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Site-directed mutagenesis studies on a novel dual domain β-galactosidase/ β-glucosidase open reading frame identified from a dairy run-off metagenome
Thesis (MScAgric)--Stellenbosch University, 2021.
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| Format: | Thesis |
| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2021
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| _version_ | 1867614094122549248 |
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| access_status_str | Open Access |
| author | Ehrenreich, Craig Leigh |
| author2 | Peters, Shaun W. |
| author_browse | Ehrenreich, Craig Leigh Peters, Shaun W. |
| author_facet | Peters, Shaun W. Ehrenreich, Craig Leigh |
| author_sort | Ehrenreich, Craig Leigh |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MScAgric)--Stellenbosch University, 2021. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/124224 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:46:33.531Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2021 |
| publishDateRange | 2021 |
| publishDateSort | 2021 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/124224 Site-directed mutagenesis studies on a novel dual domain β-galactosidase/ β-glucosidase open reading frame identified from a dairy run-off metagenome Ehrenreich, Craig Leigh Peters, Shaun W. Loedolff, Bianke Kossmann, Jens Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology. Protein modelling Metagenomics Beta-glucosidase Beta-galactosidase Site-specific mutagenesis UCTD Thesis (MScAgric)--Stellenbosch University, 2021. ENGLISH ABSTRACT: Glycosyl hydrolases (GHs, EC 3.2.1.-) are currently represented by 171 distinct families (www.CAZY.org) and catalyse the hydrolysis of glycosidic bonds present in various biological molecules termed glycosides. This generally leads to the release of a carbohydrate glycone and a carbohydrate or non-carbohydrate aglycon. Glycosidic bonds can exist between two or more carbohydrate moieties or a carbohydrate moiety and a non- carbohydrate moiety. Generally, the hydrolysis of the glycosidic bond is catalysed by two amino acid residues: a general acid/base and a nucleophile. The β-glucosidases (EC 3.2.1.21) are ubiquitous enzymes that catalyse the hydrolysis of the β(1-4)-glycosidic bond of glucose-linked carbohydrates to release non-reducing terminal glucosyl residues, glucosides, or glucooligosaccharides. β-glucosidases are a widely distributed class of hydrolytic enzymes, existing in every living domain. The GH family 1 represents the largest and most widely studied of the β-glucosidases. Sequence alignment and site-directed mutagenesis of various GH1 enzymes have revealed two highly conserved amino acid motifs present in β-glucosidases (TENG and NEP), representing the catalytic nucleophile and general acid/base catalyst, respectively. Crystallographic analyses of GH1 family β-glucosidases also indicate the presence of a TIM-barrel (β/α8) protein architecture containing the active site within a deep pocket. The β-galactosidases (EC 3.2.1.23) are ubiquitous enzymes that catalyse the hydrolysis of the β(1-4)-linked glycosidic bond of galactose-linked carbohydrates to release non-reducing terminal galactosyl residues, galactosides or galactooligosaccharides. The β-galactosidase from Escherichia coli (LacZ), a GH2 enzyme, is currently the best studied β-galactosidase. Crystallographic and structure mutation studies of the LacZ enzyme shows a tetrameric protein consisting of four polypeptides that each fold into five distinct domains of which domain three is a central TIM-barrel of roughly 300 amino acid residues containing the active site domain. β-galactosidase activity occurs when the catalytic nucleophile (E537) from one polypeptide overlaps and interacts with the general acid/base catalyst (E461) of another polypeptide within the respective third domains. This is opposed to the active site within β- glucosidases, which only requires a single polypeptide chain to achieve activity. In previous studies, a fosmid-based metagenomic library composed of 40 kB inserts was derived from the run-off of a dairy farm. Using plate-based functional screening methodologies, a discrete fosmid clone was identified that demonstrated activities for both β-galactosidase and β-glucosidase. Subsequently, a single open reading frame (ORF) was iii identified (BG3L, 1315 bp). When BG3L was isolated and sub-cloned into the bacterial expression vector pRSetA, it also presented with the dual activity previously observed on both functional screens and in vitro enzyme assays using crude extracts containing recombinant protein. In this study, BG3L was further characterized using a site-directed mutagenesis approach to determine if we could abolish each activity singly and thereby demonstrating if BG3L contains two discrete functional domains. Structure-based sequence alignments of the BG3L protein sequence revealed no obvious structural similarity to GH2 β-galactosidases but resembled