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Enhancing Maltose Affinity of Bacillus circulans 251 β-CGTase and its Application in Trehalose Preparation |
Li DU1,2,3,Ling-qia SU1,2,3,Jing WU1,2,3,**() |
1 State Key Laboratory of Food Science and Technology, Jiangnan University,Wuxi 214122,China 2 School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, Wuxi 214122, China 3 International Joint Laboratory on Food Safety,Jiangnan University,Wuxi 214122,China |
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Abstract B. circulans 251 β-CGTase was applied to trehalose preparation, and the trehalose yield was increased from 50.4% to 71.9%. In order to further improve the conversion rate of substrates, B. circulans 251 β-CGTase mutants with improved affinity for maltose as a disproportionation acceptor were screened thorugh error-prone PCR and high-throughput screening. Mutant M234I with higher affinity for maltose was selected with a low concentration of 4,6-ethylidene-p-nitrophenyl-indole-D-maltoheptaose (EPS) chromogenic method. The wild-type β-CGTase and the mutant enzyme M234I were purified, and the enzymatic properties were characterized. The specific activity of the mutant M234I was 345.25U/mg (disproportionation activity), while that of the wild type was 357.63U/mg. The maltose Km vaule of the mutant M234I was 0.258 2mmol/L, which was only 54.4% of the wild type (0.474 9mmol/L), which indicates that its affinity for maltose was significantly improved. The optimum temperature and the optimum pH of the mutant did not change much compared with those of the wild type. The mutant M234I was used in the multi-enzyme complex system to produce trehalose with maltodextrin (DE 16) as substrate, the result showed that the trehalose yield was up to 74.9%, which was 3% higher than that of the wild-type β-CGTase.
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Received: 08 October 2018
Published: 04 June 2019
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Corresponding Authors:
Jing WU
E-mail: jingwu@jiangnan.edu.cn
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