Cross- linked enzyme aggregates of recombinantxylanase for hemicelluloses to xylooligosaccharides
Hemicelluloses are heterogeneous branched polymers of sugars that exist abundantly in nature. Enzymatic hydrolysis is envisioned as a highly potential method in converting hemicelluloses into fuels and value-added chemicals. However, the use of free enzyme is hampered by low operational stability, d...
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| Format: | Thesis |
| Language: | English |
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2018
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| Online Access: | http://umpir.ump.edu.my/id/eprint/30102/ http://umpir.ump.edu.my/id/eprint/30102/1/Cross-%20linked%20enzyme%20aggregates%20of%20recombinantxylanase%20for%20hemicelluloses%20to%20xylooligosaccharides.wm.pdf |
| _version_ | 1848823434680729600 |
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| author | Shalyda, Md Shaarani |
| author_facet | Shalyda, Md Shaarani |
| author_sort | Shalyda, Md Shaarani |
| building | UMP Institutional Repository |
| collection | Online Access |
| description | Hemicelluloses are heterogeneous branched polymers of sugars that exist abundantly in nature. Enzymatic hydrolysis is envisioned as a highly potential method in converting hemicelluloses into fuels and value-added chemicals. However, the use of free enzyme is hampered by low operational stability, difficulty in recovery and non-reusability, which requires for enzyme immobilization. Carrier-bound immobilization leads to utilization of high cost matrices, clogging of filters during downstream processing and presence of large amounts of non-catalytic ballast. Therefore, cross-linked enzyme aggregates (CLEA), a carrier-free technology that combines purification (precipitation) and immobilization into a single operation and does not require purified enzymes, is the solution to these problems. In this study, a recombinant xylanase (Xyl) from Trichoderma reesei was immobilized using three approaches: Xyl-CLEA, Xyl-CLEA-BSA (bovine serum albumin) and Xyl-CLEAsilanized maghemite. The use of ethanol as precipitant (1:9 volume ratio of enzyme to precipitant), glutaraldehyde (0.2:1 of glutaraldehyde to enzyme of 100 mM concentration) as cross-linking agent and the introduction of (3-aminopropyl) triethoxysilane (APTES) silanized maghemite (0.0075:1 of silanized maghemite to enzyme) prevailed in forming xylanase CLEAs with good enzyme activity recovery (78 %), thermal stability (50 % retained activity) and reusability (50 % retained activity). The Xyl-CLEA-silanized maghemite enhanced the activity recovery 1.66- and 1.50-fold compared to Xyl-CLEA and Xyl-CLEA-BSA, respectively. At elevated temperature of 60 °C and pHs of 3.0 and 8.0, Xyl-CLEA-silanized maghemite achieved better stability compared to the other CLEAs and free enzyme. Xyl-CLEAsilanized maghemite also successfully retained more than 50 % of its activity after 6 cycles, whereas Xyl-CLEA only retained approximately 10 % after 5 cycles. Therefore, the performance of Xyl-CLEA-silanized maghemite was further investigated by xylan hydrolysis under optimised reaction conditions. Xylooligosaccharides yield was slightly improved by 1.26- fold compared to the free enzyme. Kinetic parameters confirmed that CLEA immobilization did affect the productivity of the designed biocatalyst. |
| first_indexed | 2025-11-15T02:57:05Z |
| format | Thesis |
| id | ump-30102 |
| institution | Universiti Malaysia Pahang |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-15T02:57:05Z |
