Characterization of the physicochemical properties of cross-linked levanase aggregates for levan-type fructooligosaccharides production

Levan-type fructooligosaccharide (L-FOS) are oligosaccharides that is in high demand in food-based and pharmaceutical industries and it can be produced from the levan hydrolysis. Recombinant levanase from Bacillus lehensis G1 (rlevblg1) is an enzyme that specifically converts levan to L-FOS. However...

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Main Author:	Abd. Rahman, Noor Hidayah
Format:	Thesis
Language:	English
Published:	2021
Subjects:	TP Chemical technology
Online Access:	http://eprints.utm.my/id/eprint/101928/1/HidayahAbdRahmanPSChE2021.pdf
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spelling	my-utm-ep.1019282023-07-25T09:34:22Z Characterization of the physicochemical properties of cross-linked levanase aggregates for levan-type fructooligosaccharides production 2021 Abd. Rahman, Noor Hidayah TP Chemical technology Levan-type fructooligosaccharide (L-FOS) are oligosaccharides that is in high demand in food-based and pharmaceutical industries and it can be produced from the levan hydrolysis. Recombinant levanase from Bacillus lehensis G1 (rlevblg1) is an enzyme that specifically converts levan to L-FOS. However, the use of free rlevblg1 presents a lack of stability and reusability, thus hinder the synthesis of L-FOS for continuous reactions. A carrier-free immobilization of cross-linked enzyme aggregates (CLEAs) were developed to overcome these drawbacks. However, low number of lysine residues of rlevblg1 may reduce cross-linking efficiency to form a stable and active biocatalyst. This issue can be solved by enzyme co-aggregation using additives. Moreover, the formation of CLEAs is also influenced by mass diffusion limitation as the degree of molecular cross-linking attained, significantly affects substrate accessibility especially at higher substrate concentrations. To address this problem, macromolecular cross-linker was used in the formation of CLEAs. In this study, formation of cross-linked levanase aggregates (CLLAs) was performed to improve stability and reusability of free rlevblg1. An active CLLAs using glutaraldehyde (CLLAs-GA), and with bovine serum albumin (CLLAs-GA-BSA) were obtained, and the factors affecting the formation of CLLAs were investigated. The highest activity recovery of CLLAs-GA (92.8 %; 169.5 U/mg) and CLLAs-GA-BSA (121.2 %; 221.3 U/mg) was achieved at optimized conditions. The optimum temperature of CLLAs-GA and CLLAs-GA-BSA increased to 35 °C and 40 °C, respectively, from 30 °C in its free rlevblg1. At high temperature (50 °C), the half-life of CLLAs-GA-BSA was higher than that of free rlevblg1 and CLLAs-GA. The reusability of CLLAs for 8 cycles was retained more than 50 % activity. The Vmax value of CLLAs-GA-BSA (21.97 U/mg) was increased by 14.3 % from the free rlevblg1 (19.23 U/mg). Dialdehyde starch-tapioca (DAST) was successfully developed and used to cross-link levanase to form CLLAs-DAST and CLLAs-DAST-BSA which showed activity recovery of 65.6 % (119.8 U/mg) and 81.6 % (149.0 U/mg), respectively. After DAST cross-linking, the pH and thermal stability increased, and the tolerance in organic solvents improved which resulted in an activation of CLLAs. A kinetic study revealed that CLLAs-DAST (16.72 mg/mL) and CLLAs-DAST-BSA (16.58 mg/mL) had higher affinity (Km) toward levan than that of CLLAs-GA (20.52 mg/mL) and CLLAs-GA-BSA (18.20 mg/mL). Thus, improving substrate accessibility with higher effectiveness factors especially at higher levan concentrations (10-12 mg/mL). The highest total L-FOS was achieved by CLLAs-DAST-BSA (78.9 % (w/v)), followed by CLLAs-DAST (62.4 %(w/v)), free rlevblg1 (51.2 % (w/v)), CLLAs-GA-BSA (50.1 % (w/v)) and CLLAs-GA (35.6 % (w/v)), after 3 h reaction. Although CLLAs formation using glutaraldehyde has produced an active and stable CLLAs, diffusion limitation at higher substrate concentrations reduced the L-FOS synthesis. In conclusion, DAST as a cross-linker may have application prospects as a promising and green biocatalyst for product formation such as L-FOS. 2021 Thesis http://eprints.utm.my/id/eprint/101928/ http://eprints.utm.my/id/eprint/101928/1/HidayahAbdRahmanPSChE2021.pdf application/pdf en public http://dms.library.utm.my:8080/vital/access/manager/Repository/vital:145660 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 Abd. Rahman, Noor Hidayah Characterization of the physicochemical properties of cross-linked levanase aggregates for levan-type fructooligosaccharides production
