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Production of Galactooligosaccharides Using β-Galactosidase Immobilized on Chitosan-Coated Magnetic Nanoparticles with Tris(hydroxymethyl)phosphine as an Optional Coupling Agent.

Chen SC, Duan KJ - Int J Mol Sci (2015)

Bottom Line: However, activity retention of batchwise reactions was similar for both immobilized systems.All the three enzyme systems produced GOS compound with similar concentration profiles, with a maximum GOS yield of 50.5% from 36% (w · v(-1)) lactose on a dry weight basis.The chitosan-coated magnetic Fe3O4 nanoparticles can be regenerated using a desorption/re-adsorption process described in this study.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Tatung University, Taipei 104, Taiwan. monica94707@yahoo.com.tw.

ABSTRACT
β-Galactosidase was immobilized on chitosan-coated magnetic Fe3O4 nanoparticles and was used to produce galactooligosaccharides (GOS) from lactose. Immobilized enzyme was prepared with or without the coupling agent, tris(hydroxymethyl)phosphine (THP). The two immobilized systems and the free enzyme achieved their maximum activity at pH 6.0 with an optimal temperature of 50 °C. The immobilized enzymes showed higher activities at a wider range of temperatures and pH. Furthermore, the immobilized enzyme coupled with THP showed higher thermal stability than that without THP. However, activity retention of batchwise reactions was similar for both immobilized systems. All the three enzyme systems produced GOS compound with similar concentration profiles, with a maximum GOS yield of 50.5% from 36% (w · v(-1)) lactose on a dry weight basis. The chitosan-coated magnetic Fe3O4 nanoparticles can be regenerated using a desorption/re-adsorption process described in this study.

No MeSH data available.


Effect of temperature on the enzyme activity at pH 6.0. Fe3O4-CS-immobilized enzyme (0.2 g), Fe3O4-CS-THP-immobilized enzyme (0.2 g) or the free enzyme (1 mL) was added to 100 mL of 5% (w·v−1) lactose at pH 6.0 in a 500 mL Erlenmeyer flask and incubated at various temperatures from 20 to 90 °C in an orbital shaker bath at 200 rpm for 10 min. The relative activity was determined by measuring the production of glucose. The data are averaged from three samples. (●) Fe3O4-CS-immobilized enzyme; (○) Fe3O4-CS-THP-immobilized enzyme; (▼) free enzyme.
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ijms-16-12499-f003: Effect of temperature on the enzyme activity at pH 6.0. Fe3O4-CS-immobilized enzyme (0.2 g), Fe3O4-CS-THP-immobilized enzyme (0.2 g) or the free enzyme (1 mL) was added to 100 mL of 5% (w·v−1) lactose at pH 6.0 in a 500 mL Erlenmeyer flask and incubated at various temperatures from 20 to 90 °C in an orbital shaker bath at 200 rpm for 10 min. The relative activity was determined by measuring the production of glucose. The data are averaged from three samples. (●) Fe3O4-CS-immobilized enzyme; (○) Fe3O4-CS-THP-immobilized enzyme; (▼) free enzyme.

Mentions: The optimal temperature for the two immobilized enzyme systems and the free enzyme was 50 °C (Figure 3). Immobilized enzymes were more stable than the free enzyme at temperatures higher than 60 °C. In particular, the Fe3O4-CS-THP-immobilized enzyme showed greatly increased thermal stability at 70 and 80 °C. For example, at 70 °C the Fe3O4-CS-THP-immobilized enzyme retained 70% of its maximum activity, while the Fe3O4-CS-immobilized enzyme and the free enzyme retained only 26% and 8% of their activities, respectively. Immobilization restricts flexibility, making enzymes more resistant to unfolding and denaturation at higher temperatures. Indeed, the number of covalent bonds attaching an enzyme to a solid support is correlated with a quantifiable increase in thermal stability, as shown using immobilized trypsin [35]. It is noted that the experiment for the data of Figure 3 was proceeded only 10 min. Cheng et al. reported the same free enzyme remained 40% of its initial activity after incubation at 55 °C for 2 days [5]. In order to improve a long term thermal stability, the incubator was set at 45 °C in the subsequent experiment.


