Cross-linked enzyme aggregates and entrapment immobilization of maltogenic amylase from Bacillus lehensis G1 for Maltooligosaccharides synthesis

Maltooligosaccharides (MOS) are potential oligosaccharides in food-based applications and can be synthesized through the enzymatic synthesis of maltogenic amylase from Bacillus lehensis G1 (Mag1). Although MOS can be synthesized by using free enzymes, this process is hampered by poor enzyme recovery...

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Main Author:	Nawawi, Noor Namirah
Format:	Thesis
Language:	English
Published:	2020
Subjects:	TP Chemical technology
Online Access:	http://eprints.utm.my/id/eprint/92410/1/NoornamirahNawawi2020.pdf.pdf
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spelling	my-utm-ep.924102021-09-28T07:34:18Z Cross-linked enzyme aggregates and entrapment immobilization of maltogenic amylase from Bacillus lehensis G1 for Maltooligosaccharides synthesis 2020 Nawawi, Noor Namirah TP Chemical technology Maltooligosaccharides (MOS) are potential oligosaccharides in food-based applications and can be synthesized through the enzymatic synthesis of maltogenic amylase from Bacillus lehensis G1 (Mag1). Although MOS can be synthesized by using free enzymes, this process is hampered by poor enzyme recovery and lack of enzyme stability, which makes it unrealistic for applications. To overcome these drawbacks, several optimization methods, including enzyme immobilization approach, could be applied. A carrier-free immobilization technique that uses cross-linked enzyme aggregates (CLEAs) enhances the stability of the enzymes. Indeed, a decrease in substrate accessibility of CLEAs may hinder CLEAs applications. The substrate accessibility problem of CLEAs formation was overcome by the addition of porous agents to generate porous CLEAs (p-CLEAs). However, p-CLEAs have particles that are small in size, soft and mechanically unstable, which can cause enzyme leaching and reduce the activity, as well as the performance of the enzyme. To address these problems, p-CLEAs were entrapped in calcium alginate beads (CA). In this study, a formation of cross-linked enzyme aggregates of Mag1 (Mag1-CLEAs) were carried out to improve the stability and reusability of free Mag1. The substrate accessibility problem of Mag1-CLEAs was solved by the formation of porous CLEAs of Mag1 (Mag1-p-CLEAs). All factors affecting the formation of CLEAs were investigated. The highest activity recovery of Mag1-CLEAs 58.14 % (18.6 U/mg) was obtained at 80 % (w/v) ammonium sulphate (precipitant), 0.25 % (w/v) chitosan (cross linker) with cross-linking time of 1.5 h. In comparison, Mag1-p-CLEAs prepared with 0.8 % (w/v) citrus pectin (porous agent) exhibited 91.20 % (29.2 U/mg) activity. This developed porous material exhibited larger particles size (1.60 µm) and pore size distribution of 8 – 1000 nm. Mag1-p-CLEAs noticeably retained 80 % of their activity after 30 min of incubation at 40 °C and showed longer half-life compared to free Mag1 and Mag1-CLEAs. The 1.68-fold increase in Vmax value in comparison to Mag1-CLEAs showed that the presence of pores of Mag1-p-CLEAs enhanced the beta-cyclodextrin (ß-CD) accessibility. Next, Mag1-p-CLEAs were entrapped into calcium alginate beads. Mag1-p-CLEAs-CA prepared with 2.5 % (w/v) sodium alginate and 0.6 % (w/v) calcium chloride yielded 53.16 % (17.0 U/mg) activity and showed a lower deactivation rate and longer half-life than those of entrapped free Mag1 (Mag1-CA) and entrapped non-porous Mag1-CLEAs (Mag1-CLEAs-CA). Moreover, Mag1-p-CLEAs-CA exhibited low enzyme leaching and high tolerance in various solvents compared to Mag1-p-CLEAs. A kinetic study revealed that Mag1-p-CLEAs-CA exhibited relatively high affinity towards ß-CD (Km = 0.62 mM). MOS (261.9 mg/g) were synthesized by Mag1-p-CLEAs-CA at 50 °C through hydrolysis reaction of ß-CD. Although, Mag1-p-CLEAs-CA have low transglycosylation activity, they have superior reusability and can maintain their activity for up to 11 cycles. In conclusion, the combination of CLEAs technology with entrapment approach, has proven to be a promising tool to develop stable enzymes. The developed Mag1-p-CLEAs-CA are potential biocatalyst for the continuous production of MOS. 2020 Thesis http://eprints.utm.my/id/eprint/92410/ http://eprints.utm.my/id/eprint/92410/1/NoornamirahNawawi2020.pdf.pdf application/pdf en public http://dms.library.utm.my:8080/vital/access/manager/Repository/vital:138723 phd doctoral Universiti Teknologi Malaysia Faculty of Engineering - School of Chemical & Energy Engineering
institution	Universiti Teknologi Malaysia
collection	UTM Institutional Repository
language	English
topic	TP Chemical technology
spellingShingle	TP Chemical technology Nawawi, Noor Namirah Cross-linked enzyme aggregates and entrapment immobilization of maltogenic amylase from Bacillus lehensis G1 for Maltooligosaccharides synthesis