description	Levan-type fructooligosaccharide (L-FOS) are oligosaccharides that is in high demand in food-based and pharmaceutical industries and it can be produced from the levan hydrolysis. Recombinant levanase from Bacillus lehensis G1 (rlevblg1) is an enzyme that specifically converts levan to L-FOS. However, the use of free rlevblg1 presents a lack of stability and reusability, thus hinder the synthesis of L-FOS for continuous reactions. A carrier-free immobilization of cross-linked enzyme aggregates (CLEAs) were developed to overcome these drawbacks. However, low number of lysine residues of rlevblg1 may reduce cross-linking efficiency to form a stable and active biocatalyst. This issue can be solved by enzyme co-aggregation using additives. Moreover, the formation of CLEAs is also influenced by mass diffusion limitation as the degree of molecular cross-linking attained, significantly affects substrate accessibility especially at higher substrate concentrations. To address this problem, macromolecular cross-linker was used in the formation of CLEAs. In this study, formation of cross-linked levanase aggregates (CLLAs) was performed to improve stability and reusability of free rlevblg1. An active CLLAs using glutaraldehyde (CLLAs-GA), and with bovine serum albumin (CLLAs-GA-BSA) were obtained, and the factors affecting the formation of CLLAs were investigated. The highest activity recovery of CLLAs-GA (92.8 %; 169.5 U/mg) and CLLAs-GA-BSA (121.2 %; 221.3 U/mg) was achieved at optimized conditions. The optimum temperature of CLLAs-GA and CLLAs-GA-BSA increased to 35 °C and 40 °C, respectively, from 30 °C in its free rlevblg1. At high temperature (50 °C), the half-life of CLLAs-GA-BSA was higher than that of free rlevblg1 and CLLAs-GA. The reusability of CLLAs for 8 cycles was retained more than 50 % activity. The Vmax value of CLLAs-GA-BSA (21.97 U/mg) was increased by 14.3 % from the free rlevblg1 (19.23 U/mg). Dialdehyde starch-tapioca (DAST) was successfully developed and used to cross-link levanase to form CLLAs-DAST and CLLAs-DAST-BSA which showed activity recovery of 65.6 % (119.8 U/mg) and 81.6 % (149.0 U/mg), respectively. After DAST cross-linking, the pH and thermal stability increased, and the tolerance in organic solvents improved which resulted in an activation of CLLAs. A kinetic study revealed that CLLAs-DAST (16.72 mg/mL) and CLLAs-DAST-BSA (16.58 mg/mL) had higher affinity (Km) toward levan than that of CLLAs-GA (20.52 mg/mL) and CLLAs-GA-BSA (18.20 mg/mL). Thus, improving substrate accessibility with higher effectiveness factors especially at higher levan concentrations (10-12 mg/mL). The highest total L-FOS was achieved by CLLAs-DAST-BSA (78.9 % (w/v)), followed by CLLAs-DAST (62.4 %(w/v)), free rlevblg1 (51.2 % (w/v)), CLLAs-GA-BSA (50.1 % (w/v)) and CLLAs-GA (35.6 % (w/v)), after 3 h reaction. Although CLLAs formation using glutaraldehyde has produced an active and stable CLLAs, diffusion limitation at higher substrate concentrations reduced the L-FOS synthesis. In conclusion, DAST as a cross-linker may have application prospects as a promising and green biocatalyst for product formation such as L-FOS.
format	Thesis
qualification_name	Doctor of Philosophy (PhD.)
qualification_level	Doctorate
author	Abd. Rahman, Noor Hidayah
author_facet	Abd. Rahman, Noor Hidayah
author_sort	Abd. Rahman, Noor Hidayah
title	Characterization of the physicochemical properties of cross-linked levanase aggregates for levan-type fructooligosaccharides production
title_short	Characterization of the physicochemical properties of cross-linked levanase aggregates for levan-type fructooligosaccharides production
title_full	Characterization of the physicochemical properties of cross-linked levanase aggregates for levan-type fructooligosaccharides production
title_fullStr	Characterization of the physicochemical properties of cross-linked levanase aggregates for levan-type fructooligosaccharides production
title_full_unstemmed	Characterization of the physicochemical properties of cross-linked levanase aggregates for levan-type fructooligosaccharides production
title_sort	characterization of the physicochemical properties of cross-linked levanase aggregates for levan-type fructooligosaccharides production
granting_institution	Universiti Teknologi Malaysia
granting_department	Faculty of Engineering - School of Chemical & Energy Engineering
publishDate	2021
url	http://eprints.utm.my/id/eprint/101928/1/HidayahAbdRahmanPSChE2021.pdf
_version_	1776100805385912320

Characterization of the physicochemical properties of cross-linked levanase aggregates for levan-type fructooligosaccharides production

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