Crosslinking immobilisation of surface-engineered xylanase and its hydrolysis performance of oil palm empty fruit bunches for xylooligosaccharide production
Hydrolysis of hemicellulose to sugar such as xylooligosaccharide (XOS) is an alternative method to reduce the naturally abundant lignocellulose biomass waste such as oil palm empty fruit bunches (OPEFB). To achieve this, enzymatic hydrolysis has been envisioned as a highly potential method in conver...
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| Format: | Thesis |
| Language: | English |
| Published: |
2021
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| Online Access: | http://eprints.utm.my/102125/1/MohdKhairulPSChE2021.pdf |
| _version_ | 1796867202321743872 |
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| author | Abdul Wahab, Mohd. Khairul Hakimi |
| author_facet | Abdul Wahab, Mohd. Khairul Hakimi |
| author_sort | Abdul Wahab, Mohd. Khairul Hakimi |
| collection | ePrints |
| description | Hydrolysis of hemicellulose to sugar such as xylooligosaccharide (XOS) is an alternative method to reduce the naturally abundant lignocellulose biomass waste such as oil palm empty fruit bunches (OPEFB). To achieve this, enzymatic hydrolysis has been envisioned as a highly potential method in converting hemicellulose to XOS. However, the conventional use of free enzymes is always hampered by the low stability of the enzyme, difficulty in recovery, and non-recyclability. These limitations can be solved by enzyme immobilization, such as cross-linked enzyme aggregate (CLEA), in which the immobilization occurs without a solid carrier. The interaction between the amine group at the enzyme with the crosslinker plays a significant role in determining the immobilization efficiency. Nevertheless, the low content of lysine at the surface of the enzyme could be a problem to achieve efficient cross-linking. In this study, a three-dimensional (3D) model of xylanase (rXyn) from Aspergillus fumigatus RT-1 was developed using Modeller v9, and surface analyzed using Swiss PDB Viewer. In silico mutagenesis was performed at four residues on the surface of the enzyme (mXyn) and docked with glutaraldehyde using AutoDock. Molecular Dynamic (MD) simulation was performed on all structures (rXyn, rXyn-glu, mXyn, and mXyn-glu) for 1 ns at four different temperatures, and it was found that the structures were stabilized when docked with glutaraldehyde. The recombinant xylanase (rXyn) was mutated using site-directed mutagenesis at four different residues, mainly at the back of the enzyme and away from the catalytic site. The parameters of CLEA (choice of precipitants, the concentration of precipitant, concentration of crosslinker, concentration of bovine serum albumin (BSA), and cross-linking time) were optimized. The mXyn-CLEA-BSA was found to be able to recover higher xylanase activity at 137.08 % compared to the rXyn-CLEA, rXyn-CLEA-BSA, and mXyn-CLEA, which showed lower recovery activity at 96.64%, 104.71%, and 115.48%, respectively. At 70 °C for 60 minutes, mXyn-CLEA-BSA achieved the highest stability than the other CLEAs and free enzymes. mXyn-CLEA-BSA also successfully retained more than 40% of its activity after 5 cycles, whereas in the same cycle, rXyn-CLEA lost its total activity. In comparison, rXyn-CLEA-BSA and mXyn- CLEA only retained 19.66% and 21.41% of its activity, respectively. Therefore, the performance of mXyn-CLEA-BSA was further investigated in the catalytic reaction using pre-treated OPEFB under optimized reaction conditions. Four different sizes of CLEA particles and three different sizes of OPEFB was used to study the diffusional effect. The smaller size of CLEA particle and OPEFB were found to give higher hemicellulose yield. From high performance liquid chromatography analysis, the reaction between mXyn-CLEA-BSA and OPEFB produced xylotriose and small traces of xylose, with 0.361 mg/mL and 0.044 mg/mL, respectively. These findings showed that the combination of protein surface engineering and CLEA technology could improve xylanase stability and reusability by strengthening the intermolecular linkages between xylanase and glutaraldehyde. Furthermore, the developed CLEAs offers a great advantage in synthesizing XOS from the insoluble substrate. |
| first_indexed | 2024-03-05T21:23:41Z |
| format | Thesis |
| id | utm.eprints-102125 |
| institution | Universiti Teknologi Malaysia - ePrints |
| language | English |
| last_indexed | 2024-03-05T21:23:41Z |
| publishDate | 2021 |
| record_format | dspace |
