| The sustainable development strategy of global energy puts forward an urgent need for green bioenergy production with enzyme as the core.The synthesis of hydrocarbons from oils catalyzed by the cascades of lipase and decarboxylase as a substitute for traditional fossil fuels has become a research hotspot.However,poor substrate transport efficiency and low conversion rate in the reaction process limit its downstream application and promotion.This paper focused on a protein self-assembly strategy based on Spy Tag/Spy Catcher.In the beginning,two fusion proteins,including TLL-Linker-Spy Catcher based on the lipase from Thermomyces lanuginosus and Cv FAP-Linker-Spy Tag based on the fatty acid photodecarboxylase from Chlorella variabilis were designed.Secondly,a multi-enzyme complex(TLL-Cv FAP)was formed by mixed self-assembly in vitro according to the truth that Spy Tag/Spy Catcher can form stable isopeptide bond spontaneously,and its catalytic effect on the conversion efficiency of hydrocarbon generated from oils was further investigated.The relevant results of this study further enriched the basic research on the preparation of multienzyme complex and provide a scientific basis for reducing the synthesis cost of hydrocarbon biofuels and improving the production efficiency.1.Design and construction of multienzyme complex self-assembled elements.The fusion plasmid of lipase(p ET28a-TLL-Linker-Spy Catcher)and photodecarboxylase(p ET28 aCv FAP-Linker-Spy Tag)was constructed by seamless cloning and enzyme cleavage linking techniques,and expressed by E.coli BL21(DE3)as host.The molecular weight and enzyme activity of the proteins before and after fusion were compared and analyzed.The results showed that the molecular weights of TLL and TLL-Linker-Spy Catcher were about 30 k Da and 45 k Da,respectively,and the enzyme activities were 152.11 ± 3.97U/mol and 141.45 ± 4.36 U/mol,separately.The molecular weights of Cv FAP and Cv FAP-Linker-Spy Tag were about 78 k Da and 80 k Da,and with palmitic acid as the substrate,the TON were 3480 ± 24 and 3600 ± 18,respectively.The fusion process had no significant effect on enzyme activity.2.Construction and performance characterization of multienzyme complexes by selfassembled in vitro.As self-assembly elements,the fusion proteins,including TLL-LinkerSpy Catcher and Cv FAP-Linker-Spy Tag,were mixed in vitro to construct the multi-enzyme complex TLL-Cv FAP.The assembly efficiency and enzyme activity were analyzed.The effects of temperature,p H and molar ratio of self-assembled components on assembly efficiency were investigated.The results showed that under the conditions of temperature 30 ?,p H 7.0 and fusion protein concentration ratio 1:1,the assembly efficiency of the multi-enzyme complex TLL-Cv FAP reached about 60% after 12 h of assembly,and the enzyme activity of the multienzyme complex was increased by about 50% compared with that of the double free enzyme.3.Synthesis of hydrocarbons from oils catalyzed by the multienzyme complex.To explore the catalytic effect of the above multienzyme complex on the hydrolysis and decarboxylation of soybean oil to prepare hydrocarbon biofuels,the effects of temperature,p H and the amount of the multi-enzyme complex on the conversion efficiency of hydrocarbon synthesis from soybean oil catalyzed by the multi-enzyme complex were investigated,and the universality of the multi-enzyme complex for hydrocarbon synthesis from different oils was evaluated.The experimental results showed that under the conditions of 30 ?,p H 7.0 and the addition amount of the multi-enzyme complex was 50 ?M for 12 h,the concentration of hydrocarbons obtained from 10 m M soybean oil as substrate reached 25 m M,and the conversion rate was 83.3%,which was about 50% higher than that of the free two-enzyme system.Besides,the conversion efficiency of hydrocarbon biofuels from different oils catalyzed by the multi-enzyme complex was increased,ranging from 5 to 50%. |
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