高活性和高热稳定性乙烯合成酶的筛选和鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2022-0329

摘要/Abstract

摘要：

乙烯合成酶（ethylene-forming enzyme，EFE）是潜在的乙烯生物合成关键酶，酶的高活性和高热稳定性是工业化应用的必要基础。根据进化树选取了6种乙烯合成酶候选基因，成功地进行了表达纯化并测定其活性和热稳定性。最终筛选到来源于Streptomyces bottropensis和 Streptomyces turgidiscabies的乙烯合成酶SbEFE和StEFE具有较高活性和热稳定性，酶比活分别是已报道的乙烯合成酶PsEFE的3.23和4.23倍。35℃水浴30 min后，酶活性仍可保留95.73%和88.22%，而PsEFE在相同条件下完全失活。酶动力学分析表明，SbEFE和StEFE催化反应的副产物也明显低于PsEFE。因此，SbEFE和StEFE是目前已发现的具有最高活性和热稳定性的乙烯合成酶，为深入研究乙烯合成酶的催化机制和应用工艺提供了新的基础。

关键词: 乙烯合成酶, 催化活性, 热稳定性, 乙烯, 动力学参数

Abstract:

The ethylene-forming enzyme（EFE）is the potential key enzyme for bioethylene production. Enzyme activity and thermostability are essential for the industrial application. Six candidate genes of ethylene-forming enzymes based on the evolution tree were selected, their expressions and purifications were conducted successfully, and their activity and thermostability were measured. EFEs from Streptomyces bottropensis（SbEFE）and Streptomyces turgidiscabies（StEFE）were found to have the highest activity and thermostability. The specific activities of SbEFE and StEFE were 3.23 and 4.23 times of the reported PsEFE, and enzymatic activity remained 95.73% and 88.22%, while PsEFE was completely inactivated under the same condition. Analysis of enzymatic kinetics indicated that by-product catalyzed by SbEFE and StEFE was obviously lower than that by PsEFE. Therefore, SbEFE and StEFE are the enzymes with the highest activity and thermostability in currently found EFEs, which may provide novel foundation for deeply studying catalytic mechanism of EFE and application process.

Key words: ethylene-forming enzyme, catalytic activity, thermostability, ethylene, kinetic parameters

张晨, 张佟佟, 刘海萍. 高活性和高热稳定性乙烯合成酶的筛选和鉴定[J]. 生物技术通报, 2022, 38(11): 269-276.

ZHANG Chen, ZHANG Tong-tong, LIU Hai-ping. Screening and Identification of Ethylene-forming Enzymes with High Activity and Thermostability[J]. Biotechnology Bulletin, 2022, 38(11): 269-276.

图/表 10

表1 EFE种属来源

Table 1 Species of EFEs

蛋白名称 Protein	种属名称 Species	蛋白分子量 Molecular weight/kD
CgEFE	Colletotrichum gloeosporioides	46.94
ChEFE	Colletotrichum higginsianum	47.00
GgEFE	Glomerella graminicola	46.30
SbEFF	Streptomyces bottropensis	38.52
StEFE	Streptomyces turgidiscabies	38.55
SsvEFE	Streptomyces sviceus	39.61

图1 不同种属来源EFE蛋白的SDS-PAGE电泳图

Fig. 1 SDS-PAGE of EFEs from different species

图2 各种属EFE酶比活的比较误差线表示标准差，*表示差异显著（P <0.05），***表示差异极其显著（P <0.001），下同

Fig. 2 Comparison of the specific activities of EFEs from various species The error bars represent the standard deviation，* represents significant difference（P<0.05），and *** represents extremely significant difference（P<0.001），The same below

图3 37oC处理30 min EFE酶的比活

Fig. 3 Specific activities of EFEs treated at 37oC for 30 min

图4 温度对PsEFE、SbEFE和StEFE酶活性的影响

Fig. 4 Effects of temperature on the enzymatic activities of PsEFE，SbEFE and StEFE

图5 反应温度对SbEFE和StEFE酶活性的影响

Fig. 5 Effects of reaction temperature on SbEFE and StEFE enzyme activities

图6 反应体系pH对SbEFE和StEFE酶活性的影响

Fig. 6 Effects of pH on SbEFE and StEFE enzyme activities

表2 SbEFE和StEFE对底物2-OG和L-Arg的动力学参数

Table 2 Kinetic parameters of SbEFE and StEFE for subs-trate 2-OG and L-Arg

蛋白Protein	底物 Substrate	K_m/（μmol·L^-1）	K_cat/min^-1	K_cat/K_m /（L·μmmol^-1·min^-1）
PsEFE	2-OG¹	26.41±10.06	39.55±5.86	1.50
	L-Arg¹	38.18±4.79	35.23±1.86	0.92
	L-Arg²	101.30±31.60	0.92±0.14	0.009
SbEFE	2-OG¹	116.60±23.66	145.20±15.33	1.25
	L-Arg¹	27.26±5.17	135.35±9.29	4.97
	L-Arg²	96.16±32.68	1.70±0.27	0.018
StEFE	2-OG¹	261.30±192.40	298.78±149.68	1.14
	L-Arg¹	24.04±8.29	132.82±17.54	5.52
	L-Arg²	92.89±34.28	1.61±0.28	0.017

图7 PsEFE，SbEFE和StEFE的酶活性曲线 A：PsEFE，SbEFE和StEFE随底物2-OG浓度变化生成乙烯的酶活曲线；B：PsEFE，SbEFE和StEFE随底物L-Arg浓度变化生成乙烯的酶活曲线；C：PsEFE，SbEFE和StEFE随底物L-Arg浓度变化生成P5C的酶活曲线

Fig. 7 Enzyme activities of PsEFE，SbEFE and StEFE A：Ethylene production by PsEFE，SbEFE and StEFE with different 2-OG concentration. B：Ethylene production by PsEFE，SbEFE and StEFE with different L-Arg concentration. C：P5C activity curve of PsEFE，SbEFE and StEFE with L-Arg concentration

图8 EFE蛋白序列比较 ▲表示参与金属螯合的氨基酸，●表示参与2-OG结合的氨基酸，★表示参与L-Arg结合的氨基酸

Fig. 8 Sequence alignment of EFEs ▲represents amino acids involved in metal chelation. ●represents amino acids involved in 2-OG binding，★represents amino acids involved in L-Arg binding

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