Study On The Enzymatic Properties Of Marine Microbial Novel Lipase And Its Catalytic Synthesis Of Cinnamyl Acetate

Cinnamyl acetate is a kind of important flavor ester.Because of the shortage of output extracted from natural product,most of cinnamyl acetate is synthetic with chemical methods.However,the cost of chemical methods is very high,the extract process is fussy,and chemical methods will lead to environmental pollution.Lipases not only catalyze the hydrolysis of ester bonds,also some other types of reaction,such as esterification,transesterification and alcoholysis,their application is vital in food,pharmaceuticals,fodder,biochemical engineering,detegents,textile.We could take advantage of lipases' catalytic characteristic to compound cinnamyl acetate instead of chemical methods,thus the green synthetisis will come true.This study screened 17 lipase genes from the genome of Streptomyces sp. SCSIO 13580 identified from the deep sea of the South China Sea with the analysis of bioinformatics FramePlot 4.0beta and SignalP 4.1 Server softwares,and designed two primers for every gene make use of Primer Premier 5 software.These 17 lipase genes were amplified with their primers.Then amplificons were inserted into pET-28a?+? plasmid and transformed into E. coli BL21?DE3?.It suggested that 14 lipse genes were expressed in E. coli BL21?DE3? through verification of SDS-PAGE gel.The success rate of expression reached 82.4%.The enzyme powder of 14 lipases was produced and put into organic phase to catalyze the synthesis of cinnamyil acetate.Thus we screened the Lipase L-1,whose conversion ratio is highest among the 14 lipases.And Lipase L-1 was purified using Ni-NTA affinity chromatography.Further analysis of the gene sequence of Lipase L-1 was done by the application of bioinformatics methods and tools.Lipase L-1 contains 1005 bp and encodes a putative lipase of 334 amino acids. Lipase L-1 exhibits 51% similarity with a protein in Genbank database,which is most similar to it,this shows that Lipase L-1 is a novel lipase. Alignment of the llipase protein sequences was generated with DNAMAN software.It suggested that Lipase L-1 protein sequence contains lipases' catalytic triad Ser-Asp-His and conserved GXSXG pentapeptide. A bootstrapped phylogenetic tree was built with MEGA 6 software.The enzymatic characteristics of Lipase L-1 was studied with p-nitrophenol ester method. The optimal substrate tested of L-1 was p-NPC, the optimal working pH was 8.0,but it shows highest stebility in pH buffer;the optimal working temperature was 40 ?,its enzymatic activity decreased markedly after it was disposed in the environment over 40 ?.Enzymatic activity of Lipase L-1 decreased obviously along with the rise of temperature in the range of 20-40 ?.1 mmol/L Li+ and Mg²⁺ could stimulate the activity of Lipase L-1.Lipase L-1 exhibited high activities in the presence of methanol,alcohol,isopropanol and isobutanol,it also showed nice tolerance to many other organic solvents and detergents. The hydrolysis activity of L-1 was 51.5 U/mg under optimal working conditions.The effects including cinnamyl alcohol/vinyl acetate ratio?mol/mol?, temperature, solvents and aclyl donors, on the catalytic activities were further investigated in the base of single factor test. The optimum enzymatic working conditions were obtained as follows: vinyl acetate as the acyl donor, n-hexane as the solvent, ratio of cinnamyl alcohol/vinyl acetate 1:6,reaction temperature of 40 ?,the maximum conversion rate could reach 48.3%, the activity of enzyme powder was 5.59 U/g.