Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 164, Issue -, Pages -Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2019.105478
Keywords
1,4-alpha-glucan branching enzyme; Rhodotherrnus obamensis STB05; Thermostability; pH stability; Specific activity
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Funding
- National Natural Science Foundation of China [31722040, 31771935]
- National First-class Discipline Program of Food Science and Technology [JUFSTR20180204]
- China Postdoctoral Science Foundation [2018M632233]
- Qing Lan Project
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A gene encoding 1,4-alpha-glucan branching enzyme (GBE, EC 2.4.1.18) from the extremely thermophilic bacterium Rhodothermus obamensis STB05 was successfully cloned and expressed in Escherichia colt. Extracellular expression of the recombinant enzyme (R. o-GBE) was achieved with a yield of 1080 mg/L. Then it was purified and further characterized biochemically. R.o-GBE was optimally active at pH 7.0 and 65 degrees C. It remained stable at temperatures up to 80 degrees C and had a half-life at 85 degrees C of approximately 31 min. Far-UV circular dichroism and intrinsic fluorescence analyses revealed that high temperatures reduced its activity by changing the secondary and tertiary structure of R. o-GBE. The enzyme had broad pH stability between pH 3.0 and 11.0 at 4 degrees C, and preferred weakly acidic conditions at high temperatures. None of the metal ions enhanced the activity of R.o-GBE, but Ca2+ may be required for its activity. Its specific activity with amylopectin was 6651 U/mg, which is much higher than that reported for other GBEs. Its excellent thermostability, broad pH stability, and high specific activity make R.o-GBE highly suitable for industrial applications.
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