TY - JOUR
T1 - A 4-α-Glucanotransferase from Thermus thermophilus HB8
T2 - Secretory Expression and Characterization
AU - Wan, Huihui
AU - Ouyang, Xiaoying
AU - Yang, Ting
AU - Ye, Tianyun
AU - Jin, Minfei
AU - Huang, Jing
N1 - Publisher Copyright:
© 2022, The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.
PY - 2022/7
Y1 - 2022/7
N2 - 4-α-glucanotransferase (4GT, EC 2.4.1.25) catalyzes the breakdown of the α-1,4 glycosidic bonds of the starch main chain and forms new α-1,4 glycosidic bonds in the side chain, which is often used to optimize the physical and chemical properties of starch and to improve the quality of starch-based food. However, the low enzyme activity of 4GT limits its production and widespread application. Herein, the 4GT gene encoding 500 amino acids from Thermus thermophilus HB8 was cloned and expressed in Escherichia coli. The purified 4GT exhibited maximum activity at pH 7.0 and 60 °C and had a good stability at pH 6.0–8.0 and 30–60 °C. It was confirmed that 4GT possessed the catalytic function of extending the branch length of potato starch. Furthermore, the 4GT gene was successfully expressed extracellularly in Bacillus subtilis. Then, the enzyme yield of 4GT increased by 4.1 times through screening of different plasmids and hosts. Additionally, the fermentation conditions were optimized to enhance 4GT extracellular enzyme yield. Finally, a recombinant Bacillus subtilis with 299.9 U/mL enzyme yield of 4GT was obtained under the optimized fermentation process. In conclusion, this study provides a valuable reference for characterization and expression of food-grade enzymes.
AB - 4-α-glucanotransferase (4GT, EC 2.4.1.25) catalyzes the breakdown of the α-1,4 glycosidic bonds of the starch main chain and forms new α-1,4 glycosidic bonds in the side chain, which is often used to optimize the physical and chemical properties of starch and to improve the quality of starch-based food. However, the low enzyme activity of 4GT limits its production and widespread application. Herein, the 4GT gene encoding 500 amino acids from Thermus thermophilus HB8 was cloned and expressed in Escherichia coli. The purified 4GT exhibited maximum activity at pH 7.0 and 60 °C and had a good stability at pH 6.0–8.0 and 30–60 °C. It was confirmed that 4GT possessed the catalytic function of extending the branch length of potato starch. Furthermore, the 4GT gene was successfully expressed extracellularly in Bacillus subtilis. Then, the enzyme yield of 4GT increased by 4.1 times through screening of different plasmids and hosts. Additionally, the fermentation conditions were optimized to enhance 4GT extracellular enzyme yield. Finally, a recombinant Bacillus subtilis with 299.9 U/mL enzyme yield of 4GT was obtained under the optimized fermentation process. In conclusion, this study provides a valuable reference for characterization and expression of food-grade enzymes.
UR - https://www.scopus.com/pages/publications/85130336333
U2 - 10.1007/s00284-022-02856-y
DO - 10.1007/s00284-022-02856-y
M3 - 文章
C2 - 35604453
AN - SCOPUS:85130336333
SN - 0343-8651
VL - 79
JO - Current Microbiology
JF - Current Microbiology
IS - 7
M1 - 202
ER -