Identification, Characterization, and Site-Specific Mutagenesis of a Thermostable ω-Transaminase from <i>Chloroflexi bacterium</i>

Abstract

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