Co-expression of the Thermotoga neapolitana aglB gene with an upstream 3'-coding fragment of the malG gene improves enzymatic characteristics of recombinant AglB cyclomaltodextrinase.

Abstract A cluster of Thermotoga neapolitana genes participating in starch degradation includes the malG gene of sugar transport protein and the agl B gene of cyclomaltodextrinase. The start and stop codons of these genes share a common overlapping sequence, a TGA tg. Here, we compared properties of expression products of three different constructs with agl B from T. neapolitana . The first expression vector contained the agl B gene linked to an upstream 90-bp 3′-terminal region of the mal G gene with the stop codon overlapping with the start codon of agl B. The second construct included the isolated coding sequence of agl B with two tandem potential start codons. The expression product of this construct in Escherichia coli had two tandem Met residues at its N terminus and was characterized by low thermostability and high tendency to aggregate. In contrast, co-expression of agl B and the 3′-terminal region of mal G (the first construct) resulted in AglB with only one N-terminal Met residue and a much higher specific activity of cyclomaltodextrinase. Moreover, the enzyme expressed by such a construct was more thermostable and less prone to aggregation. The third construct was the same as the second one except that it contained only one ATG start codon. The product of its expression had kinetic and other properties similar to those of the enzyme with only one N-terminal Met residue.