Cloning And Characterization Of Three Thermostable ω-transaminases

ω-transaminase belongs to the class III family of transaminases. It is the only kind of transaminase in the five classes which could transfer an amino group from a carbon atom that does not bear a carboxyl group. Chiral amines and unnatural amino acids, including β-aminoacids have been widely used in diverse sectors such as the pharmaceutical, chemical, cosmetic, food, and agricultural industries. Synthesis of chiral amines through chemical methods are normally expensive and unenvironment-friendly. Biocatalysis provides us a new way to produce these unnatural chrial amines.In this ariticle, protein sequences of three new thermostable putative co-transaminases were found out through BLAST and functionally expressed in Escherichia coli BL21 (DE3). The purified recombinant N-terminal His-tagged-TA had a dimeric structure with optimum pH and temperature of 9.0 and 65 degrees, respectively. The enzymes have excellent thermostability that lead to no activities-decreasing after incubate under 50 degrees for more than 8 h. Km values show these three enzymes are all taurine transaminase, a subgroup of co-transaminase. Substrate spectrum show that these three enzymes are all S seletive. Racemate amines don’t cause substrate inhibition in the experiment. According to the two bindng-pocket model of co-TA, the S pocket of the three enzymes have a steric constraint which could only accept a group that is not larger than an ethyl group. Amimo acceptors which bear a hydroxyl group on the a-position to the carbonyl carbon show much high activities when compared to acceptors without hydroxyl group. The reactivity of amino acceptor appears to be determined by the degree of susceptibility of the carbonyl carbon to nucleophilic attack by the amino group of PMP.