Chiral amines are the key intermediates of various pharmaceuticals [1], [2], [3], and approximately 40% of chiral drugs contain chiral amine functional groups [4]. (R)-(+)− 1-(1-naphthyl)ethylamine ((R)-NEA) is an extremely important chiral amine building block in the synthesis of Cinacalcet Hydrochloride, which is a pharmaceutical used to treat secondary hyperthyroidism and hypercalcemia [5], [6]. The synthesis of (R)-NEA by amine transaminases (ATAs) is practically useful (Scheme 1) due to its mild reaction conditions, high atom economy, excellent chemo-, regio-selectivity and sustainability [7], [8], [9].

ATAs (EC 2.6.1. X) are pyridoxal 5′-phosphate (PLP)-dependent enzymes that are capable of catalyzing the asymmetric amination from prochiral ketones to chiral amines with strict stereoselectivity and 100% theoretical yield [10], [11]. ATAs are divided into fold types I to VII, and ω-ATAs belong to the fold-type IV superfamily [11], [12]. Although various ω-ATAs exhibit potential performance in the synthesis of chiral amines, the activity, stability, and recyclability of enzymes remain key factors in their application [13]. In the catalytic application of ω-ATAs, organic solvents are often added to aid in the dissolution of unnatural organic substrates. However, the use of organic solvents can easily lead to the deactivation of ω-ATAs, which will increase the cost of enzymes [14], [15], [16]. In addition, if enzymes and other cellular impurities cannot be effectively separated from the reaction system, it can also lead to severe emulsification during product extraction, increasing the difficulty of product separation, purification, and reducing product purity [17]. Therefore, there is an urgent demand to develop a strategy for enhancing the thermostability, organic solvents tolerance and reusability.

Compared with free enzymes, immobilized enzymes exhibit favorable advantages in industrial application [18], [19], such as high operation stability, feasible immobilized enzyme recycling, improved stability, accelerated separation and purification [20]. Especially for enzymatic reactions in organic phases, it is particularly important to improve the organic solvents tolerance of enzymes through immobilization [21], [22]. Various methods, include embedding, covalent attachment, and physical adsorption are the main methods to immobilize enzymes on the solid supports [23]. Unfortunately, free enzymes are easy to leach out from the solid supports via physical adsorption [24]. For embedding method, the enzyme activity would be decreased due to the mass transfer resistance [24], [25]. In covalent attachment method, introducing functional coupling groups for surface modification may inevitably influence the enzyme’s conformation and cause extra costs and environmental pollution [24], [26]. Therefore, it is necessary to establish a new immobilization method to enhance enzyme’s performances via combining the advantages of different methods and overcoming their shortcomings.

Recently, the application of multi-walled carbon nanotubes (MWCNTs) has been used wildly and MWCNTs has emerged as a new type of nanomaterials for immobilization. MWCNTs are nanoscale circular tubes, which are formed by rotating multiple layers of graphite sheets at a certain angle along the central axis [27]. MWCNTs are molecular porous materials with strong adsorption capacity, large specific surface area, good stability, favorable biocompatibility, and controllable surface functional groups [19], [28]. MWCNTs can protect enzymes against harsh conditions, while at the same times allow the transfer of small-molecule substrates/products owing to their high chemical and structural stability [29]. Covalent binding using MWCNTs as the carriers is the mainly immobilization method, which utilizes controllable functional groups on the surface of MWCNTs to form covalent bonds with amino acid residues on the protein surface, such as amino, carboxyl, serine, and threonine hydroxyl groups [30], [31]. Tiwari et al. [32] used chitosan-SiO2-multiwall carbon nanotubes nanocomposite as the carrier to immobilize creatine amidinohydrolase, and the enzyme could be stored for 8 months at 4 °C. Ke et al. [33] improved enzyme activity and enantioselectivity of Burkholderia cepacia lipase via adsorption on modified MWCNTs. Although there have been many advances in the immobilization of enzymes using MWCNTs, their performance as biocatalysts is still relatively weak.

In our previous work, we have cloned a gene of ω-ATA from Aspergillus terreus, and a mutant D224K/V149A/L182F/L187F (designated as AtATA) was acquired [34]. AtATA as a biocatalyst could convert 20 mmol·L–1 1-acetonaphthone to the desired product ((R)-NEA) with 78% conversion and high enantiomeric purity (e.e.p > 99.5% R) within 7 h. Herein, we develop a combinatorial immobilization technique for AtATA, which is based on amino modified multi-walled carbon nanotubes (MWCNTs-NH2) adsorption and glutaraldehyde (GA) cross-linking (Fig. 1). The enzymatic properties and catalytic performance toward 1-acetonaphthone in stirring reactor (STR) of the prepared immobilized enzyme are investigated.