Oriented Immobilization of Enzymes Made Fit for Applied Biocatalysis: Non‐Covalent Attachment to Anionic Supports using Z_basic2 Module

Abstract

Immobilization of enzymes on insoluble carriers is a key technology for biocatalytic process development. [1] The main advantage of immobilized enzymes is that they are readily separated from solution and, therefore, support continuous processing in combination with an integrated re-use of the catalyst. [2] Full realization of the benefit of immobilization is often seen upon moving from the laboratory-to a larger-scale process operation, and the majority of enzymatic transformations performed on a multiton-per-year manufacturing scale employ carrier-bound catalysts. [2c, 3] There is usually a significant cost contribution of immobilization to the total costs of the catalyst. Therefore, the maximum amount of enzyme activity that can be loaded on the unit mass of a carrier is a clear target for optimization. Available methods for enzyme immobilization can be categorized according to whether positioning of the protein on the carrier surface is specific or random in orientation. [4] Specific positioning facilitates the design of the immobilization for optimum retention of the activity of the free enzyme in the carrier-bound catalyst. [4b, 5] However, specific positioning usually falls short, often by orders of magnitude, of the high protein loading capacity of common techniques of random immobilization, which represents the current industrial standard. [5b] Therefore, a broadly applicable method that combines the advantages of specific and random modes of protein binding to achieve immobilization of enzymes on carriers of industrial use would present a major advancement.The strategy presented herein was originally developed for an application in protein purification and exploits charge complementarity between the cationic "binding module" Z basic2 and the anionic supports. [6] Z basic2 fusion partners that display a negative net charge at the applied pH, will experience charge repulsion from the carrier surface (Scheme 1). Adsorption of Z basic2 fusion proteins should thus occur in a highly directed manner, driven almost exclusively by Z basic2 . By using two industrially applied enzymes, for example the d-amino acid oxidase from Trigonopsis variabilis (TvDAO, EC 1.4.3.3) [3a, 5b, 7] and sucrose phosphorylase from Leuconostoc mesenteroides (LmSPase, EC 2.4.1.7), [8] we show that protein chimeras harbor-ing Z basic2 at their respective N-terminus are bound in high density (! 200 mg protein g dry carrier À1) and yield (! 95 % of free-enzyme activity) on common porous resins displaying anionic sulfoalkyl surface groups. Non-covalent immobilization of each Z basic2 protein was highly selective from crude protein mixtures, showed useful resistance to leaching, and, because it was largely reversible upon applying a high salt concentration, allowed easy regeneration of the carrier material for multiple rounds of immobilization. For each of the two enzymes chosen, fusion to the Z basic2 module did not compromise recombinant protein production in Escherichia coli (E. coli) and was fully compatible with the catalytic ...

Strategies for the one-step immobilization–purification of enzymes as industrial biocatalysts

“…Perhaps the most popular domain is the poly-His tag, with high affinity for metal chelates (Figure 5) (e.g. for purification by IMAC) (Porath et al, 1975, Porath and Olin, 1983, Porath, 1992 but the range of these affinity peptides is huge and still growing: domains of affinity for cellulose, chitin binding domain, peptide tags, among 9 others (Arroyo et al, 2011, Bello-Gil et al, 2014, Bergeron et al, 2009, Bolivar and Nidetzky, 2012a, b, Cassimjee et al, 2011, Chern and Chao, 2005, Daunert et al, 2007, Kondo and Teshima, 1995, Kowsari et al, 2014, Kweon et al, 2005, Linder and Teeri, 1997, Martinez et al, 2000, Mateo et al, 2001b, Moldes et al, 2004a, Moldes et al, 2004b, Scaramozzino et al, 2005, Shpigel et al, 1999, Vishwanath et al, 1995, Wang et al, 2013a, Wiesbauer et al, 2011, Zhao et al, 2013. Table 1 shows a summary of the main domains used for this purpose while Table 2 shows some specific examples of uses of these domains.…”

“…These processes involve multipoint adsorption, and the anion tag permits to have a very intense one. Using cation exchangers, at pH 7 just a few proteins become adsorbed, thus a cationic domain may permit quite a selective adsorption of the tagged protein (Figure 7) (Bolivar and Nidetzky, 2012a, b, Gräslund et al, 2000, Gräslund et al, 2002a, Gräslund et al, 2002b, Hedhammar et al, 2006, Hedhammar and Hober, 2007, Nock et al, 1997, Sassenfeld and Brewer, 1984, Wiesbauer et al, 2011.…”

“…And aA two too low amount of groups in the support may produce a slow immobilization (taking many hours to have fully immobilized enzymes. Stabilization may be mainly found by preventing subunit dissociation of multimeric enzymes, the values found will depend on the free enzyme concentration sused for comparison (ranging from factors of 10 to thousands) (Bolivar and Nidetzky, 2012a, b, Gräslund et al, 2000, Gräslund et al, 2002a, Gräslund et al, 2002b, Hedhammar et al, 2006, Hedhammar and Hober, 2007, Nock et al, 1997, Sassenfeld and Brewer, 1984, Wiesbauer et al, 2011. We have not found actual uses of these strategies at industrial level, perhaps the moderate stabilization achieved did not encourage this as a protocol of for industrial enzyme immobilization, even being quite a quite simple protocol.…”

Oriented Coimmobilization of Oxidase and Catalase on Tailor-Made Ordered Mesoporous Silica

“…With the aim of comparing, the specific activity of purified Z basic2 _DAAO was 71 U/mg_protein 47 , that of Z basic2 _catalase was 60000 U/mg_protein.…”

Let the substrate flow, not the enzyme: Practical immobilization of d‐amino acid oxidase in a glass microreactor for effective biocatalytic conversions

“…Due to multiple Arg residues clustered on one of its sides, Z basic2 exposes a protein surface featuring a high positive charge density. Z basic2 adsorbs to negatively charged surfaces in a specific manner and with high affinity (Bolivar and Nidetzky, ; Wiesbauer et al, ). At neutral pH or above, glass is negatively charged.…”