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Cloning, Expression, Purification, and Characterization of Cold-Adapted α-Amylase from Pseudoalteromonas arctica GS230

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Abstract

A cold-adapted α-amylase (ParAmy) gene from Pseudoalteromonas arctica GS230 was cloned, sequenced, and expressed as an N-terminus His-tag fusion protein in E. coli. A recombinant protein was produced and purified with DEAE-sepherose ion exchange chromatography and Ni affinity chromatography. The molecular weight of ParAmy was estimated to be 55 KDa with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). With an optimum temperature for activity 30 °C, ParAmy showed 34.5% of maximum activity at 0 °C and its activity decreased sharply at above 40 °C. ParAmy was stable in the range of pH 7–8.5 at 30 °C for 1 h. ParAmy was activated by Mn2+, K+ and Na+, and inhibited by Hg2+, Cu2+, and Fe3+. N-Bromosuccinimid showed a significant repressive effect on enzyme activity. The K m and V max values of the α-amylase for soluble starch were 7.28 mg/mL and 13.07 mg/mL min, respectively. This research suggests that Paramy has a good potential to be a cold-stable and alkalitolerant amylase in detergent industry.

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Abbreviations

ParAmy:

α-Amylase from Pseudoalteromonas arctica GS230

DEAE:

Diethylaminoethyl cellulos

DTT:

Dithiothreitol

EDTA:

Ethylenediaminetetraacetic acid

LB:

Luria–Bertani

NBS:

N-Bromosuccinimide

Ni-NTA:

Ni2+-nitrilotriacetate

ORF:

Open reading frame

SDS:

Sodium dodecyl sulfate

SDS–PAGE:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

TCA:

Trichloroacetic acid

UV/Vis:

Ultraviolet/visible

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Acknowledgments

This work was financially supported by National Natural Science Foundation of China (40746030), Natural Science Foundation of the Science and Technology Department of Jiangshu (BE200830094,BE2009095), Natural Science Foundation of the Education Department of Jiangshu (09KJA170001).

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Authors and Affiliations

  1. School of Marine Science and Technology, Huaihai Institute of Technology, Lianyungang, Jiangsu, 222005, China

    Mingsheng Lu, Shujun Wang, Yaowei Fang, Shu Liu & Hongfei Liu

  2. School of Bioengineering, Jiangnan University, Wuxi, Jiangsu, 214122, China

    Huangzhong Li & Hongfei Liu

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  1. Mingsheng Lu
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  2. Shujun Wang
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  3. Yaowei Fang
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  4. Huangzhong Li
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Correspondence to Mingsheng Lu.

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Lu, M., Wang, S., Fang, Y. et al. Cloning, Expression, Purification, and Characterization of Cold-Adapted α-Amylase from Pseudoalteromonas arctica GS230. Protein J 29, 591–597 (2010). https://doi.org/10.1007/s10930-010-9290-0

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