Cellulose degradation in Glenea cantor (Fabricius) (Coleoptera: Cerambycidae): functional characterization of GcEGaseZ7 and Cellulase reveals a novel enzymatic activity

Abstract

Glenea cantor (Fabricius) is an important forest pest that mainly attacks kapok trees, breaking down cellulose and lignin through 3 enzyme activities: endoglucanase, filter paper enzyme, and cellobiase. In this study, we unveiled the cloning and expression of 10 endoglucanase genes, GcEGase5A1, GcEGase5A2, GcEGaseZ2, GcEGaseZ3, GcEGaseZ4, GcEGaseZ5, GcEGaseZ7, GcEGaseZ8, GcEGaseZ9, and Cellulase, all of which exhibit enzymatic activities in G. cantor. These findings indicated that Cellulase shares sequence homology with beetle GHF45, whereas the other 9 endoglucanase genes are homologous to beetle GHF5. GcEGaseZ4 presented the highest expression in the foregut. In contrast, GcEGase5A2 and Cellulase presented peak expression in the midgut. Furthermore, GcEGaseZ7 was identified as the most highly expressed endoglucanase in the hindgut. Functional assays confirmed the ability of GcEGaseZ7 and Cellulase to degrade cellulose, and their cellulase activities were 75.57 ± 1.21 U/mg and 344.79 ± 6.91 U/mg, respectively. These results enhance our understanding of the complex cellulase system in insects and provide insights into the efficient digestion of cellulosic materials by wood-consuming insects. This research also has potential applications in bioenergy production and the development of biomaterials from lignocellulosic biomass.

Keywords: glycosyl hydrolase; insect enzymatic activity; longhorn beetles; prokaryotic expression.

Fig. 1.

Electrophoretic map of endoglucanase genes PCR of Glenea cantor.

Fig. 2.

Multiple alignments of amino acid sequences of endoglucanase in Glenea cantor. Note: A) Multiple alignments of amino acid sequences of endoglucanase in GHF5 of G. cantor; B) Multiple alignments of amino acid sequences of endoglucanase in GHF45 of G. cantor.

Fig. 3.

Phylogenetic tree of endoglucanase from other species and Glenea cantor. Note: ‘·’: The endoglucanase of Glenea cantor. The amino acid (aa) sequence alignment of endoglucanase genes in G. cantor and the homologous sequences obtained from different beetles, fungi, bacteria, and protists were analyzed by the Clustal W method. Phylogenetic trees were constructed using the neighbor-joining method and the reliability of the tree structure was evaluated using the 1,000-fold bootstrap replication.

Fig. 4.

Relative expression of endoglucanase genes in different parts of the gut of the fourth-instar larvae of Glenea cantor. Note: The data are average ± standard error in the figure. Different lowercase letters indicate that the relative expression levels of endoglucanase genes in different intestinal positions of fourth-instar larvae are significantly different by Tukey’s HSD test (P < 0.05). A-J: the relative expression levels of GcEGase5A1, GcEGase5A2, GcEGaseZ2, GcEGaseZ3, GcEGaseZ4, GcEGaseZ5, GcEGaseZ7, GcEGaseZ8, GcEGaseZ9 and cellulase in the intestine of fourth instar larvae.

Fig. 5.

Cellulase-28a and GcEGaseZ7-28a fusion protein of soluble analysis. Note: M: Molecular weight of protein marker, size marked on the left; 1: 0.5 mg/ml BSA; 2: 0.25 mg/ml BSA; 3–5: Cellulase-28a-BL21 bacterial liquid before fragmentation, supernatant after fragmentation, and precipitate after fragmentation; 6–8: Cellulase-28a-Rossetta bacterial liquid before fragmentation, supernatant after fragmentation, and precipitate after fragmentation; 9–11: GcEGaseZ7-28a-Rossetta bacterial liquid before fragmentation, supernatant after fragmentation, and precipitate after fragmentation; 12–14: GcEGaseZ7-28a-BL21 bacterial liquid before fragmentation, supernatant after fragmentation, and precipitate after fragmentation.

Fig. 6.

Plate verification of recombinant protein expression strains of Cellulase-28a and GcEGaseZ7-28a. Note: A-B: the sterilized water control and the control of PET-28a-B3 coating plate; C-F: plate verification of the recombinant expression strains Cellulase-28a-R3, Cellulase-28a-B3, GcEGaseZ7-28a-R3, and GcEGaseZ7-28a-B3.

Fig. 7.

Purification of Cellulase-28a and GcEGaseZ7-28a. Note: M: Molecular weight of protein marker, size marked on the left; A) 1–2: Uninduced and induced expression bacteria of Cellulase-28a; 3–4: The fragmented supernatant of Cellulase-28a; 5–8: Undiluted, 5-fold dilution, 10-fold dilution, and 20-fold dilution of Cellulase-28a precipitates after fragmentation; 9–11: 0.2 mg/ml, 0.3 mg/ml, and 0.4 mg/ml of BSA. B) 1–4: Undiluted, 5-fold dilution, 10-fold dilution, and 20-fold dilution of Cellulase-28a dissolved in 8M urea and precipitated after fragmentation; 5–6: 0.2 mg/ml and 0.4 mg/ml of BSA. C) 1–3: Untreated, the supernatant of GcEGaseZ7-28a purified by His tag and the eluate of the supernatant of GcEGaseZ7-28a fragmented and purified by His tag; 4–7: Untreated, 5-fold dilution, 10-fold dilution, and 20-fold dilution after dialysis and concentration of the eluted protein obtained from the supernatant of GcEGaseZ7-28a purified by His tag; 8–10: 5-fold dilution, 10-fold dilution, and 20-fold dilution of GcEGaseZ7-28a precipitates after fragmentation; 11–13: 0.2 mg/ml, 0.3 mg/ml, and 0.4 mg/ml BSA. D) 1–3: 5-fold dilution, 10-fold dilution, and 20-fold dilution of GcEGaseZ7-28a dissolved in 8M urea and precipitated after fragmentation; 4–6: 0.2 mg/ml 0.3 mg/ml and 0.4 mg/ml BSA.