LaaA, a novel high-active alkalophilic alpha-amylase from deep-sea bacterium Luteimonas abyssi XH031T

• LaaA is the first reported alkophilic alpha-amylase from the deep-sea genus Luteimonas.
• As a novel alkophilic alpha-amylase, the activity can reach upto 8881 U/mg which is considerable higher than other reported alpha-amylase.
• LaaA possesses higher active in low temperature.
• Moreover, we found the Arg227, Asp229, Glu256, and Asp328 are total conserved and essential for the activity of alpha-amylase LaaA by site-directed mutagenesis.
• It’s a potential alkali-tolerant and high-active amylase used in detergent industry.

Alpha-amylase is a kind of broadly used industrial enzymes, most of which have been exploited from terrestrial organism. Comparatively, alpha-amylase from marine environment was largely undeveloped. In this study, a novel alkalophilic alpha-amylase with high activity, Luteimonas abyssi alpha-amylase (LaaA), was cloned from deep-sea bacterium L. abyssi XH031T and expressed in Escherichia coli BL21. The gene has a length of 1428 bp and encodes 475 amino acids with a 35-residue signal peptide. The specific activity of LaaA reached 8881 U/mg at the optimum pH 9.0, which is obvious higher than other reported alpha-amylase. This enzyme can remain active at pH levels ranging from 6.0 to 11.0 and temperatures below 45 °C, retaining high activity even at low temperatures (almost 38% residual activity at 10 °C). In addition, 1 mM Na+, K+, and Mn2+ enhanced the activity of LaaA. To investigate the function of potential active sites, R227G, D229K, E256Q/H, H327V and D328V mutants were generated, and the results suggested that Arg227, Asp229, Glu256 and Asp328 were total conserved and essential for the activity of alpha-amylase LaaA. This study shows that the alpha-amylase LaaA is an alkali-tolerant and high-active amylase with strong potential for use in detergent industry.

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