| publishDate | 2018 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | ump-301022023-05-30T07:47:46Z http://umpir.ump.edu.my/id/eprint/30102/ Cross- linked enzyme aggregates of recombinantxylanase for hemicelluloses to xylooligosaccharides Shalyda, Md Shaarani QK Botany TP Chemical technology Hemicelluloses are heterogeneous branched polymers of sugars that exist abundantly in nature. Enzymatic hydrolysis is envisioned as a highly potential method in converting hemicelluloses into fuels and value-added chemicals. However, the use of free enzyme is hampered by low operational stability, difficulty in recovery and non-reusability, which requires for enzyme immobilization. Carrier-bound immobilization leads to utilization of high cost matrices, clogging of filters during downstream processing and presence of large amounts of non-catalytic ballast. Therefore, cross-linked enzyme aggregates (CLEA), a carrier-free technology that combines purification (precipitation) and immobilization into a single operation and does not require purified enzymes, is the solution to these problems. In this study, a recombinant xylanase (Xyl) from Trichoderma reesei was immobilized using three approaches: Xyl-CLEA, Xyl-CLEA-BSA (bovine serum albumin) and Xyl-CLEAsilanized maghemite. The use of ethanol as precipitant (1:9 volume ratio of enzyme to precipitant), glutaraldehyde (0.2:1 of glutaraldehyde to enzyme of 100 mM concentration) as cross-linking agent and the introduction of (3-aminopropyl) triethoxysilane (APTES) silanized maghemite (0.0075:1 of silanized maghemite to enzyme) prevailed in forming xylanase CLEAs with good enzyme activity recovery (78 %), thermal stability (50 % retained activity) and reusability (50 % retained activity). The Xyl-CLEA-silanized maghemite enhanced the activity recovery 1.66- and 1.50-fold compared to Xyl-CLEA and Xyl-CLEA-BSA, respectively. At elevated temperature of 60 °C and pHs of 3.0 and 8.0, Xyl-CLEA-silanized maghemite achieved better stability compared to the other CLEAs and free enzyme. Xyl-CLEAsilanized maghemite also successfully retained more than 50 % of its activity after 6 cycles, whereas Xyl-CLEA only retained approximately 10 % after 5 cycles. Therefore, the performance of Xyl-CLEA-silanized maghemite was further investigated by xylan hydrolysis under optimised reaction conditions. Xylooligosaccharides yield was slightly improved by 1.26- fold compared to the free enzyme. Kinetic parameters confirmed that CLEA immobilization did affect the productivity of the designed biocatalyst. 2018-03 Thesis NonPeerReviewed pdf en http://umpir.ump.edu.my/id/eprint/30102/1/Cross-%20linked%20enzyme%20aggregates%20of%20recombinantxylanase%20for%20hemicelluloses%20to%20xylooligosaccharides.wm.pdf Shalyda, Md Shaarani (2018) Cross- linked enzyme aggregates of recombinantxylanase for hemicelluloses to xylooligosaccharides. PhD thesis, Universiti Teknologi Malaysia (Contributors, Thesis advisor: Md. Illias, Rosli). |
| spellingShingle | QK Botany TP Chemical technology Shalyda, Md Shaarani Cross- linked enzyme aggregates of recombinantxylanase for hemicelluloses to xylooligosaccharides |
| title | Cross- linked enzyme aggregates of recombinantxylanase for hemicelluloses to xylooligosaccharides |
| title_full | Cross- linked enzyme aggregates of recombinantxylanase for hemicelluloses to xylooligosaccharides |
| title_fullStr | Cross- linked enzyme aggregates of recombinantxylanase for hemicelluloses to xylooligosaccharides |
| title_full_unstemmed | Cross- linked enzyme aggregates of recombinantxylanase for hemicelluloses to xylooligosaccharides |
| title_short | Cross- linked enzyme aggregates of recombinantxylanase for hemicelluloses to xylooligosaccharides |
| title_sort | cross- linked enzyme aggregates of recombinantxylanase for hemicelluloses to xylooligosaccharides |
| topic | QK Botany TP Chemical technology |
| url | http://umpir.ump.edu.my/id/eprint/30102/ http://umpir.ump.edu.my/id/eprint/30102/1/Cross-%20linked%20enzyme%20aggregates%20of%20recombinantxylanase%20for%20hemicelluloses%20to%20xylooligosaccharides.wm.pdf |