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

Zeineb Baklouti,Slim Abdelkafi,Pascal Dubessay,Cédric Delattre,Philippe Michaud,Guillaume Pierre,Christine Gardarin

doi:10.3390/life10100234

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).

Highlights

The filamentous fungi belonging to Trichoderma genus have been considered with interest for their use as enzymatic producer for biocontrol strategies [1]
Many efforts have been made in the development of fusion enzymes by genetic engineering to optimize the degradation of plants cell wall polymers
We focused on the analysis of the functionality of different chimeric constructions, combining the fusion of a glucuronan lyase and a chitinase acting independently on two structural polymers of cell-wall of Mucorales fungi, and the determination of the benefits related to biochemical and catalytic properties, gained by the fusion

Summary

Introduction

The filamentous fungi belonging to Trichoderma genus have been considered with interest for their use as enzymatic producer for biocontrol strategies [1]. The production and secretion by Trichoderma of Cell-Wall Degrading Enzymes (CWDEs), among which chitinases [4,5], glucanases [6,7,8] and proteases [9,10,11] are the most representative, playing a major and significant role in targeted host invasion [12,13]. Chitinases are polysaccharide hydrolases categorized into glycoside hydrolase (GH) 18, 19, 20, 23 and 48 families of the CAZy database (http://www.cazy.org/). Linkages in chitin (Figure 1), an abundant polysaccharide, found as a major structural compound of fungi cell wall [14,15]. Chitins are polymers composed of β(1,4) linked N-acetyl-d-glucosamine (GlcpNAc)

Methods

Results

Conclusion