Novel methods to prepare cross-linked enzyme aggregates (CLEA) challenging immobilization models-urease and pepsin
Akkaş, Tuğçe (2015) Novel methods to prepare cross-linked enzyme aggregates (CLEA) challenging immobilization models-urease and pepsin. [Thesis]
The common goal of various protein immobilization techniques has been to bypass the intrinsic drawbacks of utilizing free enzymes as catalytic materials in industry. Crosslinked enzyme aggregates (CLEAs), one of the most successful, easily and widely applicable techniques developed so far, has greatly improved the storage and operational stability of enzyme preparations as well as permitted their easy recovery and thus reuse. Involving the seemingly simple semi-specific chemical cross linking of protein aggregates forced out of solution, the general applicability of typical CLEA methods has occasionally been challenged by protein-specific anomalies, reflecting intrinsic structural and functional traits, altering the effectiveness of aggregation and crosslinkability, as well as the resultant bioactivity of the material. In this work, the described limitations, have been addressed using two particularly CLEA-unfriendly protein starting materials, namely, native pepsin and urease. In case of urease, conventional CLEA methods led to dramatically low aggregation and cross linking yields, and displayed statistically insignificant catalytic activity in the immobilized product. Critical breakthrough was achieved by enforcing protein aggregation via lyophilization as opposed to routine precipitation. The subsequent crosslinking of the lyophilizate (yielding a CLEL) in a suitable antisolvent led to a much improved crosslinking yield and catalytic activity. In case of pepsin, the problematic step was achieving covalent crosslinking by conventional CLEA methods, as pepsin bears a single surface lysyl residue and predictably was relatively unresponsive to all crosslinking attempts of surface amino groups. The problem was alleviated by appropriate choice of a rather large crosslinker, i.e., dextran polyaldehyde, and the use of the subzero crosslinking temperatures, therefore permitting the formation of the first ever catalytically competent pepsin CLEA. Novel immobilized formulations presented herein, are expected to contribute as alternatives to many established industrially important applications, involving challenging protein systems. Furthermore, these also could be utilized to prompt greener processes, such as the syntheses of industrially important commodity compounds.
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