Comparative Characterization of Aspergillus Pectin Lyases by Discriminative Substrate Degradation Profiling
Fungal genomes often contain several copies of genes that encode carbohydrate active enzymes having similar activity. The copies usually have slight sequence variability, and it has been suggested that the multigenecity represents distinct reaction optima versions of the enzyme. Whether the copies r...
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Frontiers Media S.A.
2020-07-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fbioe.2020.00873/full |
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| author | Birgitte Zeuner Thore Bach Thomsen Mary Ann Stringer Kristian B. R. M. Krogh Anne S. Meyer Jesper Holck |
| author_facet | Birgitte Zeuner Thore Bach Thomsen Mary Ann Stringer Kristian B. R. M. Krogh Anne S. Meyer Jesper Holck |
| author_sort | Birgitte Zeuner |
| collection | DOAJ |
| description | Fungal genomes often contain several copies of genes that encode carbohydrate active enzymes having similar activity. The copies usually have slight sequence variability, and it has been suggested that the multigenecity represents distinct reaction optima versions of the enzyme. Whether the copies represent differences in substrate attack proficiencies of the enzyme have rarely been considered. The genomes of Aspergillus species encode several pectin lyases (EC 4.2.2.10), which all belong to polysaccharide lyase subfamily PL1_4 in the CAZy database. The enzymes differ in terms of sequence identity and phylogeny, and exhibit structural differences near the active site in their homology models. These enzymes catalyze pectin degradation via eliminative cleavage of the α-(1,4) glycosidic linkages in homogalacturonan with a preference for linkages between methyl-esterified galacturonate residues. This study examines four different pectin lyases (PelB, PelC, PelD, and PelF) encoded by the same Aspergillus sp. (namely A. luchuensis), and further compares two PelA pectin lyases from two related Aspergillus spp. (A. aculeatus and A. tubingensis). We report the phylogeny, enzyme kinetics, and enzymatic degradation profiles of the enzymes’ action on apple pectin, citrus pectin, and sugar beet pectin. All the pectin lyases exerted highest reaction rate on apple pectin [degree of methoxylation (DM) 69%, degree of acetylation (DAc) 2%] and lowest reaction rate on sugar beet pectin (DM 56%, DAc 19%). Activity comparison at pH 5–5.5 produced the following ranking: PelB > PelA > PelD > PelF > PelC. The evolution of homogalacturonan-oligomer product profiles during reaction was analyzed by liquid chromatography with mass spectrometry (LC-MS) detection. This analyses revealed subtle differences in the product profiles indicating distinct substrate degradation preferences amongst the enzymes, notably with regard to acetyl substitutions. The LC-MS product profiling analysis thus disclosed that the multigenecity appears to provide the fungus with additional substrate degradation versatility. This product profiling furthermore represents a novel approach to functionally compare pectin-degrading enzymes, which can help explain structure-function relations and reaction properties of disparate copies of carbohydrate active enzymes. A better understanding of the product profiles generated by pectin modifying enzymes has significant implications for targeted pectin modification in food and biorefinery processes. |
| first_indexed | 2024-12-11T09:05:05Z |
| format | Article |
| id | doaj.art-261afd7a64ca4bd89edb881b46fddf68 |
| institution | Directory Open Access Journal |
| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-12-11T09:05:05Z |
| publishDate | 2020-07-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-261afd7a64ca4bd89edb881b46fddf682022-12-22T01:13:38ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852020-07-01810.3389/fbioe.2020.00873551028Comparative Characterization of Aspergillus Pectin Lyases by Discriminative Substrate Degradation ProfilingBirgitte Zeuner0Thore Bach Thomsen1Mary Ann Stringer2Kristian B. R. M. Krogh3Anne S. Meyer4Jesper Holck5Protein Chemistry and Enzyme Technology, Department of Biotechnology and Biomedicine, Technical University of Denmark, Lyngby, DenmarkProtein Chemistry and Enzyme Technology, Department of Biotechnology and Biomedicine, Technical University of