Gene Cloning,Expression And Characteristics Of Feruloyl Esterases From Bacillus Pumilus W3

Danshensu(Salvianic acid A,DHPLA)and caffeic acid(Caffeic acid,CA)have significant pharmacological activities,especially in the treatment of cardiovascular diseases,and are the core compounds in the metabolism of many drugs.As the aging of the population has intensified in recent years,the prevention and treatment of cardiovascular diseases is also particularly important.Therefore,the synthesis of bioactive molecules such as DHPLA and CA has important research significance.However,the content of plant extracts is too low to meet market demand.Studies have shown that rosmarinic acid(RA)can form DHPLA and CA through a one-step enzymatic reaction of ferulyl esterases(FAEs),and RA has a wide range of plant sources in nature.The content can reach 20%.However,information on the enzymes or microorganisms that hydrolyze RA is very limited.Therefore,it is of great significance to develop FAEs with high RA hydrolysis activity.In this study,through strain screening,genome information mining,bioinformatics analysis,and clone expression verification,a new FAEs with strong RA hydrolysis activity was obtained,and its enzymatic properties and whole-cell catalysis applications were carried out.The main research results are as follows:(1)The Bacillus pumilus W3 with high RA hydrolysis activity was screened.A total of16 esterase genes were obtained from its genome,which were numbered as bpfae01-16.It was cloned and expressed in E.coli BL21(DE3),and pure enzymes were used to verify the hydrolytic activity of RA,which proved that only Bp Fae12 can hydrolyze RA to generate CA and DHPLA.The results of chiral analysis showed that the DHPLA produced by Bp Fae12hydrolysis of RA was D-DHPLA.(2)Sequence alignment and conserved domain analysis showed that Bp Fae12 has a typical conserved sequence of FAEs catalytic activity:Gly¹⁴³-His-Ser-Arg-Gly¹⁴⁷;Phylogenetic tree results show that Bp Fae12 is compatible with Lactobacillus fermentum FAEs are on the same branch,and the sequence similarity was up to 76%.(3)Enzymatic properties studies show that the optimal reaction p H of Bp Fae12 is 8.0,the optimal reaction temperature is 50°C,and the stability is good at p H 5.0-8.0.Kinetics results show that Bp Fae12 can effectively hydrolyze the four model substrates of FAEs(methyl ferulate,methyl p-coumarate,methyl caffeate,methyl sinamate).It further proves that Bp Fae12is a new FAEs.Among them,the K_m of Bp Fae12 to methyl ferulate,methyl p-coumarate,methyl caffeate and methyl sinapate are 0.64,0.38,0.62,0.75 mmol·L^-1,respectively;k_cat/K_mare 898.63,1801.75,544.82,25.61 mmol·L^-1·s^-1,respectively.Bp Fae12 shows good catalytic activity on RA and its structurally similar substrate chlorogenic acid.The K_m and k_cat/K_m for RA are 0.41 mmol·L^-1 and 16.21 mmol·L^-1·s^-1,respectively;The K_m and k_cat/K_m of chlorogenic acid are 3.9 mmol·L^-1 and 46.53 mmol·L^-1·s^-1,respectively.The metal ions Co²⁺?Ni²⁺?Zn ²⁺?Cu²⁺?K⁺?Fe³⁺?Mn²⁺?Ca²⁺and Fe²⁺have varying degrees of inhibition on the catalytic activity of Bp Fae12.The esterase inhibitor PMSF inhibits the activity significantly.DMSO promotes the activity of Bp Fae12.(4)The conditions of recombinant E.coli BL21/p Cold II-bpfae12 whole-cell transformation to RA were optimized,and the optimal reaction temperature,p H,and the amount of whole-cell catalyst added for whole-cell catalysis were optimized.The results proved that the optimal reaction temperature was 40°C,p H 8.0.The addition amount of the whole cell catalyst is 20 g·L^-1,after 4 hours of reaction,the RA conversion rate can reach 99.74%.