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 ...

No abstract

The inside cover picture shows the structural model of sucrose phosphorylase from Leuconostoc mesenteroides fused to a positively charged Zbasic2 module that is placed next to a carrier surface showing the opposing (negative) charge. In their Communication on , B. Nidetzky et al., describe that non‐covalent attachment to ionic supports is a very useful approach for a reversible oriented immobilization of a number of enzymes. High protein loading and excellent catalytic effectiveness of the carrier‐bound enzymes can be achieved.

Various bioactive natural products, like the aminocoumarin antibiotics novobiocin and coumermycin, exhibit an aromatic C-methyl group adjacent to a glycosylated phenolic hydroxyl group. Therefore, tailoring of basic phenolic scaffolds to contain the intricate C-methyl/O-glycosyl motif is of high interest for structural and functional diversification of natural products. We demonstrate site-selective 8-C-methylation and 7-O-β-D-glucosylation of 4,5,7-trihydroxy-3-phenyl-coumarin (1) by S-adenosyl-L-methionine dependent C-methyltransferase (from Streptomyces niveus) and uridine 5'-diphosphate glucose dependent glycosyltransferase from apple (Malus × domestica). Both enzymes were characterized and shown to react readily with underivatized 1. However, glucosylation of the ortho-hydroxyl group prevented C-methylation, probably by precluding an essential substrate activation through deprotonation of 7-OH. Therefore, dual modification was only feasible when C-methylation occurred strictly before O-glucosylation. The target product was synthesized in near quantitative yield (98% conversion) from 500 µM 1 and its structure was confirmed by NMR. Combination of C-methyltransferase and O-glycosyltransferase reactions for synthetic tailoring of a natural product through biocatalysis was demonstrated for the first time.