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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2021, Vol. 41 Issue (11): 55-63    DOI: 10.13523/j.cb.2107034
技术与方法     
基于光谱法-图像灰度法高通量筛选高效固定CO2的苯甲酸脱羧酶*
范雁1,2,杨淼1,薛松3,**()
1 中国科学院大连化学物理研究所 大连 116023
2 中国科学院大学 北京 100049
3 大连理工大学生物工程学院 大连 116022
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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摘要：

目的:苯甲酸脱羧酶能够催化羧化反应固定CO2,为了获得高效的苯甲酸脱羧酶,需要利用高通量分子克隆与突变体筛选系统对产生的大量突变体进行筛选,因此建立、开发高效的筛选评价方法对于获得高羧化效率的突变体至关重要。方法:利用2,3-二羟基苯甲酸脱羧酶催化邻苯二酚固定CO2的反应体系,建立了光谱法-图像灰度法高通量筛选和评价固定CO2的苯甲酸脱羧酶突变体。利用分光光度法在308 nm快速定量羧化产物2,3-二羟基苯甲酸。同时利用高效液相色谱(HPLC)法对分光光度法的测定结果进行了校正,确定了分光光度法估算的2,3-二羟基苯甲酸浓度与HPLC方法测定的准确浓度之间具有良好的线性关系(R2 = 0.996)。利用HPLC-分光光度法的相关性可以获得实际样品中准确的2,3-二羟基苯甲酸浓度。利用Image J软件获得蛋白质标准品和突变体的灰度均值,根据灰度法定量蛋白质标准品的标准曲线计算突变体的蛋白质表达量。利用单位酶量催化邻苯二酚获得2,3-二羟基苯甲酸的浓度比较突变体的催化活性。结果:纯酶和粗酶体系下HPLC法测定的2,3-二羟基苯甲酸浓度与分光光度法测定的吸光度值的关系分别为C1=0.500A1-0.010(R2 = 0.996)和C2=1.458A2+0.431 9(R2 = 0.991)。从13个突变体中获得了两个正向突变体,羧化活性分别是WT的3.5倍和1.7倍。结论:基于光谱法-图像灰度法可以实现高通量筛选固定CO2的苯甲酸脱羧酶,该方法可用于具有相似功能的苯甲酸脱羧酶对其他取代基的酚类和水杨酸类似物的底物选择性筛选。
关键词： 光谱法-图像灰度法;  CO2固定;  2;  3-二羟基苯甲酸;  高通量筛选;  苯甲酸脱羧酶    
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.
Key words: Spectroscopy-image grayscale method    CO2 fixation    2    3-dihydroxybenzoic acid    High-throughput screening    Benzoate decarboxylase
收稿日期: 2021-07-14 出版日期: 2021-12-01
ZTFLH:  Q819  
基金资助: * 国家自然科学基金面上项目(21877110)
通讯作者: 薛松     E-mail: xuesong@dlut.edu.cn
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引用本文:

范雁,杨淼,薛松. 基于光谱法-图像灰度法高通量筛选高效固定CO2的苯甲酸脱羧酶*[J]. 中国生物工程杂志, 2021, 41(11): 55-63.

FAN Yan,YANG Miao,XUE Song. High-throughput Screening of Benzoate Decarboxylase for High-efficiency Fixation of CO2 Based on Spectroscopy-image Grayscale Method. China Biotechnology, 2021, 41(11): 55-63.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2107034    或    https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I11/55

图1  全波长扫描
图2  分光光度法测定2,3-DHBA标准曲线的绘制
图3  混合标准品的全波长扫描
图4  羧化反应体系的全波长扫描
图5  分光光度法和HPLC法测定样品2,3-DHBA浓度的关系
样品 样品信息 2,3-DHBA浓度/(mmol/L) 相对偏差/%
酶量 /μg 反应时间/min 分光光度法计算值a HPLC法实际测定值b
1 50 10 1.10 ± 0.04 1.07 ± 0.04 2.8
2 50 20 1.30 ± 0.09 1.40 ± 0.05 7.0
3 200 10 1.72 ± 0.02 2.01 ± 0.09 14.2
4 200 15 2.34 ± 0.07 2.70 ± 0.11 13.3
表1  HPLC法和分光光度法测定不同样品中2,3-DHBA浓度的结果对比
图6  粗酶体系下分光光度法和HPLC法测定2,3-DHBA浓度的关系
图7  SDS-PAGE图
图8  2,3-DHBD_Ao蛋白标准品灰度均值的标准曲线
图9  不同取代基的酚和水杨酸类似物的全波长扫描
样品
名称		 2,3-DHBA浓度
/(mmol/L)		 灰度
均值		 蛋白质含量
/g		 酶活/[mmol/
(L·g)]
WT 3.21 34 752 0.36 8.94
I38L 1.98 12 809 0.06 31.51
D293S 1.74 70 070 0.84 2.09
A63W 1.77 49 343 0.56 3.19
A63E 1.82 43 383 0.48 3.83
F296N 2.10 75 395 0.91 2.31
A63I 1.80 16 765 0.12 15.47
A63T 1.88 60 292 0.70 2.67
F296P - 11 953 0.05 -
A63Q - 9 768 0.02 -
A63L 1.83 23 458 0.21 8.88
D293E 1.86 25 079 0.23 8.12
A63F 1.92 38 592 0.41 4.68
F296K 2.19 63 227 0.74 2.95
表2  高通量筛选突变体的酶活测定结果
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