Identification of archaeon-producing hyperthermophilic alpha-amylase and characterization of the alpha-amylase.
The extremely thermophilic anaerobic archaeon strain, HJ21, was isolated from a deep-sea hydrothermal vent, could produce hyperthermophilic alpha-amylase, and later was identified as Thermococcus from morphological, biochemical, and physiological characteristics and the 16S ribosomal RNA gene sequence. The extracellular thermostable alpha-amylase produced by strain HJ21 exhibited maximal activity at pH 5.0. The enzyme was stable in a broad pH range from pH 5.0 to 9.0. The optimal temperature of alpha-amylase was observed at 95 degrees C. The half-life of the enzyme was 5 h at 90 degrees C. Over 40% and 30% of the enzyme activity remained after incubation at 100 degrees C for 2 and 3 h, respectively. The enzyme did not require Ca(2+) for thermostability. This alpha-amylase gene was cloned, and its nucleotide sequence displayed an open reading frame of 1,374 bp, which encodes a protein of 457 amino acids. Analysis of the deduced amino acid sequence revealed that four homologous regions common in amylases were conserved in the HJ21 alpha-amylase. The molecular weight of the mature enzyme was calculated to be 51.4 kDa, which correlated well with the size of the purified enzyme as shown by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Wang
Shujun
S
College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, People's Republic of China.
Lu
Zhaoxin
Z
Lu
Mingsheng
M
Qin
Song
S
Liu
Hongfei
H
Deng
Xiangyuan
X
Lin
Qian
Q
Chen
Jianan
J
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D019641:DNA, Archaeal / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012623:Seawater / Q000382:microbiology*; D016415:Sequence Alignment; D013696:Temperature; D019712:Thermococcus / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification*; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D000516:alpha-Amylases