Regulation of Catalytic Activity of Recombinant L-Asparaginase from Rhodospirillum Rubrum by Conjugation with a PEG-Chitosan Copolymer

A new approach for the regulation of catalytic properties of the medically significant enzyme L-asparaginase is suggested based on the formation of conjugates with PEG-chitosan (chitoPEGylation). The efficiency of this approach is demonstrated using recombinant L-asparaginase from Rhodospirillum rubrum (RrA). This preparation is immunologically different from the one used in medical practice preparations of L-asparaginase from E. coli, which offers a promising alternative for applications in the case of hypersentsitivity development. The low level of activity of RrA towards L-glutamine, which decreases significantly the chance of side effects developing, is an advantage of RrA. The technique for the synthesis of the RrA conjugates with PEG-chitosan (chitoPEGylation) of a varying modification degree is developed. It is established that conjugation of RrA with PEG-chitosan increased the specific activity of the enzyme in comparison with the native one. The activity changes from 56 IU/mg (for the native enzyme) to 61–72 IU/mg (for the conjugates) depending on the degree of chitosan PEGylation. The secondary structure of the Rhodospirillum rubrum asparaginase conjugates with PEG-chitosan is examined using CD- and IR-spectroscopy. It is found that the enzyme structure changed only slightly as a result of conjugation with PEG-chitosan: the content of α-helices changed from 36% (for the native enzyme) to 30–33% (for the conjugates). The content of β-structures changed from 15% (for the native enzyme) to 18% (for the conjugate). The obtained data open new opportunities for the synthesis of L-asparaginase preparations with improved biocatalytic properties.