Effect of metal ions on the activity and stability of recombinant cold-adapted α-amylase LamA from Luteimonas abyssi

Abstract:

[Background] Luteimonas abyssi XH031 is one kind of marine bacteria with strong starch degradation ability. Previous studies showed that LamA, a cold-adapted α-amylase identified from strain XH031, kept high activity under low temperature. LamA will have great application prospects if the high temperature tolerance is improved. [Objective] To determine the calcium ion-dependent thermo-stability enhancement mechanism of LamA, site-directed mutagenesis of key amino acids in the calcium ion binding region was constructed. [Methods] The thermo-stability of LamA was measured in the presence of different chemicals. The amino acid sites that affect calcium ion binding and thermal stability were searched by bioinformatics analysis. Furthermore, the mutant proteins were constructed by the site-directed mutagenesis method and then overexpressed and purified. [Results] Under the calcium-free conditions, LamA was completely inactivated after incubation at 65 °C for 30 minutes. However, in the presence of 5 mmol/L calcium ion, LamA still had 36% of the activity after incubation at 65 °C for 30 min. The result showed that calcium ions can significantly improve the thermo-stability of LamA. The D200R and H233D/M234C mutant proteins completely lost starch degradation activities. Moreover, the activities of N120D, Q185E and T224D mutant proteins were decreased. However, the mutant proteins kept similar stability compared with the wild-type enzyme under the high temperature and calcium-free conditions. N120D mutant protein preserved only 27.8% residual activity compared with the wild-type enzyme at 65 °C supplemented with the calcium ion. Through molecular biology experiments and protein structure simulation, we speculated that the calcium ion binding to the Asn120 site stabilized the structure of LamA under high temperatures. [Conclusion] This study preliminarily clarified the mechanism of calcium ion-dependent thermo-stability enhancement of the cold-adapted α-amylase LamA and provided a theoretical basis for the engineering transformation of related enzymes.