Epoxidation Of Limonene And Decarboxylation Of Fatty Acids Catalyzed Via Enzymatic Cascades

Olefins and fatty acids are naturally abundant in agricultural by-products and some subsidiary processing products.These compounds are important raw materials for industrial chemical production and synthesis.Currently,there is a clear interest in the utilization of olefins and fatty acids for high-value and sustainable chemical industry.Compared to traditional chemical processes,enzymatic catalysis has been recognized as an eco-friendly technology due to low energy consumption,low pollution,mild reaction condition and high stereo-and regio-selectivity.However,due to the substrate specifity,it is difficult to deal with complex reactions by a single enzyme,which limits the application of enzymes.Moreover,enzymatic cascade catalysis has shown prominent advantages in simplifying procedure,improving yields,and reducing costs and pollution.Therefore,it has been generally acknowledged that the enzymatic cascade catalysis serves as one of the most promising directions in the field of“Green Chemistry”.Nevertheless,the use of enzymatic cascade in the field of high value utilization of olefins and fatty acids is still in its infancy,and it is in urgent need of deep and systematic development.This study aims at valorization of olefins and fatty acids by constructing cascade reactions.Attentions were paid to the key enzymes capable of catalyzing conversions of olefins and fatty acids,therefore,ipase-coupled choline oxidase?AnChOx?and photodecarboxylase?CvFAP?were used.Much a.This study is expected to help solve the problems of low efficiency,high carbon emissions and high energy consumptions during processing of agricultural by-products and waste resources.Main experimental results are shown as follows:1.Preparation of choline oxidase An ChOx and its catalytic properties in natural deep eutectic solvents?NADES?were investigated.Firstly,Arthrobacter nicotianae Choline oxidase?AnChOx?was expressed in E.coli BL21?DE3?and purified by affinity chromatography.The results showed that the molecular weight of recombinant An ChOx was59.1 kDa,the protein expression level was 90 mg L^-1,and the specific activity of catalytic oxidation of choline chloride?ChCl?was 4.70 U mg^-1.Secondly,the catalytic ability of AnChOx for production of hydrogen peroxide in NADES system was studied.The results showed that the concentration of hydrogen peroxide produced by AnChOx with ChCl as substrates in various NADES system could reach 6.41-11.36 mM under the conditions of NADES system pH 7.0,50%water content,40^oC,5?M AnChOx,12h.2.The epoxidation of limonene from orange peels catalyzed by lipase-AnChOx cascade was studied.Based on previous studies,this section focused on in situ extraction of limonene from orange peels by NADES,and the efficiency of cascade catalytic epoxidation of limonene.Firstly,limonene in orange peels was extracted in situ with NADES.Meanwhile,ChCl in NADES also served as the substrate for in situ production of hydrogen peroxide.Secondly,lipase catalyzed the oxidation of octanoic acid with hydrogen peroxide to produce peroxy acid.Finally,the limonene was oxidized to epoxy limonene.The in situ production of hydrogen peroxide and one-pot one-step synthesis of epoxy limonene were realized in a synergistic manner.The cascade catalytic reactions of 12 lipases coupled with AnChOx were studied in detail.The results demonstrated that epoxy limonene could be readily obtained.The cascade catalysis of immobilized lipase Novozyme 435 and AnChOx enabled a limonene conversion rate of 32.94%after 24 h under the applied conditions.3.Preparation of photodecarboxylase CvFAP and its catalytic performance were studied.Firstly,the fatty acid photodecarboxylase?CvFAP?from Chlorella variabilis was expressed in E.coli BL21?DE3?.The results showed that the molecular weight of recombinant CvFAP was 77.0 kDa.The total cell yields was 30 g L^-1?wet weight?,and the crude enzyme expression level was 380 mg L^-1.Secondly,the catalytic abilities of CvFAP for decarboxylation of fatty acids by crude enzyme and whole cell were studied,respectively.The results showed that under the conditions of pH 8.5,30 ^oC and 0.35 g mL^-11 whole cell CvFAP,the decarboxylation rates of fatty acid with different chain length were in the range of 18.34%and 87.62%after 24 h.The corresponding product concentration was 9.17-43.81 mM,the fatty acid decarboxylation concentration of crude enzymes was 2.11-17.61 mM.The efficiency of whole cell CvFAP on fatty acid decarboxylation was significantly higher than CvFAP crude enzyme?p<0.05?.Finally,the catalytic specificity of CvFAP on substrates was studied.The results indicated that CvFAP could also catalyze decarboxylation of hydroxyl fatty acids to produce aliphatic alcohols.Key parameters were investigated.The optimal reaction conditions were 30^o C,pH8.5 and 0.35 g mL^-11 whole cell CvFAP obtained.The cascade catalysis of lipase coupled with CvFAP displayed great potential for production of high-value products such as fatty alcohols.4.Alkanes were prepared by cascade catalysis of lipase-coupled photodecarboxylase CvFAP for in situ hydrolysis and decarboxylation of oils.In this section,3 lipases coupled with whole cell CvFAP were studied to convert natural/waste oils into a new generation of biofuels?alkanes/alkenes?.The highest concentration of alkanes?alkenes?could reach 74.08mM with a conversion rate of 49.39%after 24 h under the conditions of 30?,pH 8.5,100mg mL^-11 immobilized lipase ROL and 0.35 g mL^-11 whole cell CvFAP.5.Aliphatic alcohols were prepared by cascade catalysis of lipase-coupled photodecarboxylase CvFAP for in situ hydrolysis and decarboxylation of castor oil.Cascade catalytic hydrolysis and decarboxylation by immobilized lipase ROL and whole cell CvFAP were evaluated for converting abundant low-value castor oil into unsaturated fatty alcohols.The results showed that the concentration of unsaturated fatty alcohols reached 53.17 mM with a conversion rate of 41.70%after 12 h under the conditions of 30^oC,pH 8.5,50 mg mL^-1immobilized lipase ROL and 0.35 g mL^-11 whole cell CvFAP.In summary,cascade catalytic systems based on“lipase-AnChOx”and“lipase-CvFAP”were established in this study.Through the cascade reaction,efficient conversion of fatty acids and olefins from agricultural by-products and their subsidiary processing products was successfully achieved.Pleasantly,this study enabled an efficient and high-value utilization of agricultural by-products.At the same time,this novel protocol could facilitate the reduction of environmental pollution caused by agricultural wastes,thereby contributing to increasing agricultural incomes.