Enantiomerically pure chiral amines are important intermediates in agrochemical and chemical industry, and play an increasingly important role as chiral blocks for the preparation of pharmaceutical agents, such as (R)-α-phenylethylamine (1-(R)-PEA) and (R)-(+)-1-(1-naphthyl)ethylamine (R-NEA) (Blaser, 2003, Ghislieri and Turner, 2014, Nugent and El-Shazly, 2010, Thie et al., 2008). As a resolving agent, 1-(R)-PEA is used in the optical resolution of p-chloromandelic acid (He et al. 2009). Similarly, as a chiral block, (R)-NEA is commonly used in the synthesis of Cinacalcet Hydrochloride for the treatment of secondary hyperthyroidism and hypercalcemia (Julia and Lesley, 2005, Vázquez et al., 2016). Over the past two decades, biocatalysis has emerged as an attractive alternative to the conventional chemical methods in generating enantiomerically pure chiral amine compounds (Bezborodov and Zagustina, 2016, Huisman and Collier, 2013), by the application of transaminases (Fuchs et al., 2015, Kelly et al., 2020), imine reductases (Leipold et al., 2013, Li et al., 2017), amine dehydrogenases (Franklin et al. 2020), and oxidoreductase (Patil et al. 2018). Among many industrial enzymes, transaminases have the advantages of low-price raw material (isopropyl amine or alanine) and strictly stereoselective chiral amine products, indicating that these are promising biocatalysts with great potential for synthetic applications (Slabu et al., 2017, Rong et al., 2020).

Transaminases are typical of pyridoxal-5’-phosphate (PLP)-dependent enzymes with broad substrate specificity and high enantioselectivity, and the structure were divided into fold I to fold IV. The overall catalytic reaction of transaminase in different structure proceeds through a ping-pong bi-bi mechanism including two half-reactions, catalyzing the oxidative deamination of the amino donor and the reductive ammonization of the amino receptor (Cassimjee et al., 2011, Slabu et al., 2017). Compared with the (S)-selective transaminases, the (R)-selective have been in greater demand for industrial applications in recent years (Gao et al., 2017, Kim et al., 2018, Tang et al., 2019, Voss et al., 2020). This is highlighted by the application of engineered (R)-selective transaminases (ATA117-Rd11) in the biocatalysis process for production of sitagliptin, which is widely used for the treatment of type 2 diabetes mellitus (Guan et al. 2015). While the thermostability of (R)-selective transaminases are not enough to compare with the (S)-selective (Supplemental Table S1). Therefore, improving the thermostability of transaminases through protein engineering has become a research hotspot (Xie et al., 2020, Liu et al., 2021).

Several protein engineering strategies have been successfully applied to redesign the transaminases for enhanced thermostability (Supplemental Table S2), including (i) semi-rational mutagenesis (Buß et al., 2017, Börner et al., 2017, Xie et al., 2019, Meng et al., 2020, Xie et al., 2020, Cao et al., 2021); (ii) rational mutagenesis (Xie et al., 2017, Heckmann et al., 2020); (iii) non-rational mutagenesis (Martin et al. 2007). Recently, synthetic shuffling based on DNA assembly technology has been greatly advanced in protein engineering (Ness et al., 2002, Young, 2004, Zhang et al., 2013, Jin et al., 2016). It generates multiple random combinatorial mutations via reassembling DNA oligonucleotide fragments and introducing abundant diversity mutations at different target sites (Jones et al. 2017). In contrast of disordered recombination of traditional DNA shuffling (Stemmer 1994), synthetic shuffling greatly reduces the possibility of mismatches between primers, which obtains the target mutation library more accurately (Fig. 1). In this study, a directed evolution method of synthetic shuffling was used to modify the thermostability of a transaminase from Aspergillus terreus (At-ATA). The results showed that 30 out of 5000 mutants had improved thermostability after screening, among which mutants with residual enzyme activity higher than 50% at 45 °C for 10 min were selected for further enzyme property analysis. Also, acetophenone and 1-acetonaphthone were used to evaluate the enzyme catalytic ability of the best mutants at high temperature.