Heterologous expression of <Emphasis Type="Italic">Thermobifida fusca</Emphasis> thermostable alpha-amylase in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> and its application in boiling stable resistant sago starch preparation |
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Authors: | Chao-Hsun Yang Yu-Chun Huang Cheng-Yu Chen Chia-Ying Wen |
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Institution: | (1) Department of Cosmetic Science, Providence University, Taichung, 43301, Taiwan |
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Abstract: | A gene encoding the thermostable α-amylase in Thermobifida fusca NTU22 was amplified by PCR, sequenced, and cloned into Yarrowia lipolytica P01g host strain using the vector pYLSC1 allowing constitutive expression and secretion of the protein. Recombinant expression
resulted in high levels of extracellular amylase production, as high as 730 U/l in the Hinton flask culture broth. It is higher
than that observed in P. pastoris expression system and E. coli expression system. The purified amylase showed a single band at about 65 kDa by SDS-polyacrylamide gel electrophoresis and
this agrees with the predicted size based on the nucleotide sequence. About 70% of the original activity remained after heat
treatment at 60°C for 3 h. The optimal pH and temperature of the purified amylase were 7.0 and 60°C, respectively. The purified
amylase exhibited a high level of activity with raw sago starch. After 72-h treatment, the DP
w
of raw sago starch obviously decreased from 830,945 to 237,092. The boiling stable resistant starch content of the sago starch
increased from 8.3 to 18.1%. The starch recovery rate was 71%. |
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