[Expression of acidophilic alpha-amylase from Alicyclobacillus acidocaldarius].
The alpha-amylase (EC 3.2.1.1) from the Gram-positive Alicyclobacillus acidocaldarius was one kind of thermoacidophilic enzyme, with optimal temperature and pH of 75 degrees C and 3, respectively. The nucleotide sequence of the gene amy was cloned by PCR. The gene amy was 3901bp long, comprising one open reading frame encoding a polypeptide of 1301 amino acids. The calculated molecular weight of the alpha-amylase AMY was about 140kD. The gene amy was expressed in E. coli BL21 (DE3) and Pichia pastoris respectively, and both of the cloned proteins had bioactivity. The activity of amylase expressed in P. pastoris was further testified by amylase activity staining. The alpha-amylase expressed in P. pastoris had been purified and characterized. The apparent molecular weight of that was about 160kD according to SDS-PAGE. The optimum of pH for the enzyme was pH 3.2 as the native enzyme was; but the optimum of temperature was 65 degrees C and a little lower than that of the native enzyme. Above 50% of relative activity remained after incubation for 30 minutes in 70 degrees C. So the enzyme expressed by P. pastoris was also thermoacidophilic. Moreover some sequence was cloned by PCR, which ranged from + 1174 bp to + 3288 bp in the gene amy, encoding 705 amino acids with the calculated molecular weight of 79kD. The truncated gene amy' was expressed in E. coli BL21 (DE3) induced by 1 mmol/L IPTG, and the expressed enzyme also retained alpha-amylase activity.
Yuan
Tie-Zheng
TZ
Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Yao
Bin
B
Luo
Hui-Ying
HY
Wang
Ya-Ru
YR
Wu
Ning-Feng
NF
Fan
Yun-Liu
YL
D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D000516:alpha-Amylases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D000516:alpha-Amylases