Magnetization of active inclusion bodies: comparison with centrifugation in repetitive biotransformations

Abstract

BackgroundPhysiological aggregation of a recombinant enzyme into enzymatically active inclusion bodies could be an excellent strategy to obtain immobilized enzymes for industrial biotransformation processes. However, it is not convenient to recycle “gelatinous masses” of protein inclusion bodies from one reaction cycle to another, as high centrifugation forces are needed in large volumes. The magnetization of inclusion bodies is a smart solution for large-scale applications, enabling an easier separation process using a magnetic field.ResultsMagnetically modified inclusion bodies of UDP–glucose pyrophosphorylase were recycled 50 times, in comparison, inclusion bodies of the same enzyme were inactivated during ten reaction cycles if they were recycled by centrifugation. Inclusion bodies of sialic acid aldolase also showed good performance and operational stability after the magnetization procedure.ConclusionsIt is demonstrated here that inclusion bodies can be easily magnetically modified by magnetic iron oxide particles prepared by microwave-assisted synthesis from ferrous sulphate. The magnetic particles stabilize the repetitive use of the inclusion bodies .Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0987-7) contains supplementary material, which is available to authorized users.

An Enzymatic 2‐Step Cofactor and Co‐Product Recycling Cascade towards a Chiral 1,2‐Diol. Part II: Catalytically Active Inclusion Bodies

“…Further experiments would be needed to optimize the stability of the PfBAL-CatIBs, which could be achieved e. g. by cross-linking [17] or magnetization. [18] Experimental Section Materials Chemicals and enzymes were purchased from Sigma-Aldrich, Fluka, Roth, Biosolve, Alfa Aesar, AppliChem, Merck, and Thermo Scientific (Waltham, MA, USA). Enantiopure (R)-(3,3',5,5')-tetramethoxy benzoin (TMBZ) was taken from a stock prepared as described elsewhere.…”

Catalytically-active inclusion bodies for biotechnology—general concepts, optimization, and application

“…Several of these were selected due to their well-known aggregation tendency, e.g., cellulosebinding domains (CBDs, (Nahalka 2008;Koszagova et al 2018;Choi et al 2011;Nahalka and Nidetzky 2007;Nahalka and Patoprsty 2009;Nahalka et al 2008)). Two different CBDs have been tested for CatIB induction: the rather small 108 amino acid long CBDcell from Cellulomonas fimi (Choi et al 2011), as well as the 156 amino acid long CBDclos from Clostridium cellulovorans (Nahalka 2008;Koszagova et al 2018;Nahalka and Nidetzky 2007;Nahalka and Patoprsty 2009;Nahalka et al 2008). Most of the CBD-derived CatIBs were only used for proof-of-concept All structures are shown in cartoon representation in gray with the Rosetta-identified hydrophobic surface patches shown as blue surfaces (Kuhlman and Baker 2000;Rohl et al 2004).…”

“…Due to their purity, consisting predominately of the aggregating target protein, they have traditionally been used for refolding studies, in which they served as an easy to separate source of pure target protein (Singh et al 2015). This long-held misconception has been challenged in recent years as more and more studies have revealed the dynamic, heterogeneous nature of bacterial IBs, which alongside of misfolded protein also contain protein species with amyloid structure as well as native-like and correctly folded protein (Garcia-Fruitos et al 2005;Park et al 2012;Jäger et al 2019a;Jäger et al 2018;Jäger et al 2019b;Kloss et al 2018a, b;Lamm et al 2020;Zhou et al 2012;Wang et al 2015;Jiang et al 2019;Wu et al 2011;Lin et al 2013;Diener et al 2016;Choi et al 2011;Nahalka and Nidetzky 2007;Nahalka et al 2008;Nahalka 2008;Nahalka and Patoprsty 2009;Koszagova et al 2018;Huang et al 2013;Arie et al 2006). Thus, more and more evidence suggests that those properties are to a certain degree an inherent feature of all IBs and that all cytoplasmic proteins exist in a conformational equilibrium between soluble-folded, partially misfolded, and insoluble aggregates.…”

“…The lower right panel depicts a close-up view to better visualize polar localization of the produced CatIBs. The upper right panel depicts a scanning electron microscopy image of isolated CatIB particles Nahalka and Patoprsty 2009;Koszagova et al 2018;Huang et al 2013;Arie et al 2006) (Fig. 1c).…”

Active tyrosine phenol-lyase aggregates induced by terminally attached functional peptides in Escherichia coli

“…Besides the soluble expression of TPL, insoluble TPL aggregates without enzyme activity also existed, which might be resulted from undesirable misfolded polypeptides. As the waste byproducts during protein expression, IBs are recognized as the major bottleneck in recombinant protein expression [19] and are discarded from further processing or are eventually used as a pure protein by in vitro refolding and recovery [37]. As whole-cell biocatalyst, L-DOPA titer could be seriously affected by TPL IBs without activity.…”