several GH1 enzyme architectures. Molecular models of BG3L were constructed for comparative structural and docking analysis against lactose, cellobiose and synthetic 4-nitrophenyl glycosides (artificial substrates). The in silico models were validated against the available crystal structure of a metagenomic β-glucosidase, PDB: 5XGZ. The validated models were used to identify amino acid residues potentially involved in enzymatic activities with Q23, E25, H123 and E399 being identified. A PCR-based site-directed mutagenesis methodology was then used to abolish either β-glucosidase or β-galactosidase activity. Plate-based functional screening indicated the abolishment of β-galactosidase activity (E25S) or both activities (E25Q, H123G). None of the mutations led to an abolishment of only the β-glucosidase activity. The optimal β-glucosidase activity of the E25S mutated enzyme (lacking β-galactosidase activity) was observed at pH 6.0 and 45 °C, which is similar to the WT BG3L (unmutated). The E25S mutation also led to improved affinity for cellobiose over WT BG3L. iii identified (BG3L, 1315 bp). When BG3L was isolated and sub-cloned into the bacterial expression vector pRSetA, it also presented with the dual activity previously observed on both functional screens and in vitro enzyme assays using crude extracts containing recombinant protein. In this study, BG3L was further characterized using a site-directed mutagenesis approach to determine if we could abolish each activity singly and thereby demonstrating if BG3L contains two discrete functional domains. Structure-based sequence alignments of the BG3L protein sequence revealed no obvious structural similarity to GH2 β-galactosidases but resembled several GH1 enzyme architectures. Molecular models of BG3L were constructed for comparative structural and docking analysis against lactose, cellobiose and synthetic 4-nitrophenyl glycosides (artificial substrates). The in silico models were validated against the available crystal structure of a metagenomic β-glucosidase, PDB: 5XGZ. The validated models were used to identify amino acid residues potentially involved in enzymatic activities with Q23, E25, H123 and E399 being identified. A PCR-based site-directed mutagenesis methodology was then used to abolish either β-glucosidase or β-galactosidase activity. Plate-based functional screening indicated the abolishment of β-galactosidase activity (E25S) or both activities (E25Q, H123G). None of the mutations led to an abolishment of only the β-glucosidase activity. The optimal β-glucosidase activity of the E25S mutated enzyme (lacking β-galactosidase activity) was observed at pH 6.0 and 45 °C, which is similar to the WT BG3L (unmutated). The E25S mutation also led to improved affinity for cellobiose over WT BG3L. To our knowledge, our approach has identified the E25S as the first such mutation within GH1 enzymes which indicates that the glutamic acid plays a role in modulating β-galactoside specificity. It has also revealed that the BG3L gene encodes for a unique enzyme which presents two discrete enzyme activities from a single active domain. AFRIKAANSE OPSOMMING: Geen opsomming beskikbaar National Research Foundation (NRF) Masters 2021-12-05T15:32:25Z 2022-02-22T10:17:23Z 2022-06-08T03:00:14Z 2021-12 Thesis http://hdl.handle.net/10019.1/124224 en_ZA Stellenbosch University x, 75 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Protein modelling Metagenomics Beta-glucosidase Beta-galactosidase Site-specific mutagenesis UCTD Ehrenreich, Craig Leigh Site-directed mutagenesis studies on a novel dual domain β-galactosidase/ β-glucosidase open reading frame identified from a dairy run-off metagenome |
| title | Site-directed mutagenesis studies on a novel dual domain β-galactosidase/ β-glucosidase open reading frame identified from a dairy run-off metagenome |
| title_full | Site-directed mutagenesis studies on a novel dual domain β-galactosidase/ β-glucosidase open reading frame identified from a dairy run-off metagenome |
| title_fullStr | Site-directed mutagenesis studies on a novel dual domain β-galactosidase/ β-glucosidase open reading frame identified from a dairy run-off metagenome |
| title_full_unstemmed | Site-directed mutagenesis studies on a novel dual domain β-galactosidase/ β-glucosidase open reading frame identified from a dairy run-off metagenome |
| title_short | Site-directed mutagenesis studies on a novel dual domain β-galactosidase/ β-glucosidase open reading frame identified from a dairy run-off metagenome |
| title_sort | site directed mutagenesis studies on a novel dual domain β galactosidase β glucosidase open reading frame identified from a dairy run off metagenome |
| topic | Protein modelling Metagenomics Beta-glucosidase Beta-galactosidase Site-specific mutagenesis UCTD |
| url | http://hdl.handle.net/10019.1/124224 |
| work_keys_str_mv | AT ehrenreichcraigleigh sitedirectedmutagenesisstudiesonanoveldualdomainbgalactosidasebglucosidaseopenreadingframeidentifiedfromadairyrunoffmetagenome |