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| High-throughput Screening of Benzoate Decarboxylase for High-efficiency Fixation of CO2 Based on Spectroscopy-image Grayscale Method |
FAN Yan1,2,YANG Miao1,XUE Song3,**( ) |
1 Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
2 University of Chinese Academy of Sciences, Beijing 100049, China
3 School of Bioengineering, Dalian University of Technology, Dalian 116024, China |
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Abstract Objective: Benzoic acid decarboxylase can catalyze the carboxylation reaction to fix CO2. In order to obtain a benzoate decarboxylase that can efficiently fix CO2, it is required to screen a large number of mutants based on the high-throughput molecular cloning and mutant screening systems. Thus, it is essential to develop an efficient screening-evaluation method for obtaining mutants with high-efficiency of carboxylation. Methods: 2,3-dihydroxybenzoic acid decarboxylase catalyzes catechol with CO2 to produce 2,3-dihydroxybenzoic acid. A spectrometry-image grayscale method was established to high-throughput screen and evaluate the activities of mutants. The concentration of 2,3-dihydroxybenzoic acid was quickly quantified by spectrophotometry at 308 nm. Further, the spectrophotometric results were corrected using the high performance liquid chromatography (HPLC) method. The 2,3-dihydroxybenzoic acid concentration by the spectroscopy method was linearly correlated with the accurate concentration determined by the HPLC method (R2 = 0.996). The 2,3-dihydroxybenzoic acid concentration of the actual sample was determined by the correlation of HPLC-spectrometry. The grayscale average of protein standards and mutants were obtained by Image J software. The protein expression level of mutants was calculated by the standard curve using the grayscale method. The enzyme activities of mutants were compared based on the 2,3-dihydroxybenzoic acid concentration. Results: The 2,3-dihydroxybenzoic acid concentration was quantified by the absorbance value using linear equation C1=0.500A1-0.010 (R 2=0.996, purified enzyme) and C2=1.458A2+0.431 9 (R 2 =0.991, crude enzyme). Two mutants were screened out from 13 mutants, which carboxylation activities were 3.5-fold and 1.7-fold of WT, respectively. Conclusion: The high-throughput screening of benzoate decarboxylase for fixing CO2 can be achieved by the spectroscopy-image grayscale method. This method is suitable for screening of substrate selectivity, i.e. phenols with other substituents and salicylic acid analogs, which is catalyzed by benzoate decarboxylase with similar functions.
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Received: 14 July 2021
Published: 01 December 2021
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Corresponding Authors:
Song XUE
E-mail: xuesong@dlut.edu.cn
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