(567bm) Cross-Linked Enzyme Aggregates: Improvement and Application

Authors: 
Qi, W., Chemical Engineering Research Center, School of Chemical Engineering and Technology, Tianjin University
Wang, M., Chemical Engineering Research Center, School of Chemical Engineering and Technology, Tianjin University
Su, R., Chemical Engineering Research Center, School of Chemical Engineering and Technology, Tianjin University
He, Z., Tianjin University


Cross-linked enzyme aggregates (CLEAs) is a novel non-carrier immobilized enzyme technology which was firstly developed in 2000 [Cao, 2000]. The simple preparing process consists of precipitation and cross-linking without purification or crystallization from crude enzyme. Although CLEAs have been applied to many biocatalytic reactions [van Pelt, 2007], some undesirable properties limited its further application: (1) the small particle size hampers the recovery operation; (2) the poor compressive resistance causes the decrease of catalytic efficiency; and (3) the inner steric hindrance limites the application in macromolecular substrates reaction. To overcome above drawbacks, three improved technology of CLEAs were developed. Enbeded CLEAs of papain in commercial macroporous silica gel (MSG) were prepared through simple adsorption, precipitation and one-step-cross-linking. Compared with CLEAs, the optimal temperature of CLEAs-MSG was broadened to 40-90 ºC, and the optimal pH was increased to 7.0. The storage stability and thermal stability were also obviously enhanced. Due to the suitable and adjustable size and the excellent compressive strength of silica gel pellets, CLEAs-MSG can be easily recovered by filtration or centrifugation. By using CLEAs-MSG of papain as biocatalyst, the kinetically controlled synthesis of z-Ala-Gln was achieved with the yield of 32.9%, which was almost equal to that by using free papain as biocatalyst. Porose CLEAs (P-CLEAs) of papain were prepared through adding starch (pore-making agent) into enzyme solution, and then co-precipitating, co-cross-linking and finally removing the starch by amylase. Compared with CLEAs, the catalytic efficiency of P-CLEAs on macromolecules such as BSA and ovalbumin were obviously increased 9.0 and 9.4 times respectively. Encapsulated-CLEAs (E-CLEAs) of penicillin G acylase were prepared through adding CLEAs and calcium chloride into a gently stirring alginate solution. The formed hollow E-CLEAs with adjustable and uniform size had excellent mobility and dispersibility which prevented CLEAs from mechanical inactivation caused by centrifugation and filtration. Additionally, a high-throughput proteomic analysis strategy was developed by incorporating on-plate digestion with trypsin-CLEAs, which showed excellent proteolysis efficiency, improved thermal stability, reduced enzyme autolysis and highly-accurate MALDI-FTMS peptide mass fingerprinting. The average sequence coverage of 46% was obtained by digestion of seven protein standards for 5 min at 77 ºC. (1)Cao L. Q., van Rantwijk F. and Sheldon R. A. Cross-linked enzyme aggregates: A simple and effective method for the immobilization of penicillin acylase, Organic Letters, 2, 1361-1364, 2000. (2)van Pelt S., Quignard S., Kubac D., Dimitry Y. S. B., van Rantwijk F. and Sheldon R. A. Nitrile hydratase CLEAs: The immobilization and stabilization of an industrially important enzyme. Green Chemistry, 10, 395-400, 2007.

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