Preparation Of 10-Oxostearic Acid By Cascade Catalysis Of Oleate Hydratase With 10-Hydroxyacid Dehydrogenase

The oleic acid derived from plant oil is catalyzed by oleate hydratase to generate 10-hydroxy stearic acid.And the cascade of oleate hydratase and 10-hydroxy stearic acid dehydrogenase is a new way to transform oleic acid into 10-oxostearic acid,with the advantages of mild reaction conditions and high space-time yield.Further cleavage of 10-oxostearic acid,various high-value added functional chemicalscan be synthesized,such as dicarboxylic acid,hydroxy acid and omega-amino acids.The research contents are divided into three parts:(1)Screening of oleate hydratase.According to the reported protein sequences of oleate hydratases,the potential oleate hydratases were screened from the genomic database and were cloned from different strains and genomes.The target genes were expressed inEscherichia coli to determine the catalytic activity.As a result,an oleate hydratase(PaOH)from Paracoccus aminophilus showed a relatively higher activity.(2)Characterization of PaOH and the optimization of hydration reaction.PaOH showed the highest activity at pH 6.5,30? and in 4%(v/v)DMSO.The specific activity of PaOH under the optimal conditions was 5.21 U mg-1.PaOH gives the highest catalytic efficiency constant(kcat/KM),10,700 min-1·mM-1.The half-lives of PaOH are 4.05 h and 3.02 h at 25?and 30?.Substrate loading and catalyst loading of the hydration reaction was optimized.With the addition of 90 g·L-1 oleic acid and 10 g·L-1 lyophilized enzyme,the conversion reached 96.1%after 10 h,and the space time yield was 552 g L-1· d-1.The yield of 10-hydroxy stearic acid is 67.9%(mol/mol)and the purity is 99.3%.(3)Cascade catalysis of hydratase and dehydrogenase.Since the oxidation reaction of MIDH requires expensive coenzyme NAD+ as the auxiliary substrate,it is necessary to construct a coenzyme recycling system.After comparison,lactate dehydrogenase(LDH)was used to realize the coenzyme regeration.In order to maximize the catalytic capacity of PaOH,the cascade reaction was carried out step by step in one pot.With the addition of 90 g·L-1 oleic acid,10 g·L-1 lyophilized enzyme of PaOH,O.1mM NAD+,and 0.5 g·L-1 lyophilized enzyme MIDH and LDH,the conversion reached 95.0%after 10 h,and the space time yield was 217 g·L-1·d-1.The final yield of 10-oxostearic acidwas 51.9%(mol/mol),with a purity of 99.0%.