Identification, Characterization, and Site-Specific Mutagenesis of a Thermostable ω-Transaminase from Chloroflexi bacterium.

In the present study, we have identified an ω-transaminase (ω-TA) from Chloroflexi bacterium from the genome database by using two ω-TA sequences (ATA117 Arrmut11 from Arthrobacter sp. KNK168 and amine transaminase from Aspergillus terreus NIH2624) as templates in a BLASTP search and motif sequence alignment. The protein sequence of the ω-TA from C. bacterium (CbTA) shows 38% sequence identity to that of ATA117 Arrmut11. The gene sequence of CbTA was inserted into pRSF-Duet1 and functionally expressed in Escherichia coli BL21(DE3). The results showed that the recombinant CbTA has a specific activity of 1.19 U/mg for (R)-α-methylbenzylamine [(R)-MBA] at pH 8.5 and 45 °C. The substrate acceptability test showed that CbTA has significant reactivity to aromatic amino donors and amino receptors. More importantly, CbTA also exhibited good affinity toward some cyclic substrates. The homology model of CbTA was built by Discovery Studio, and docking was performed to describe the relative activity toward some substrates. CbTA evolved by site-specific mutagenesis and found that the Q192G mutant increased the activity to (R)-MBA by around 9.8-fold. The Q192G mutant was then used to convert two cyclic ketones, N-Boc-3-pyrrolidinone and N-Boc-3-piperidone, and both the conversions were obviously improved compared to that of the parental CbTA.