Expression of Thermobifida fusca thermostable raw starch digesting alpha-amylase in Pichia pastoris and its application in raw sago starch hydrolysis.
A gene encoding the thermostable raw starch digesting alpha-amylase in Thermobifida fusca NTU22 was amplified by PCR, sequenced and cloned into Pichia pastoris X-33 host strain using the vector pGAPZalphaA, allowing constitutive expression and secretion of the protein. Recombinant expression resulted in high levels of extracellular amylase production, as high as 510 U/l in the Hinton flask culture broth. The purified amylase showed a single band at about 65 kDa by SDS-polyacrylamide gel electrophoresis after being treated with endo-beta-N-acetylglycosaminidase H, and this agrees with the predicted size based on the nucleotide sequence. About 75% 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 48-h treatment, the DPw of raw sago starch obviously decreased from 830,945 to 378,732. The surface of starch granules was rough, and some granules displayed deep cavities.
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* / Q000235:genetics; D003001:Cloning, Molecular; D029784:Cycas / Q000737:chemistry; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics*; D055550:Protein Stability; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D017422:Sequence Analysis, DNA; D013213:Starch / Q000378:metabolism*; D000516:alpha-Amylases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
D004271:DNA, Fungal; D005656:Fungal Proteins; D011994:Recombinant Proteins; D013213:Starch; D000516:alpha-Amylases