description	Maltooligosaccharides (MOS) are potential oligosaccharides in food-based applications and can be synthesized through the enzymatic synthesis of maltogenic amylase from Bacillus lehensis G1 (Mag1). Although MOS can be synthesized by using free enzymes, this process is hampered by poor enzyme recovery and lack of enzyme stability, which makes it unrealistic for applications. To overcome these drawbacks, several optimization methods, including enzyme immobilization approach, could be applied. A carrier-free immobilization technique that uses cross-linked enzyme aggregates (CLEAs) enhances the stability of the enzymes. Indeed, a decrease in substrate accessibility of CLEAs may hinder CLEAs applications. The substrate accessibility problem of CLEAs formation was overcome by the addition of porous agents to generate porous CLEAs (p-CLEAs). However, p-CLEAs have particles that are small in size, soft and mechanically unstable, which can cause enzyme leaching and reduce the activity, as well as the performance of the enzyme. To address these problems, p-CLEAs were entrapped in calcium alginate beads (CA). In this study, a formation of cross-linked enzyme aggregates of Mag1 (Mag1-CLEAs) were carried out to improve the stability and reusability of free Mag1. The substrate accessibility problem of Mag1-CLEAs was solved by the formation of porous CLEAs of Mag1 (Mag1-p-CLEAs). All factors affecting the formation of CLEAs were investigated. The highest activity recovery of Mag1-CLEAs 58.14 % (18.6 U/mg) was obtained at 80 % (w/v) ammonium sulphate (precipitant), 0.25 % (w/v) chitosan (cross linker) with cross-linking time of 1.5 h. In comparison, Mag1-p-CLEAs prepared with 0.8 % (w/v) citrus pectin (porous agent) exhibited 91.20 % (29.2 U/mg) activity. This developed porous material exhibited larger particles size (1.60 µm) and pore size distribution of 8 – 1000 nm. Mag1-p-CLEAs noticeably retained 80 % of their activity after 30 min of incubation at 40 °C and showed longer half-life compared to free Mag1 and Mag1-CLEAs. The 1.68-fold increase in Vmax value in comparison to Mag1-CLEAs showed that the presence of pores of Mag1-p-CLEAs enhanced the beta-cyclodextrin (ß-CD) accessibility. Next, Mag1-p-CLEAs were entrapped into calcium alginate beads. Mag1-p-CLEAs-CA prepared with 2.5 % (w/v) sodium alginate and 0.6 % (w/v) calcium chloride yielded 53.16 % (17.0 U/mg) activity and showed a lower deactivation rate and longer half-life than those of entrapped free Mag1 (Mag1-CA) and entrapped non-porous Mag1-CLEAs (Mag1-CLEAs-CA). Moreover, Mag1-p-CLEAs-CA exhibited low enzyme leaching and high tolerance in various solvents compared to Mag1-p-CLEAs. A kinetic study revealed that Mag1-p-CLEAs-CA exhibited relatively high affinity towards ß-CD (Km = 0.62 mM). MOS (261.9 mg/g) were synthesized by Mag1-p-CLEAs-CA at 50 °C through hydrolysis reaction of ß-CD. Although, Mag1-p-CLEAs-CA have low transglycosylation activity, they have superior reusability and can maintain their activity for up to 11 cycles. In conclusion, the combination of CLEAs technology with entrapment approach, has proven to be a promising tool to develop stable enzymes. The developed Mag1-p-CLEAs-CA are potential biocatalyst for the continuous production of MOS.
format	Thesis
qualification_name	Doctor of Philosophy (PhD.)
qualification_level	Doctorate
author	Nawawi, Noor Namirah
author_facet	Nawawi, Noor Namirah
author_sort	Nawawi, Noor Namirah
title	Cross-linked enzyme aggregates and entrapment immobilization of maltogenic amylase from Bacillus lehensis G1 for Maltooligosaccharides synthesis
title_short	Cross-linked enzyme aggregates and entrapment immobilization of maltogenic amylase from Bacillus lehensis G1 for Maltooligosaccharides synthesis
title_full	Cross-linked enzyme aggregates and entrapment immobilization of maltogenic amylase from Bacillus lehensis G1 for Maltooligosaccharides synthesis
title_fullStr	Cross-linked enzyme aggregates and entrapment immobilization of maltogenic amylase from Bacillus lehensis G1 for Maltooligosaccharides synthesis
title_full_unstemmed	Cross-linked enzyme aggregates and entrapment immobilization of maltogenic amylase from Bacillus lehensis G1 for Maltooligosaccharides synthesis
title_sort	cross-linked enzyme aggregates and entrapment immobilization of maltogenic amylase from bacillus lehensis g1 for maltooligosaccharides synthesis
granting_institution	Universiti Teknologi Malaysia
granting_department	Faculty of Engineering - School of Chemical & Energy Engineering
publishDate	2020
url	http://eprints.utm.my/id/eprint/92410/1/NoornamirahNawawi2020.pdf.pdf
_version_	1747818594387886080

Cross-linked enzyme aggregates and entrapment immobilization of maltogenic amylase from Bacillus lehensis G1 for Maltooligosaccharides synthesis

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