Enzymes are often immobilized to allow their recovery and reuse from batch reactors or so that they can be used as heterogeneous catalysts in continuous flow operation [1], [2], [3], [4]. Continuous flow is attractive for biocatalytic manufacturing, conferring advantages over batch-mode catalysis in physical footprint, safety and operational control, and being compatible with modular compartmentalization allowing the rapid prototyping of new multi-enzyme cascades [5], [6], [7]. There is an array of technical solutions for protein immobilization, developed for several application areas (e.g., protein affinity purification)[8], [9], [10]. Interest in improving these technologies is growing, with a focus on developing low-cost, generalizable protein immobilization technologies [2], [11], [12], [13].

Although not true in every circumstance, random attachment by chemical cross-linking can lead to a marked reduction in enzyme activity [14], [15]. Site-specific interactions between dedicated attachment sites on an enzyme and the target surface are often sought to reduce enzyme inactivation during immobilization. One mechanism by which site specificity can be introduced is via the modification of the carrier’s surface to allow orthogonal interactions with a tailored enzyme. The introduction of genetically encoding tags enables covalent or non-covalent immobilization of proteins on supports. The histidine tag is a classic example of this, in which the enzyme of choice is modified to carry series of consecutive histidine residues (often six) that interact with metal ions chelated to a surface (e.g., nickel, cobalt or iron ions)[12], [16]. Other non-covalent tags include silicon binding tags[17], [18], [19], carbohydrate binding modules[20], [21], polystyrene binding peptides[22], leucine zippers[23], [24], [25]. Although non-covalent, these interactions are high avidity within their operation pH range [26].Covalent orthogonal attachment is also possible, often achieved by using an industrially relevant enzyme fused to a second sacrificial enzyme that forms a covalent intermediate during their reaction cycle. This covalent attachment has been demonstrated using enzymes such as a variant haloalkane dehalogenase or esterase with surfaces modified to display suicide inhibitors [27], [28], [29].

Chemical modification of surfaces to enable enzyme immobilization adds additional cost to processes (e.g., continuous flow biocatalysis). Ideally, high avidity, targeted interaction between protein tags/binding domains and unmodified materials would be used in enzyme immobilization, offering a low-cost alternative to modified surfaces [22], [30], [31], [32]. Cellulose and silicon are abundant and inexpensive materials for which binding domains and tags have been identified, such as the silicon binding coat protein from Bacillus cereus (CotB) and carbohydrate binding module (CBMs) from some cellulases [30], [31], [33], [34], [35]

The arginine rich polypeptide CotB1p, isolated from the CotB protein of Bacillus cereus was described as an alternative tag for purification of a recombinant protein [30]. Due to its small size and its high avidity for silica (reported Kd of 1.24 nM), the CotB1p tag has been proposed as a potentially generic tool not only for use as an alternative low-cost method of affinity protein purification, but also for enzyme immobilization [30]. CBMs have also been shown to be suitable candidates for application as affinity tags [36], [37], [38], with dissociation constants for different carbohydrates in the milli- to nanomolar range [39], [40]. Moreover, the available biochemical, functional and structural data for a range of CBMs should facilitate the rational design of enzyme fusions [41].

Here, we investigated CotB1p and a series of CBMs as potential high avidity binding modules in continuous flow biocatalysis. We used fluorescent proteins to characterize their ability to mediate interactions with surfaces suitable for the construction of continuous flow reactors. We focused enzymes that catalyze the synthesis of amine primary and secondary amines, as they are broadly employed in the biocatalytic manufacturing of advanced pharmaceutical intermediates (APIs)[42]. Immobilized amine biocatalytic syntheses have been explored extensively in the literature, employing a range of covalent and non-covalent immobilization methods[12], [43], [44], [45]. In the study presented here, we initially used transaminases from Chromobacterium violaceum and Vibrio fluvialis [46] to investigate the use of these binding tags in a simple (one enzyme) biocatalytic reaction. Later, we used these tags with an NADP(H)-dependent fusion, using imine-reductase from Verrucosispora maris (GenBank accession number: WP_013733165.1)[47] and a cofactor recycling enzyme (glucose dehydrogenase from Bacillus megaterium, GenBank accession number: WP_013084087.1). The performance of immobilized catalytic fusions in repetitive batch and in continuous flow reactions was compared to demonstrate the utility of the CBD immobilization system in biocatalysis.