Heterologous expression and biochemical characterization of a novel cold-active α-amylase from the Antarctic bacteria Pseudoalteromonas sp. 2-3

被引:19
|
作者
Sanchez, Anamaria C. [1 ]
Ravanal, Maria Cristina [2 ]
Andrews, Barbara A. [1 ]
Asenjo, Juan A. [1 ]
机构
[1] Univ Chile, Ctr Biotechnol & Bioengn CeBiB, Dept Chem Engn Biotechnol & Mat, Beauchef 851, Santiago, Chile
[2] Univ Austral Chile, Fac Ciencias Agr, Inst Ciencia & Tecnol Alimentos ICYTAL, Avda Julio Sarrazin S-N, Valdivia, Chile
关键词
alpha-Amylase; Psychrophilic; Antarctic bacteria; Pseudoalteromonas sp. 2-3; Heterologous expression; Starch; SOLID-STATE FERMENTATION; MODERATE HALOPHILE; ASPERGILLUS-NIGER; PURIFICATION; CLONING; ENZYMES; GLYCOSIDE; SEQUENCE; CLASSIFICATION; IDENTIFICATION;
D O I
10.1016/j.pep.2018.11.009
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
alpha-Amylase is an endo-acting enzyme which catalyzes random hydrolysis of starch. These enzymes are used in various biotechnological processes including the textile, paper, food, biofuels, detergents and pharmaceutical industries. The use of active enzymes at low temperatures has a high potential because these enzymes would avoid the demand for heating during the process thereby reducing costs. In this work, the gene of alpha-amylase from Pseudoalteromonas sp. 2-3 (Antarctic bacteria) has been sequenced and expressed in Escherichia coli BL21(DE3). The ORF of the alpha-amylase gene cloned into pET22b(+) is 1824 bp long and codes for a protein of 607 amino acid residues including a His(6)-tag. The mature protein has a calculated molecular mass of 68.8 kDa. Recombinant alpha-amylase was purified with Ni-NTA affinity chromatography. The purified enzyme is active on potato starch with a K-m of 6.94 mg/ml and V-max of 0.27 mg/ml*min. The pH optimum is 8.0 and the optimal temperature is 20 degrees C. This enzyme was strongly activated by Ca2+; results consistent with other alpha-amylases. To the best of our knowledge, this enzyme has the lowest temperature optimum so far reported for alpha-amylases.
引用
收藏
页码:78 / 85
页数:8
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