The single mutations that produced the highest glucose specificity were combined, leading to the creation of the S326Q/S365Y double mutant, which was virtually nonreactive to maltose while retaining high glucose dehydrogenase activity.
To assess elongation arrest-dependent decline in the β-galactosidase expression from the fusion protein, we also constructed their arrest-defective versions by introducing the quadruple alanine substitution mutation at the PTC-proximal residues 86 89 (AAAA mutant), which was shown previously to abolish the arrest21.
Hence, the BL21 (DE3) luxS mutant, which was incapable of producing endogenous AI-2, but could bind to exogenous AI-2.
We found that the product yield with respect to substrate was 0.88 in the mutant which was higher when compared to 0.62 for wild strain.
Hence, the BL21 (DE3) luxS mutant, which was incapable of producing endogenous AI-2, was constructed to express the recombinant protein LsrB and eliminate the interference from endogenous AI-2.
Since the BL21∆luxS mutant eliminated endogenous AI-2, the LsrB (BL21∆luxS) mutant, which was overproduced in strain BL21∆luxS, had no AI-2 activity upon denaturation, but could bind to exogenous AI-2.
Furthermore, the pept-1 mutant, which was identified as a low fat mutant in a Nile Red screen [26], actually has very high fat stores.
This phenotype is similar to that in alp5-1134, a histone acetyltransferase component mutant, which was reported to be required for silencing at the central core [56].
We also included as control the NEM316ΔsodA mutant which was previously shown to display increased susceptibility to bacterial killing by macrophages [32].
All mutants did bind 125I-MLT and melatonin with high affinity with the exception of the MT1-I49N mutant, which was devoid of any binding activity.
As discussed above, the UBD located in the C-terminal region is intact in the L157A mutant, which was found ubiquitinated (data not shown).
