A Simple Technique of Preparing Stable CLEAs of Phenylalanine Ammonia Lyase Using Co-aggregation with Starch and Bovine Serum Albumin

Abstract

Cross-linked enzyme aggregates (CLEAs) have been recently proposed as an alternative to conventional immobilization methods on solid carriers. However, the low cross-linking efficiency causes the major activity loss and instability in the conventional protocol for CLEA preparation. Herein, the effects of bovine serum albumin and starch addition on the cross-linking efficiency of CLEAs of phenylalanine ammonia lyase (PAL) from Rhodotorula glutinis were evaluated. A co-aggregation strategy was developed to improve cross-linking efficiency by adding starch and bovine serum albumin (BSA). CLEAs of PAL prepared in the presence of BSA and starch (PSB-CLEAs) retained 36 % activity, whereas CLEAs prepared without BSA and starch (PAL-CLEAs) retained only 8 % activity of the starting enzyme preparation. Compared with PAL-CLEAs, the thermal stability of PSB-CLEAs has improved considerably, maintaining 30 % residual activity after 4 h of incubation at 70 °C, whereas the PAL-CLEAs have only 13 % residual activity. PSB-CLEAs also exhibited the expected increased stability of PAL against hydrophilic organic solvents, superior operability, and higher storage stability. The proposed technique of preparing CLEAs using co-aggregation with starch and BSA would rank among the potential strategies for efficiently preparing robust and highly stable enzyme aggregates.

Hybrid Magnetic Cross-Linked Enzyme Aggregates of Phenylalanine Ammonia Lyase from Rhodotorula glutinis

“…The enhanced reusability of HM-PAL-CLEAs was speculated partially benefited from the milder operation of magnetic separating operation than centrifugation performed on PAL-CLEAs. The number of surface amine groups of PAL was not enough to achieve efficient cross-linking of all the enzyme molecules in CLEAs [18], [33], therefore leaking of enzyme from CLEAs would be a major reason for the loss in PAL activity. This problem will be more serous for CLEAs that need vigorous centrifugation for separation and shaking for re-suspension.…”

Characterization of a Novel Cross-Linked Lipase: Impact of Cross-Linking on Solubility and Release from Drug Product

“…This relationship between enzyme activity and cross-linking has been previously observed. 20,21 Importantly, subsequent dissolution evaluations with enzyme activity end points were normalized to the total lipase activity content of each capsule based on the specific activity of each lot and the mass of lipase-CLEC in each capsule. Dissolution Characterization and Product Performance.…”

Benzoate Surfactants for Enhancing the Activity of Lipoprotein Lipase from Burkholderia Species in Organic Solvent

“…They include the immobilization of enzymes on solid supporters [6][7][8][9][10][11][12] and the use of additives such as inorganic salts, [13][14][15][16][17] ionic liquids, [18][19][20][21][22] or surfactants. They include the immobilization of enzymes on solid supporters [6][7][8][9][10][11][12] and the use of additives such as inorganic salts, [13][14][15][16][17] ionic liquids, [18][19][20][21][22] or surfactants.…”