Biochemical Characterization of a Bifunctional Enzyme Constructed by the Fusion of a Glucuronan Lyase and a Chitinase from Trichoderma sp

Abstract

Bifunctional enzymes created by the fusion of a glucuronan lyase (TrGL) and a chitinase (ThCHIT42) from Trichoderma sp. have been constructed with the aim to validate a proof of concept regarding the potential of the chimera lyase/hydrolase by analyzing the functionality and the efficiency of the chimeric constructions compared to parental enzymes. All the chimeric enzymes, including or nor linker (GGGGS), were shown functional with activities equivalent or higher to native enzymes. The velocity of glucuronan lyase was considerably increased for chimeras, and may involved structural modifications at the active site. The fusion has induced a slightly decrease of the thermostability of glucuronan lyase, without modifying its catalytic activity regarding pH variations ranging from 5 to 8. The biochemical properties of chitinase seemed to be more disparate between the different fusion constructions suggesting an impact of the linkers or structural interactions with the linked glucuronan lyase. The chimeric enzymes displayed a decreased stability to temperature and pH variations, compared to parental one. Overall, TrGL-ThCHIT42 offered the better compromise in terms of biochemical stability and enhanced activity, and could be a promising candidate for further experiments in the field of fungi Cell Wall-Degrading Enzymes (CWDEs).

Keywords: bifunctional enzyme; chitinase; enzymatic efficiency; glucuronan lyase; polysaccharide.

Figure 1

Chitinase and glucuronan lyase mechanism (with R = H and/or acetyl group).

Figure 2

Schematic representation of chimeric constructions with their respective junctions (linkers, underlined) (a), and SDS-PAGE gel (12%) analysis of recombinant parental and fused enzymes after purification by Chromatography (IMAC) (b).

Figure 3

Specific activities for glucuronan lyase obtained for different substrate concentrations (a), and for chitinase using three substrates to test endo-type ((β-d-N,N’,N’’-triacetylchitotriose) and exo-type (N-acetyl-β-d-glucosaminidase and N,N’-diacetyl-β-d-chitobioside) chitinase activity (b). All the values were issue from three independent experiments (n = 3).

Figure 4

Determination of optimal temperatures of parental and chimeric constructions for glucuronan lyase using deacetylated glucuronan as substrate (a) and chitinase (b) using colloidal chitin. The values are the average of three independent repeated experiments.

Figure 5

Analysis of thermostability and pH stability of both parental and the fusions. The thermostability of glucuronan lyase (a) and chitinase (b) was estimated within a range of 25–65 °C, and the residual activity was calculated to the temperature reference (100% at 25 °C). The pH stability was analyzed within the pH range 3–9 for glucuronan lyase (c) and chitinase (d), and the residual activity was determined using pH optimum (5.5 for glucuronan lyase and 6 for chitinase) as reference (100%). All assays were carried out in triplicate.

Figure 6

Overall 3D structure simulation of TrGL and chimeric enzymes (left panel) and the conformational structure of the cleft of glucuronan lyase (right panel). The amino acids His53, Glu55, Gln91, Tyr200 and Asp206 are catalytic residues. The residues shown in red are located at the lid of the cleft. The enlargement of the cleft (at the lid level) is indicated by a double arrow (↔), with the distance indicated in Angstrom. A curvature of the chain near the residue Asp104 is shown by a green arrow.