Tuning the catalytic performances of a sucrose isomerase for production of isomaltulose with high concentration
Applied Microbiology and Biotechnology(2022)
摘要
Obtaining a sucrose isomerase (SIase) with high catalytic performance is of great importance in industrial production of isomaltulose (a reducing sugar). In order to obtain such SIase mutant, a high-throughput screening system in microtiter plate format was developed based on a widely used 2,4-dinitrosalicylic acid (DNS) method for determination of reducing sugar. An SIase from Erwinia sp. Ejp617 ( Er SIase) was selected to improve its catalytic efficiency. After screening of ~ 8000 mutants from a random mutagenesis library, Q209 and R456 were identified as beneficial positions. Saturation mutagenesis of the two positions resulted in a double-site mutant Er SIase_Q209S-R456H that showed the highest catalytic efficiency, and its specific activity reached 684 U/mg that is 17.5-fold higher than that of the wild-type Er SIase. By employing the lyophilized Escherichia coli ( E. coli ) cells harboring Er SIase_Q209S-R456H, a high space–time yield (STY = 3.9 kg/(L·d)) was achieved toward 600 g/L sucrose. Furthermore, the in silico analysis suggested that the hydrogen bond network was improved and steric hindrance was reduced due to the beneficial substitutions. Key points • A sucrose isomerase mutant with high catalytic efficiency was obtained . • The highest space–time yield was achieved toward high-concentration sucrose . • The optimized H-bond network contributed to the enhanced catalytic efficiency .
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关键词
Sucrose isomerase, Isomaltulose, Direct evolution, Protein engineering
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