Heterologous expression of Thermobifida fusca thermostable alpha-amylase in Yarrowia lipolytica and its application in boiling stable resistant sago starch preparation.
A gene encoding the thermostable alpha-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 degrees C for 3 h. The optimal pH and temperature of the purified amylase were 7.0 and 60 degrees 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%.
Yang
Chao-Hsun
CH
Department of Cosmetic Science, Providence University, Taichung 43301, Taiwan. chyang@pu.edu.tw
Huang
Yu-Chun
YC
Chen
Cheng-Yu
CY
Wen
Chia-Ying
CY
D000192:Actinomycetales / Q000201:enzymology*; D003001:Cloning, Molecular; D015870:Gene Expression; D007218:Industrial Microbiology / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D013213:Starch / Q000096:biosynthesis*; D025062:Yarrowia / Q000378:metabolism; D000516:alpha-Amylases / Q000096:biosynthesis* / Q000235:genetics
D011994:Recombinant Proteins; D013213:Starch; D000516:alpha-Amylases