Discovery and characterization of fungal xyloglucanases for citrus peel valorization

Kai Li*

*Corresponding author for this work

Research output: Book/ReportPh.D. thesis

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Abstract

In this thesis, three novel xyloglucanases from Penicillium sp. were expressed and investigated. The Penicillium sp. fungal strains were selected as the source of xyloglucanase discovery based on its primary natural habitat in citrus fruit. Xyloglucan was chemically extracted from citrus peel which has been currently regarded as an undervalued byproduct in industrial juice production. Four xyloglucanases, including PiGH12A and PiGH12B from Penicillium italicum, PdGH12 from Penicillium digitatum, and AnGH12 from Aspergillus nidulans, were successfully expressed in Pichia pastoris X-33. The optimal reaction conditions and molecular weight (MW) were almost identical in Penicillium sp. xyloglucanases, which were also highly temperature-sensitive. In comparison, AnGH12 exhibited greater thermal stability and more suitable optimum reaction conditions with high reaction rate, and hence has been chosen for further investigation.

The product from AnGH12 treated citrus peel xyloglucan was separated using a 2 kDa membrane into ASP-R (MW > 2 kDa) and ASP-P (MW < 2 kDa) fractions with different molecular weights distribution and degrees of polymerization. Various monosaccharide compositions have been identified in ASP-R and ASP-P fractions, revealing the presence of fucosylated xyloglucan in citrus peel xyloglucan. In vitro growth assays using eight strains were applied for evaluating the prebiotic potential activities of ASP-R and ASP-P fractions. In the growth of Bifidobacterium longum Bl-05, B. longum 232 and B. infantis 1497 strains, ASP-R fraction had a significantly (p < 0.05) higher prebiotic potential than other groups. Additionally, both of ASP-R and ASP-P fractions has exhibited the ability to inhibit the growth of Clostridium perfringens, highlighting their powerful antimicrobial activity against pathogenic pathogens.

The xyloglucan oligomer releases of GH12s xyloglucanases were determined in two substrates (tamarind seed xyloglucan and citrus peel xyloglucan), all of which were hydrolyzed into substantially identical xyloglucan oligomer patterns. Three xyloglucanases (PdGH12, PiGH12A and AnGH12) may not have a toxic peptides fragment, however PiGH12B was suspected to be a toxic enzyme due to the presence of one toxic peptides fragment. As with other xyloglucanases in GH12 family, the homology models of GH12s xyloglucanases had a similar β-jelly roll fold structure with an unique active site pocket filled with negatively charged amino acids. Homology models of xyloglucan-xyloglucanases complex together with protein sequence alignment were applied to reveal the catalytic residues and binding sites for xyloglucanase. Surprisingly, the binding site for PiGH12B at -4 position was tyrosine instead of tryptophan, which may explain why this enzyme had the highest Km value. All of GH12s xyloglucanases have exhibited a similar hydrolytic mechanism, cleaving only after unsubstituted glucose residues in the xyloglucan backbone. Finally, the interactions between amino acids and xyloglucan were analyzed: glutamic acid or methionine at cleavage site, tryptophan or tyrosine at negative subsites, and phenylalanine or glycine at positive subsites.

In conclusion, the substrate-enzyme relationship of four xyloglucanases from GH12 family were probed by homology models of xyloglucan-xyloglucanases complex, which also revealed details of molecular interactions. The homology models of xyloglucan-iii xyloglucanases complex in combination with xyloglucanases hydrolytic action mode were provided a deeper understanding of how xyloglucanases recognize and degrade xyloglucan, as well as to increase productivity of fucose-containing xyloglucan oligomers, which may have potential applications in the production of human milk oligosaccharide. As a typical xyloglucanases in GH12 family, the AnGH12 was considered a viable candidate for enzymatic food processing for reasons of energy conservation and cost reduction in commercial food industries.
Original languageEnglish
Place of PublicationKgs. Lyngby, Denmark
PublisherDTU Bioengineering
Number of pages131
Publication statusPublished - 2022

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