title: LaaA, a novel high-active alkalophilic alpha-amylase from deep-sea bacterium Luteimonas abyssi XH031(T).
abstract: 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 XH031(T) and expressed in Escherichia coli BL21. The gene has a length of 1428bp and encodes 475 amino acids with a 35-residue signal peptide. The specific activity of LaaA reached 8881U/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, 1mM Na(+), K(+), and Mn(2+) 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.
authors.affiliation: College of Marine Life Sciences, Ocean University of China, Qingdao, 266003, China.
authors.forename: Qinghao
authors.initials: Q
authors.lastname: Song
authors.affiliation: College of Marine Life Sciences, Ocean University of China, Qingdao, 266003, China. Electronic address: wangy12@ouc.edu.cn.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: College of Marine Life Sciences, Ocean University of China, Qingdao, 266003, China.
authors.forename: Chong
authors.initials: C
authors.lastname: Yin
authors.affiliation: College of Marine Life Sciences, Ocean University of China, Qingdao, 266003, China. Electronic address: xhzhang@ouc.edu.cn.
authors.forename: Xiao-Hua
authors.initials: XH
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012623:Seawater / Q000382:microbiology; D017386:Sequence Homology, Amino Acid; D044166:Xanthomonadaceae / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D000516:alpha-Amylases
keywords: Alkalophilic alpha-amylase; High-active; Luteimonas abyssi; Site-specific mutagenesis