PMID- 34064736 OWN - NLM STAT- PubMed-not-MEDLINE LR - 20210605 IS - 2079-4983 (Print) IS - 2079-4983 (Electronic) IS - 2079-4983 (Linking) VI - 12 IP - 2 DP - 2021 May 11 TI - Enhanced Stability of Long-Living Immobilized Recombinant beta-d-N-Acetyl-Hexosaminidase A on Polylactic Acid (PLA) Films for Potential Biomedical Applications. LID - 10.3390/jfb12020032 [doi] LID - 32 AB - beta-d-N-acetyl-hexosaminidase (Hex, EC 3.2.1.52) is an acid hydrolase that catalyzes the cleavage of the beta-1,4 bond in N-acetyl-d-galactosamine (Gal-NAc) and N-acetyl-d-glucosamine (Glc-NAc) from the non-reducing end of oligosaccharides and glycoconjugates. It is widely expressed in both the prokaryotic and eukaryotic world, where it performs multiple and important functions. Hex has antifungal activity in plants, is capable of degrading many biological substrates, and can play an important role in the biomedical field for the treatment of Tay-Sachs and Sandhoff diseases. With the aim being able to obtain a device with a stable enzyme, a method of covalent immobilization on polylactic acid (PLA) films was developed for the A isoform of the beta-d-N-acetyl-hexosaminidase enzyme (HexA), produced in a recombinant way from Human Embryonic Kidney-293 (HEK-293) cells and suitably purified. An in-depth biochemical characterization of the immobilized enzyme was carried out, evaluating the optimal temperature, thermal stability, pH parameters, and Km value. Moreover, the stability of the enzymatic activity over time was assessed. The results obtained showed an improvement in terms of kinetic parameters and stability to heat for the enzyme following immobilization and the presence of HexA in two distinct immobilized forms, with an unexpected ability for one of them to maintain its functionality for a long period of time (over a year). The stability and functionality of the enzyme in its immobilized form are therefore extremely promising for potential biotechnological and biomedical applications. FAU - Calzoni, Eleonora AU - Calzoni E AD - Department of Chemistry, Biology and Biotechnology, University of Perugia, 06123 Perugia, Italy. FAU - Cesaretti, Alessio AU - Cesaretti A AUID- ORCID: 0000-0001-5814-8524 AD - Department of Chemistry, Biology and Biotechnology, University of Perugia, 06123 Perugia, Italy. AD - Center of Excellence on Innovative Nanostructured Materials-CEMIN, University of Perugia, 06123 Perugia, Italy. FAU - Montegiove, Nicolo AU - Montegiove N AUID- ORCID: 0000-0002-0678-0824 AD - Department of Chemistry, Biology and Biotechnology, University of Perugia, 06123 Perugia, Italy. FAU - Di Michele, Alessandro AU - Di Michele A AUID- ORCID: 0000-0002-0357-2608 AD - Department of Physics and Geology, University of Perugia, 06123 Perugia, Italy. FAU - Emiliani, Carla AU - Emiliani C AD - Department of Chemistry, Biology and Biotechnology, University of Perugia, 06123 Perugia, Italy. AD - Center of Excellence on Innovative Nanostructured Materials-CEMIN, University of Perugia, 06123 Perugia, Italy. LA - eng PT - Journal Article DEP - 20210511 PL - Switzerland TA - J Funct Biomater JT - Journal of functional biomaterials JID - 101570734 PMC - PMC8162980 OTO - NOTNLM OT - biocatalysis OT - biopolymers OT - enzymatic replacement therapy OT - enzyme immobilization OT - enzyme recyclability OT - enzyme stability OT - lysosomal storage diseases COIS- The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. EDAT- 2021/06/03 06:00 MHDA- 2021/06/03 06:01 PMCR- 2021/05/11 CRDT- 2021/06/02 01:10 PHST- 2021/03/19 00:00 [received] PHST- 2021/04/27 00:00 [revised] PHST- 2021/05/06 00:00 [accepted] PHST- 2021/06/02 01:10 [entrez] PHST- 2021/06/03 06:00 [pubmed] PHST- 2021/06/03 06:01 [medline] PHST- 2021/05/11 00:00 [pmc-release] AID - jfb12020032 [pii] AID - jfb-12-00032 [pii] AID - 10.3390/jfb12020032 [doi] PST - epublish SO - J Funct Biomater. 2021 May 11;12(2):32. doi: 10.3390/jfb12020032.