| spelling | utm.eprints-1021252023-08-05T02:42:32Z http://eprints.utm.my/102125/ Crosslinking immobilisation of surface-engineered xylanase and its hydrolysis performance of oil palm empty fruit bunches for xylooligosaccharide production Abdul Wahab, Mohd. Khairul Hakimi TP Chemical technology Hydrolysis of hemicellulose to sugar such as xylooligosaccharide (XOS) is an alternative method to reduce the naturally abundant lignocellulose biomass waste such as oil palm empty fruit bunches (OPEFB). To achieve this, enzymatic hydrolysis has been envisioned as a highly potential method in converting hemicellulose to XOS. However, the conventional use of free enzymes is always hampered by the low stability of the enzyme, difficulty in recovery, and non-recyclability. These limitations can be solved by enzyme immobilization, such as cross-linked enzyme aggregate (CLEA), in which the immobilization occurs without a solid carrier. The interaction between the amine group at the enzyme with the crosslinker plays a significant role in determining the immobilization efficiency. Nevertheless, the low content of lysine at the surface of the enzyme could be a problem to achieve efficient cross-linking. In this study, a three-dimensional (3D) model of xylanase (rXyn) from Aspergillus fumigatus RT-1 was developed using Modeller v9, and surface analyzed using Swiss PDB Viewer. In silico mutagenesis was performed at four residues on the surface of the enzyme (mXyn) and docked with glutaraldehyde using AutoDock. Molecular Dynamic (MD) simulation was performed on all structures (rXyn, rXyn-glu, mXyn, and mXyn-glu) for 1 ns at four different temperatures, and it was found that the structures were stabilized when docked with glutaraldehyde. The recombinant xylanase (rXyn) was mutated using site-directed mutagenesis at four different residues, mainly at the back of the enzyme and away from the catalytic site. The parameters of CLEA (choice of precipitants, the concentration of precipitant, concentration of crosslinker, concentration of bovine serum albumin (BSA), and cross-linking time) were optimized. The mXyn-CLEA-BSA was found to be able to recover higher xylanase activity at 137.08 % compared to the rXyn-CLEA, rXyn-CLEA-BSA, and mXyn-CLEA, which showed lower recovery activity at 96.64%, 104.71%, and 115.48%, respectively. At 70 °C for 60 minutes, mXyn-CLEA-BSA achieved the highest stability than the other CLEAs and free enzymes. mXyn-CLEA-BSA also successfully retained more than 40% of its activity after 5 cycles, whereas in the same cycle, rXyn-CLEA lost its total activity. In comparison, rXyn-CLEA-BSA and mXyn- CLEA only retained 19.66% and 21.41% of its activity, respectively. Therefore, the performance of mXyn-CLEA-BSA was further investigated in the catalytic reaction using pre-treated OPEFB under optimized reaction conditions. Four different sizes of CLEA particles and three different sizes of OPEFB was used to study the diffusional effect. The smaller size of CLEA particle and OPEFB were found to give higher hemicellulose yield. From high performance liquid chromatography analysis, the reaction between mXyn-CLEA-BSA and OPEFB produced xylotriose and small traces of xylose, with 0.361 mg/mL and 0.044 mg/mL, respectively. These findings showed that the combination of protein surface engineering and CLEA technology could improve xylanase stability and reusability by strengthening the intermolecular linkages between xylanase and glutaraldehyde. Furthermore, the developed CLEAs offers a great advantage in synthesizing XOS from the insoluble substrate. 2021 Thesis NonPeerReviewed application/pdf en http://eprints.utm.my/102125/1/MohdKhairulPSChE2021.pdf Abdul Wahab, Mohd. Khairul Hakimi (2021) Crosslinking immobilisation of surface-engineered xylanase and its hydrolysis performance of oil palm empty fruit bunches for xylooligosaccharide production. PhD thesis, Universiti Teknologi Malaysia. http://dms.library.utm.my:8080/vital/access/manager/Repository/vital:145667 |
| spellingShingle | TP Chemical technology Abdul Wahab, Mohd. Khairul Hakimi Crosslinking immobilisation of surface-engineered xylanase and its hydrolysis performance of oil palm empty fruit bunches for xylooligosaccharide production |
| title | Crosslinking immobilisation of surface-engineered xylanase and its hydrolysis performance of oil palm empty fruit bunches for xylooligosaccharide production |
| title_full | Crosslinking immobilisation of surface-engineered xylanase and its hydrolysis performance of oil palm empty fruit bunches for xylooligosaccharide production |
| title_fullStr | Crosslinking immobilisation of surface-engineered xylanase and its hydrolysis performance of oil palm empty fruit bunches for xylooligosaccharide production |
| title_full_unstemmed | Crosslinking immobilisation of surface-engineered xylanase and its hydrolysis performance of oil palm empty fruit bunches for xylooligosaccharide production |
| title_short | Crosslinking immobilisation of surface-engineered xylanase and its hydrolysis performance of oil palm empty fruit bunches for xylooligosaccharide production |
| title_sort | crosslinking immobilisation of surface engineered xylanase and its hydrolysis performance of oil palm empty fruit bunches for xylooligosaccharide production |
| topic | TP Chemical technology |
| url | http://eprints.utm.my/102125/1/MohdKhairulPSChE2021.pdf |
| work_keys_str_mv | AT abdulwahabmohdkhairulhakimi crosslinkingimmobilisationofsurfaceengineeredxylanaseanditshydrolysisperformanceofoilpalmemptyfruitbunchesforxylooligosaccharideproduction |