title: Mesoporous phenylalanine ammonia lyase microspheres with improved stability through calcium carbonate templating.
abstract: Cross-linked enzyme aggregates (CLEAs) have recently emerged as a promising method for enzyme immobilization due to its simplicity and low cost. However, a lack of good size and morphological control over the as-prepared CLEAs has limited their practical applications in some cases. Here, monodisperse spherical CLEAs of phenylalanine ammonia lyase (PAL microspheres) were prepared based on CaCO3microtemplates. The preparation procedure involves filling porous CaCO3microtemplates with the protein by salt precipitation, glutaraldehyde crosslinking, and dissolution of the microtemplates. The formulation of CaCO3templates with controlled size was studied in detail. Characterization of the prepared PAL microspheres was investigated. The results showed that the PAL microspheres with high immobilization efficiency (79%) exhibited excellent stability, including increased tolerance to proteolysis, low pH, and denaturants, and excellent mechanical properties. For example, free PAL almost lost all activity after they were incubated in the presence of trypsin for 2min, whereas PAL microspheres still retained 95% of their initial activity. Moreover, scanning electron microscope, transmission electron microscope, and N2adsorption-desorption isotherms revealed that the resultant PAL microspheres possessed good monodispersity and mesoporous structure instead of the amorphous clusters of conventional CLEAs with few pores. Compared with conventional CLEAs, the monodisperse PAL microspheres with mesoporous make them more potentially useful for biomedical and biotechnological applications.
authors.affiliation: Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin University of Science and Technology, No 29, 13th, Avenue, Tianjin Economic and Technological Development Area (TEDA), Tianjin 300457, PR China; Research Center for Fermentation Engineering of Hebei, College of Bioscience and Bioengineering, Hebei University of Science and Technology, 26 Yuxiang Street, Shijiazhang 050000, PR China. Electronic address: cjd007cn@163.com.
authors.forename: Jiandong
authors.initials: J
authors.lastname: Cui
authors.affiliation: Research Center for Fermentation Engineering of Hebei, College of Bioscience and Bioengineering, Hebei University of Science and Technology, 26 Yuxiang Street, Shijiazhang 050000, PR China.
authors.forename: Yamin
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin University of Science and Technology, No 29, 13th, Avenue, Tianjin Economic and Technological Development Area (TEDA), Tianjin 300457, PR China.
authors.forename: Zhilei
authors.initials: Z
authors.lastname: Tan
authors.affiliation: Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin University of Science and Technology, No 29, 13th, Avenue, Tianjin Economic and Technological Development Area (TEDA), Tianjin 300457, PR China.
authors.forename: Cheng
authors.initials: C
authors.lastname: Zhong
authors.affiliation: Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin University of Science and Technology, No 29, 13th, Avenue, Tianjin Economic and Technological Development Area (TEDA), Tianjin 300457, PR China.
authors.forename: Peipei
authors.initials: P
authors.lastname: Han
authors.affiliation: Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin University of Science and Technology, No 29, 13th, Avenue, Tianjin Economic and Technological Development Area (TEDA), Tianjin 300457, PR China. Electronic address: jiashiru@tust.edu.cn.
authors.forename: Shiru
authors.initials: S
authors.lastname: Jia
mesh_terms: D001709:Biotechnology; D002119:Calcium Carbonate / Q000138:chemical synthesis / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000138:chemical synthesis / Q000737:chemistry*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000138:chemical synthesis / Q000737:chemistry; D007700:Kinetics; D008863:Microspheres; D010650:Phenylalanine Ammonia-Lyase / Q000138:chemical synthesis / Q000737:chemistry*; D016062:Porosity; D013696:Temperature
chemical_list: D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D010650:Phenylalanine Ammonia-Lyase; D002119:Calcium Carbonate
keywords: CLEAs; CaCO(3) microtemplates; Immobilization; PAL microspheres