleoptera clades. (C) 2014 Elsevier Ltd. All rights reserved. (AU)

• Processo FAPESP: 98/14138-2 - Center for Structural Molecular Biotechnology
• Beneficiário: Glaucius Oliva
• Modalidade de apoio: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs