Ultra-high-throughput screening of beta glucoside kinase random mutagenesis library for phosphorus-sulfur bond Formation
Abstract. In the continual quest of novel biocatalysts with improved properties that will fulfil the raising industrial demands, directed evolution appears to be one of the most powerful tools in generating novel enzymes. Error -Prone PCR is a customized PCR protocol that will randomly introduce mut...
| Main Authors: | , |
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| Format: | Proceeding Paper |
| Language: | English English English |
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2017
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| Online Access: | http://irep.iium.edu.my/61791/ http://irep.iium.edu.my/61791/1/ARCSB%202017%20slides%20Bglk.pdf http://irep.iium.edu.my/61791/2/ARCSB%202017%20Timetable%209%20November%20%20%20%202017%20Bglk.pdf http://irep.iium.edu.my/61791/3/ARCSB2017%20-%20announcement.pdf |
| _version_ | 1848785734294568960 |
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| author | Ibrahim Ali, Noorbatcha Hamzah, Mohd. Salleh |
| author_facet | Ibrahim Ali, Noorbatcha Hamzah, Mohd. Salleh |
| author_sort | Ibrahim Ali, Noorbatcha |
| building | IIUM Repository |
| collection | Online Access |
| description | Abstract. In the continual quest of novel biocatalysts with improved properties that will fulfil the raising industrial demands, directed evolution appears to be one of the most powerful tools in generating novel enzymes. Error -Prone PCR is a customized PCR protocol that will randomly introduce mutations creating a variant library. Beta glucoside kinase (bglk) is an enzyme that catalyzes the phosphor transformation to the 6’ C in a glucose molecule. The aim of the presented research is to engineer the bglk enzyme for phosphorus-sulfur bond formation. Two bglk libraries were created, one with a low mutation rate (0-4 mutation/kb) and the second with high mutation rate (9-16 mutation/kb) and was screened with Fluorescent Activated Cell Sorting (FACS) using a 6’thio-glucose-BODIPY as fluorescent substrate. Prior to this, a Glucose-BODIPY substrate was used with the wild type enzyme in order to test the cell incubation conditions with the fluorescent substrate. The positive clones collected from these two libraries, will be sequenced to verify the type and location of the mutations. |
| first_indexed | 2025-11-14T16:57:51Z |
| format | Proceeding Paper |
| id | iium-61791 |
| institution | International Islamic University Malaysia |
| institution_category | Local University |
| language | English English English |
| last_indexed | 2025-11-14T16:57:51Z |
| publishDate | 2017 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | iium-617912018-02-03T08:55:09Z http://irep.iium.edu.my/61791/ Ultra-high-throughput screening of beta glucoside kinase random mutagenesis library for phosphorus-sulfur bond Formation Ibrahim Ali, Noorbatcha Hamzah, Mohd. Salleh TP248.13 Biotechnology Abstract. In the continual quest of novel biocatalysts with improved properties that will fulfil the raising industrial demands, directed evolution appears to be one of the most powerful tools in generating novel enzymes. Error -Prone PCR is a customized PCR protocol that will randomly introduce mutations creating a variant library. Beta glucoside kinase (bglk) is an enzyme that catalyzes the phosphor transformation to the 6’ C in a glucose molecule. The aim of the presented research is to engineer the bglk enzyme for phosphorus-sulfur bond formation. Two bglk libraries were created, one with a low mutation rate (0-4 mutation/kb) and the second with high mutation rate (9-16 mutation/kb) and was screened with Fluorescent Activated Cell Sorting (FACS) using a 6’thio-glucose-BODIPY as fluorescent substrate. Prior to this, a Glucose-BODIPY substrate was used with the wild type enzyme in order to test the cell incubation conditions with the fluorescent substrate. The positive clones collected from these two libraries, will be sequenced to verify the type and location of the mutations. 2017-11-22 Proceeding Paper PeerReviewed application/pdf en http://irep.iium.edu.my/61791/1/ARCSB%202017%20slides%20Bglk.pdf application/pdf en http://irep.iium.edu.my/61791/2/ARCSB%202017%20Timetable%209%20November%20%20%20%202017%20Bglk.pdf application/pdf en http://irep.iium.edu.my/61791/3/ARCSB2017%20-%20announcement.pdf Ibrahim Ali, Noorbatcha and Hamzah, Mohd. Salleh (2017) Ultra-high-throughput screening of beta glucoside kinase random mutagenesis library for phosphorus-sulfur bond Formation. In: KUALA LUMPUR, Putrajaya Marriott Hotel, Malaysia. (Unpublished) |
| spellingShingle | TP248.13 Biotechnology Ibrahim Ali, Noorbatcha Hamzah, Mohd. Salleh Ultra-high-throughput screening of beta glucoside kinase random mutagenesis library for phosphorus-sulfur bond Formation |
| title | Ultra-high-throughput screening of beta glucoside kinase random mutagenesis library for phosphorus-sulfur bond Formation |
| title_full | Ultra-high-throughput screening of beta glucoside kinase random mutagenesis library for phosphorus-sulfur bond Formation |
| title_fullStr | Ultra-high-throughput screening of beta glucoside kinase random mutagenesis library for phosphorus-sulfur bond Formation |
| title_full_unstemmed | Ultra-high-throughput screening of beta glucoside kinase random mutagenesis library for phosphorus-sulfur bond Formation |
| title_short | Ultra-high-throughput screening of beta glucoside kinase random mutagenesis library for phosphorus-sulfur bond Formation |
| title_sort | ultra-high-throughput screening of beta glucoside kinase random mutagenesis library for phosphorus-sulfur bond formation |
| topic | TP248.13 Biotechnology |
| url | http://irep.iium.edu.my/61791/ http://irep.iium.edu.my/61791/1/ARCSB%202017%20slides%20Bglk.pdf http://irep.iium.edu.my/61791/2/ARCSB%202017%20Timetable%209%20November%20%20%20%202017%20Bglk.pdf http://irep.iium.edu.my/61791/3/ARCSB2017%20-%20announcement.pdf |