Immobilization of Lipase Through Cross-linking of Polyethylene Glycol Diglycidyl Ether with Amino Carrier LX-1000EA

doi:10.13523/j.cb.1904041

China Biotechnology

2020, Vol. 40

Issue (1-2): 124-132 DOI: 10.13523/j.cb.1904041

Orginal Article

Immobilization of Lipase Through Cross-linking of Polyethylene Glycol Diglycidyl Ether with Amino Carrier LX-1000EA

ZHU Heng^1,²,ZHANG Ji-fu³,ZHANG Yun¹,SUN Ai-jun¹,HU Yun-feng^1,^**(

)

1 CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China
2 University of Chinese Academy of Sciences,Beijing 100049,China
3 Guangdong Provincial Hospital of Chinese Medicine, Guangzhou 510120,China

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Abstract

Polyethylene glycol diglycidyl ether (PEGDGE), a double-functional epoxy reagent, was used to covalently immobilize marine lipase through cross-linking amino carrier LX-1000EA. The treated carrier behaved good effect in the covalent immobilization of lipase. After single factor screening and orthogonal experiment, the best conditions of crosslinking and immobilized is 0.75% concentration of crosslinking agent, crosslinking temperature 35℃, crosslinking time 3h, carrier quantity 1.25g, pH 9.0 and immobilization temperature 55℃, immobilization time of 1h. The enzymatic properties of LX-1000EA- PEGDGE immobilized enzyme were compared with that of free enzyme and glutaraldehyde crosslinked immobilized enzyme LX-1000HA-GA. Compared with free enzymes, the optimal reaction temperature of LX-1000EA-PEGDGE immobilized enzyme did not change; and similar with LX-1000HA-GA, the optimal reaction pH increased from 7.0 to 8.0. Under the optimal condition, the enzyme activity of LX-1000EA-PEGDGE reached 78.84U/g. Immobilization changed the acid-base tolerance of lipase, and the thermal stability and the operation stability were improved compared with free enzyme and LX-1000HA-GA immobilized enzyme. The immobilized enzyme exhibited excellent thermal stability,retaining 90% initial enzyme activity after 3h incubation at 60℃; the residual enzyme activity retained 50% of its original activity after 5 times of operation; the residual enzyme activity retained 60% of its original activity after preserved for 30 days at 4℃. The new crosslinking agent PEGDGE and organic amino carrier were combined for the first time to immobilize lipase,which provided technical support for more effective immobilization methods. It was also found that the cross-linking agent had a great impact on the properties of the immobilized enzyme.

Key words： Polyethylene glycol diglycidyl ether Amino-carrier LX-1000EA Lipase Immobilization Enzymatic property

Received: 19 April 2019 Published: 27 March 2020

ZTFLH:

Q814

Corresponding Authors: Yun-feng HU E-mail: yunfeng.hu@scsio.ac.cn

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	Heng ZHU
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ZHU Heng,ZHANG Ji-fu,ZHANG Yun,SUN Ai-jun,HU Yun-feng. Immobilization of Lipase Through Cross-linking of Polyethylene Glycol Diglycidyl Ether with Amino Carrier LX-1000EA. China Biotechnology, 2020, 40(1-2): 124-132.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.1904041 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I1-2/124

Fig.1 Poly (ethylene glycol) diglycidyl ether

Fig.2 Preparation of immobilized lipase

Table 1 Orthogonal experimental design

Fig.3 Cross-linking and immobilization conditions were determined by single-factor experiments (a) The effect of pH on the activity of immobilized enzymes (b) The effect of the concentration of the crosslinking agent on the activity of the immobilized enzyme (c) The effect of carrier volume on the activity of the immobilized enzyme (d) The effect of cross-linking temperature on the enzyme activity of the immobilized enzyme (e) The effect of immobilization temperature on the enzyme activity of the immobilized enzyme (f) The effect of crosslinking time on the activity of the immobilized enzyme (g) The effect of immobilization time on the activity of immobilized enzyme

Table 2 Results and analysis of orthogonal experiments for lipase immobilized condition optimization

Table 3 Calculation process

Fig.4 Comparison of the enzymatic properties of free enzyme and immobilized enzyme LX-1000EA-PEGDGE and LX-1000HA-GA (a)Comparison of the optimal reaction temperature of free enzyme, immobilized enzyme LX-1000EA-PEGDGE and LX-1000HA-GA (b) Comparison of the optimal pH of free enzyme, immobilized enzyme LX-1000EA-PEGDGE and LX-1000HA-GA (c) Comparison of the pH stability of free enzyme, immobilized enzyme LX-1000EA-PEGDGE and LX-1000HA-GA (d) Comparison of the thermal stability between free enzyme and immobilized enzyme LX-1000EA-PEGDGE and LX-1000HA-GA (e) The operation stability of immobilized enzyme LX-1000EA-PEGDGE and LX-1000HA-GA (f) The storage stability of free enzyme and immobilized enzyme LX-1000EA-PEGDGE, LX-1000HA-GA at 4℃


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