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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2019, Vol. 39 Issue (5): 96-104    DOI: 10.13523/j.cb.20190511
    
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.

Key words： β-CGTase      Error-prone PCR      Molecular modification      Affinity      Trehalose conversion     
Received: 08 October 2018      Published: 04 June 2019
ZTFLH:  Q819  
Corresponding Authors: Jing WU     E-mail: jingwu@jiangnan.edu.cn
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Li DU
Ling-qia SU
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Cite this article:

Li DU,Ling-qia SU,Jing WU. Enhancing Maltose Affinity of Bacillus circulans 251 β-CGTase and its Application in Trehalose Preparation. China Biotechnology, 2019, 39(5): 96-104.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20190511     OR     https://manu60.magtech.com.cn/biotech/Y2019/V39/I5/96

Mutant Mutated nucleotide bases Mutated amino acids
H-7 T624C /G702A/ C1110T M234I
Table 1 Sequencing results of the mutant
Fig.1 SDS-PAGE analysis of wild-type and the mutant M:Protein marker (high); 1:Purified wild type β-CGTase; 2:Purified mutant M234I
Enzyme Purification steps Total
protein(mg)		 Total
activity(U)		 Specific
activity(U/mg)		 Recovery
rate(%)		 Purification
fold
M234I β-CGTase Crude extract 329.5 10 972.3 33.30 100.0 1.00
Salt precipitation 151.1 7 768.4 50.41 70.8 1.51
Mono Q 10/100 GL 2.8 966.7 345.25 12.4 10.37
Wild-type β-CGTase Crude extract 401.1 14 086.6 35.12 100.0 1.00
Salt precipitation 179.1 10 621.3 59.30 75.4 1.69
Mono Q 10/100 GL 8.6 3 075.6 357.63 28.9 10.05
Table 2 The protein purified results of wild-type and the mutant (M234I) β-CGTase
Enzyme kcat
(/s)		 Km
(mmol/L)		 kcat/Km
[mmol/(L·s)]
Wild type 488.7 0.474 9 1 029.2
M234I 453.2 0.258 2 1 755.4
Table 3 Kinetic parameters of wild-type and the mutant M234I β-CGTase
Fig.2 The optimum temperature of wild-type and the mutant M234I β-CGTase
Fig.3 The stability of wild-type and the mutant M234I β-CGTase over temperature (a)45℃ (b)50℃ (c)55℃ (d) 60℃
Fig.4 The optimum pH of wild-type and the mutant M234I β-CGTase
Fig.5 The stability of wild-type and the mutant M234I β-CGTase over pH
Fig.6 Trehalose yield under different enzyme dosages
Enzyme Glucose
(g/L)		 Maltose
(g/L)		 Trehalose
(g/L)
Wild type 18.1 28.1 138.3
M234I 19.2 23.5 142.6
Table 4 Glucose, maltose and trehalose content in the reaction system before saccharification
Fig.7 The average structure of the wild-type and mutant M234I β-CGTase kinetic trajectories,the green part is the wild type loop region, and the blue part is the loop region of the mutant M234I
Fig.8 Model of B. circulans 251 CGTase variants containing a docked structure of maltose (a)M234I (b)Wild type
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