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Protein Expr Purif. 2019 Dec;164:105478. doi: 10.1016/j.pep.2019.105478. Epub 2019 Aug 14.

Expression and characterization of an extremely thermophilic 1,4-α-glucan branching enzyme from Rhodothermus obamensis STB05.

Author information

1
School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, PR China.
2
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, PR China; School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, PR China; Collaborative Innovation Center of Food Safety and Quality Control, Jiangnan University, Wuxi, Jiangsu, 214122, PR China.
3
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, PR China; School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, PR China.
4
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, PR China; School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, PR China; Collaborative Innovation Center of Food Safety and Quality Control, Jiangnan University, Wuxi, Jiangsu, 214122, PR China. Electronic address: zfli@jiangnan.edu.cn.

Abstract

A gene encoding 1,4-α-glucan branching enzyme (GBE, EC 2.4.1.18) from the extremely thermophilic bacterium Rhodothermus obamensis STB05 was successfully cloned and expressed in Escherichia coli. 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 °C. It remained stable at temperatures up to 80 °C and had a half-life at 85 °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 °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.

KEYWORDS:

1,4-α-glucan branching enzyme; Rhodothermus obamensis STB05; Specific activity; Thermostability; pH stability

PMID:
31421223
DOI:
10.1016/j.pep.2019.105478

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