Screening and Identification of Ethylene-forming Enzymes with High Activity and Thermostability

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

Abstract

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

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.

Figures/Tables 10

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

Fig. 1 SDS-PAGE of EFEs from different species

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

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

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

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

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

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

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

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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