Dataset Information

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).

SUBMITTER: Baklouti Z

PROVIDER: S-EPMC7601620 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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

Baklouti Zeineb Z Delattre Cédric C Pierre Guillaume G Gardarin Christine C Abdelkafi Slim S Michaud Philippe P Dubessay Pascal P

Life (Basel, Switzerland) 20201008 10

Bifunctional enzymes created by the fusion of a glucuronan lyase (TrGL) and a chitinase (ThCHIT42) from <i>Trichoderma</i> 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 v ...[more]

PMID: 33049934

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Project description:Bifunctional alginate lyases can efficiently degrade alginate comprised of mannuronate (M) and guluronate (G), but their substrate-degrading modes have not been thoroughly elucidated to date. In this study, we present Aly1 as a novel bifunctional endolytic alginate lyase of the genus Flammeovirga The recombinant enzyme showed optimal activity at 50°C and pH 6.0. The enzyme produced unsaturated disaccharide (UDP2) and trisaccharide fractions as the final main alginate digests. Primary substrate preference tests and further structure identification of various size-defined final oligosaccharide products demonstrated that Aly1 is a bifunctional alginate lyase and prefers G to M. Tetrasaccharide-size fractions are the smallest substrates, and M, G, and UDP2 fractions are the minimal product types. Remarkably, Aly1 can vary its substrate-degrading modes in accordance with the terminus types, molecular sizes, and M/G contents of alginate substrates, producing a series of small size-defined saturated oligosaccharide products from the nonreducing ends of single or different saturated sugar chains and yielding unsaturated products in distinct but restricted patterns. The action mode changes can be partially inhibited by fluorescent labeling at the reducing ends of oligosaccharide substrates. Deletion of the noncatalytic region (NCR) of Aly1 caused weak changes of biochemical characteristics but increased the degradation proportions of small size-defined saturated M-enriched oligosaccharide substrates and unsaturated tetrasaccharide fractions without any size changes of degradable oligosaccharides, thereby enhancing the M preference and enzyme activity. Therefore, our results provided insight into the variable action mode of a novel bifunctional endolytic alginate lyase to inform accurate enzyme use.IMPORTANCE The elucidated endolytic alginate lyases usually degrade substrates into various size-defined unsaturated oligosaccharide products (≥UDP2), and exolytic enzymes yield primarily unsaturated monosaccharide products. However, it is poorly understood whether endolytic enzymes can produce monosaccharide product types when degrading alginate. In this study, we demonstrated that Aly1, a bifunctional alginate lyase of Flammeovirga sp. strain MY04, is endolytic and monosaccharide producing. Using various sugar chains as testing substrates, we also proved that key factors causing Aly1's action mode changes are the terminus types, molecular sizes, and M/G contents of substrates. Furthermore, the NCR fragment's effects on Aly1's biochemical characteristics and alginate-degrading modes and corresponding mechanisms were discovered by gene truncation and enzyme comparison. In summary, this study provides a novel bifunctional endolytic tool and a variable action mode for accurate use in alginate degradation.

Dataset Information

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

Publications

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

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