Enhancement of Activity of Cross-Linked Enzyme Aggregates by a Novel Sugar-Assisted Precipitation Strategy

Enhancement of activity of cross-linked enzyme aggregates by a novel sugar-assisted precipitation strategy

Mengfan Wang², Wei Qi¹, Rongxin Su² and Zhimin He¹,

1) State Key Laboratory of Chemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, China. 2) Chemical Engineering Research Center, School of Chemical Engineering and Technology, Tianjin University, Tianjin, China.

*Corresponding Author's E-mail: qiwei@tju.edu.cn

Using enzymes as biocatalysts in chemical reactions has numerous advantages including selectivity, specificity, mild reaction conditions, stereoselectivity with pure enantiomers as products, and environmental friendly. However, the enzymes are sensitive and fragile to temperature, system pH and reaction medium. Recently, cross-linked enzyme aggregates (CLEAs) were developed as an efficient approach to obtain immobilized enzymes without the use of any pre-existing carriers [Sheldon, 2005]. CLEAs are stable over a wide pH and temperature ranges, tolerant to organic medium, reusable, and cost-effective. The preparation of CLEAs includes two steps: precipitation (usually using water-miscible organic solvents) and cross-linking. However, the water-miscible organic solvents generally induce the denaturation of enzyme [Bellezza, 2009], which leads to the activity loss of enzyme and finally lowers the activity yield of resulting CLEAs.

It is well known that sugar is an ideal stabilizer of various proteins in natural dehydrated environments as well as during lyophilization process. Therefore, in this work, glucose, sucrose, or trehalose was firstly introduced in the precipitation step to prepare the sugar-assisted CLEAs. For sugar-assisted CLEAs of penicillin G acylase (PGA), 79.3% of the activity yield was obtained by adding 20% trehalose and using acetone as the precipitant, which was 30% activity higher than that of CLEAs prepared without sugar. With the increasing of trehalose concentration, the final activity of trehalose-assisted CLEAs was increased and the aggregates size became larger according to scanning electron microscope (SEM) images. Confocal laser scanning microscopy (CLSM) and fourier transform infrared spectroscopy (FTIR) studies showed that the polar microenvironment and the secondary structure of native PGA were preserved to some extent when CLEAs were prepared with this novel sugar-assisted strategy (as Figure 1 A-C showed) . The kinetic studies also indicated the enhanced affinity to substrate, higher resist-inhibition capacity and catalysis efficiency of sugar-assisted CLEAs. Moreover, sugar-assisted CLEAs were significantly more stable when incubated at high temperature for a long time. The strategy may provide a feasible and efficient solution to improve the properties of CLEAs so as to broaden their application in biocatalysis and biotransformation.

Figure 1 (A) SEM, (B) CLSM, (C) FTIR of sugar-free and sugar-assisted CLEAs.

Acknowledgement

The authors acknowledge the financial supports received from the Program for New Century Excellent Talents in Chinese University (NCET-08-0386), the Key Project of Chinese Ministry of Education (108031), the Natural Science Foundation of China (20976125, 20806057, 31071509) and Tianjin (10JCYBJC05100), and the Program of Introducing Talents of Discipline to Universities of China (B06006).

References

(1) Sheldon R.A., Schoevaart R. and Van Langen L.M. Cross-linked enzyme aggregates (CLEAs): A novel and versatile method for enzyme immobilization (a review), Biocatalysis and Biotransformation, 23, 141-147, 2005.

(2) Bellezza F., Cipiciani A., Cinelli S. and Onori G. Influence of alcohols and osmolytes on thermal stability and catalytic activity of myoglobin: Co-solvent clustering effects, Chemical Physics Letters, 482, 139-142, 2009.