Production of Galactooligosaccharides Using β-Galactosidase Immobilized on Chitosan-Coated Magnetic Nanoparticles with Tris(hydroxymethyl)phosphine as an Optional Coupling Agent.

Chen SC, Duan KJ - Int J Mol Sci (2015)

Effect of temperature on the enzyme activity at pH 6.0. Fe3O4-CS-immobilized enzyme (0.2 g), Fe3O4-CS-THP-immobilized enzyme (0.2 g) or the free enzyme (1 mL) was added to 100 mL of 5% (w·v−1) lactose at pH 6.0 in a 500 mL Erlenmeyer flask and incubated at various temperatures from 20 to 90 °C in an orbital shaker bath at 200 rpm for 10 min. The relative activity was determined by measuring the production of glucose. The data are averaged from three samples. (●) Fe3O4-CS-immobilized enzyme; (○) Fe3O4-CS-THP-immobilized enzyme; (▼) free enzyme.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4490457&req=5

ijms-16-12499-f003: Effect of temperature on the enzyme activity at pH 6.0. Fe3O4-CS-immobilized enzyme (0.2 g), Fe3O4-CS-THP-immobilized enzyme (0.2 g) or the free enzyme (1 mL) was added to 100 mL of 5% (w·v−1) lactose at pH 6.0 in a 500 mL Erlenmeyer flask and incubated at various temperatures from 20 to 90 °C in an orbital shaker bath at 200 rpm for 10 min. The relative activity was determined by measuring the production of glucose. The data are averaged from three samples. (●) Fe3O4-CS-immobilized enzyme; (○) Fe3O4-CS-THP-immobilized enzyme; (▼) free enzyme.
Mentions: The optimal temperature for the two immobilized enzyme systems and the free enzyme was 50 °C (Figure 3). Immobilized enzymes were more stable than the free enzyme at temperatures higher than 60 °C. In particular, the Fe3O4-CS-THP-immobilized enzyme showed greatly increased thermal stability at 70 and 80 °C. For example, at 70 °C the Fe3O4-CS-THP-immobilized enzyme retained 70% of its maximum activity, while the Fe3O4-CS-immobilized enzyme and the free enzyme retained only 26% and 8% of their activities, respectively. Immobilization restricts flexibility, making enzymes more resistant to unfolding and denaturation at higher temperatures. Indeed, the number of covalent bonds attaching an enzyme to a solid support is correlated with a quantifiable increase in thermal stability, as shown using immobilized trypsin [35]. It is noted that the experiment for the data of Figure 3 was proceeded only 10 min. Cheng et al. reported the same free enzyme remained 40% of its initial activity after incubation at 55 °C for 2 days [5]. In order to improve a long term thermal stability, the incubator was set at 45 °C in the subsequent experiment.

Bottom Line: However, activity retention of batchwise reactions was similar for both immobilized systems.All the three enzyme systems produced GOS compound with similar concentration profiles, with a maximum GOS yield of 50.5% from 36% (w · v(-1)) lactose on a dry weight basis.The chitosan-coated magnetic Fe3O4 nanoparticles can be regenerated using a desorption/re-adsorption process described in this study.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Tatung University, Taipei 104, Taiwan. monica94707@yahoo.com.tw.

ABSTRACT
β-Galactosidase was immobilized on chitosan-coated magnetic Fe3O4 nanoparticles and was used to produce galactooligosaccharides (GOS) from lactose. Immobilized enzyme was prepared with or without the coupling agent, tris(hydroxymethyl)phosphine (THP). The two immobilized systems and the free enzyme achieved their maximum activity at pH 6.0 with an optimal temperature of 50 °C. The immobilized enzymes showed higher activities at a wider range of temperatures and pH. Furthermore, the immobilized enzyme coupled with THP showed higher thermal stability than that without THP. However, activity retention of batchwise reactions was similar for both immobilized systems. All the three enzyme systems produced GOS compound with similar concentration profiles, with a maximum GOS yield of 50.5% from 36% (w · v(-1)) lactose on a dry weight basis. The chitosan-coated magnetic Fe3O4 nanoparticles can be regenerated using a desorption/re-adsorption process described in this study.

No MeSH data available.