Encapsulation of Trigonopsis variabilis D‐amino acid oxidase and fast comparison of the operational stabilities of free and immobilized preparations of the enzyme

Abstract

A one-step procedure of immobilizing soluble and aggregated preparations of D-amino acid oxidase from Trigonopsis variabilis (TvDAO) is reported where carrier-free enzyme was entrapped in semipermeable microcapsules produced from the polycation poly(methylene-co-guanidine) in combination with CaCl2 and the polyanions alginate and cellulose sulfate. The yield of immobilization, expressed as the fraction of original activity present in microcapsules, was approximately 52 +/- 5%. The effectiveness of the entrapped oxidase for O2-dependent conversion of D-methionine at 25 degrees C was 85 +/- 10% of the free enzyme preparation. Because continuous spectrophotometric assays are generally not well compatible with insoluble enzymes, we employed a dynamic method for the rapid in situ estimation of activity and relatedly, stability of free and encapsulated oxidases using on-line measurements of the concentration of dissolved O2. Integral and differential modes of data acquisition were utilized to examine cases of fast and slow inactivation of the enzyme, respectively. With a half-life of 60 h, encapsulated TvDAO was approximately 720-fold more stable than the free enzyme under conditions of bubble aeration at 25 degrees C. The soluble oxidase was stabilized by added FAD only at temperatures of 35 degrees C or greater.

Surface Cell Growth Engineering Assisted by a Novel Bacterial Nanomaterial

“…[3] While in the past IBs were believed to be formed by unfolded or largely misfolded polypeptide chains and therefore biologically inert, [5] recent insights show them to be constituted by folded and biofunctional protein species, [6] whose presence is allowed by a particular amyloid-like organization. [7][8][9] Therefore, IBs formed by enzymes such as b-galactosidase, D-amino acid oxidase, maltodextrin phosphorylase, sialic acid aldolase, and polyphosphate kinase [8,[10][11][12][13] can be used as catalysts in different processes. On the other hand, the in vivo formation of IBs is regulated by several cellular genes (mainly encoding proteases and chaperones), which makes the genetic manipulation of their nanometer-scale properties feasible.…”

The Functional Quality of Soluble Recombinant Polypeptides Produced in Escherichia coli Is Defined by a Wide Conformational Spectrum

“…In inclusion bodies formed by enzymes, the associated enzymatic activity is sufficient for efficient in situ substrate processing. The proposal of biologically active inclusion bodies being usable as catalyzers (10) has resulted in the incorporation of diverse enzymes, in the form of inclusion bodies (including ␤-galactosidase, D-amino acid oxidase, maltodextrin phosphorylase, sialic acid aldolase, and polyphosphate kinase) (6,(11)(12)(13)(14)(15), into different types of enzymatic processes.The molecular organization and quality of the soluble protein population, which is generally believed to adopt the native, functional conformation, have been studied much less. Therefore, in this conventional view, soluble proteins are expected to show a rather narrow conformational spectrum and to be highly functional.…”

A novel bio-functional material based on mammalian cell aggresomes

“…The unexpected application of IBs as versatile, functional and biocompatible materials paves the road for their use as self-immobilized catalysts when formed by enzymes like reductases (Garcia-Fruitos et al 2005), oxidases (Nahalka and Nidetzky, 2007;Nahalka et al 2008a), kinases (Nahalka et al 2006;Nahalka and Patoprsty, 2009), phosphorylases (Nahalka, 2008) and aldolases (Nahalka et al 2008b), or as therapeutic agents in protein replacement therapies when formed by therapeutic proteins (Cano-Garrido et al 2013;Seras-Franzoso et al 2013a;Seras-Franzoso et al 2013b;Seras-Franzoso et al 2014;Vazquez et al 2012). Mammalian cells instead of bacteria are preferred factories for eukaryotic protein production, because of their ability to perform proper post-translational modifications.…”