Denmark, Lyngby, DenmarkNovozymes A/S, Lyngby, DenmarkNovozymes A/S, Lyngby, DenmarkProtein Chemistry and Enzyme Technology, Department of Biotechnology and Biomedicine, Technical University of Denmark, Lyngby, DenmarkProtein Chemistry and Enzyme Technology, Department of Biotechnology and Biomedicine, Technical University of Denmark, Lyngby, DenmarkFungal genomes often contain several copies of genes that encode carbohydrate active enzymes having similar activity. The copies usually have slight sequence variability, and it has been suggested that the multigenecity represents distinct reaction optima versions of the enzyme. Whether the copies represent differences in substrate attack proficiencies of the enzyme have rarely been considered. The genomes of Aspergillus species encode several pectin lyases (EC 4.2.2.10), which all belong to polysaccharide lyase subfamily PL1_4 in the CAZy database. The enzymes differ in terms of sequence identity and phylogeny, and exhibit structural differences near the active site in their homology models. These enzymes catalyze pectin degradation via eliminative cleavage of the α-(1,4) glycosidic linkages in homogalacturonan with a preference for linkages between methyl-esterified galacturonate residues. This study examines four different pectin lyases (PelB, PelC, PelD, and PelF) encoded by the same Aspergillus sp. (namely A. luchuensis), and further compares two PelA pectin lyases from two related Aspergillus spp. (A. aculeatus and A. tubingensis). We report the phylogeny, enzyme kinetics, and enzymatic degradation profiles of the enzymes’ action on apple pectin, citrus pectin, and sugar beet pectin. All the pectin lyases exerted highest reaction rate on apple pectin [degree of methoxylation (DM) 69%, degree of acetylation (DAc) 2%] and lowest reaction rate on sugar beet pectin (DM 56%, DAc 19%). Activity comparison at pH 5–5.5 produced the following ranking: PelB > PelA > PelD > PelF > PelC. The evolution of homogalacturonan-oligomer product profiles during reaction was analyzed by liquid chromatography with mass spectrometry (LC-MS) detection. This analyses revealed subtle differences in the product profiles indicating distinct substrate degradation preferences amongst the enzymes, notably with regard to acetyl substitutions. The LC-MS product profiling analysis thus disclosed that the multigenecity appears to provide the fungus with additional substrate degradation versatility. This product profiling furthermore represents a novel approach to functionally compare pectin-degrading enzymes, which can help explain structure-function relations and reaction properties of disparate copies of carbohydrate active enzymes. A better understanding of the product profiles generated by pectin modifying enzymes has significant implications for targeted pectin modification in food and biorefinery processes.https://www.frontiersin.org/article/10.3389/fbioe.2020.00873/fullpectin lyasemultigenecitysugar beet pectinapple pectincitrus pectinproduct profiling |
| spellingShingle | Birgitte Zeuner Thore Bach Thomsen Mary Ann Stringer Kristian B. R. M. Krogh Anne S. Meyer Jesper Holck Comparative Characterization of Aspergillus Pectin Lyases by Discriminative Substrate Degradation Profiling Frontiers in Bioengineering and Biotechnology pectin lyase multigenecity sugar beet pectin apple pectin citrus pectin product profiling |
| title | Comparative Characterization of Aspergillus Pectin Lyases by Discriminative Substrate Degradation Profiling |
| title_full | Comparative Characterization of Aspergillus Pectin Lyases by Discriminative Substrate Degradation Profiling |
| title_fullStr | Comparative Characterization of Aspergillus Pectin Lyases by Discriminative Substrate Degradation Profiling |
| title_full_unstemmed | Comparative Characterization of Aspergillus Pectin Lyases by Discriminative Substrate Degradation Profiling |
| title_short | Comparative Characterization of Aspergillus Pectin Lyases by Discriminative Substrate Degradation Profiling |
| title_sort | comparative characterization of aspergillus pectin lyases by discriminative substrate degradation profiling |
| topic | pectin lyase multigenecity sugar beet pectin apple pectin citrus pectin product profiling |
| url | https://www.frontiersin.org/article/10.3389/fbioe.2020.00873/full |
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