Molecular characterization, gene expression analysis and biochemical properties of alpha-amylase from the disk abalone, Haliotis discus discus.
The present study reports the molecular characterization, cloning, expression, and biochemical characterization of alpha-amylase identified from the disk abalone, Haliotis discus discus cDNA library. The full length of the alpha-amylase cDNA was 1650 bp, and it encoded a polypeptide of 511 amino acids. The predicted HdAmyI molecular mass of mature protein was 54 kDa and the estimated isoelectric point (pI) was 8.3. The alpha-amylase gene showed its characteristic motifs, catalytic sites, substrate binding sites and conserved regions with other known species of alpha-amylases. Purified recombinant HdAmyI exhibited a relatively low activity of 0.1 U/mg protein towards 1% starch. HdAmyI had an optimum temperature and pH of 50 degrees C and 6.5, respectively. It also demonstrated stability in a wide range of temperatures and pH. Tissue-specific mRNA expression results showed that HdAmyI is expressed only in the digestive tract and hepatopancreas, with the highest levels in the hepatopancreas. Over 8 weeks of starvation, alpha-amylase transcription was decreased significantly relative to basal levels. However, after starvation, mRNA transcription was increased and returned to normal level by the 2nd week of feeding, suggesting that the alpha-amylase mRNA expression changes according to variations in food availability at the transcriptional level in disk abalone.
Nikapitiya
Chamilani
C
Department of Biotechnology, Cheju National University, 66 Jejudaehakno, Ara-Dong, Jeju, Republic of Korea.
Oh
Chulhong
C
Whang
Ilson
I
Kim
Choong-Gon
CG
Lee
Youn-Ho
YH
Kim
Sang-Jin
SJ
Lee
Jehee
J
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D049851:Gastropoda / Q000201:enzymology* / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D015723:Gene Library; D008969:Molecular Sequence Data; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D014158:Transcription, Genetic / Q000235:genetics; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism*
D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000516:alpha-Amylases