Study On Biochemical Properties,Crystal Structure And Site-directed Mutagenesis Of A Lipase From Aspergillus Oryzae

Mono-and diacylglycerol lipases(DAGLs)are a small group of lipases that almost only accept mono-and diacylglycerides(MAGs and DAGs)as substrates.As a result,they are potentially great biocatalysts towards the synthesis of DAGs and MAGs with excellent purity.To get comprehensive understanding between the structure and catalytic performance of an enzyme,crystallographic study is one of the most reliable techniques that are frequently used.However,there are only a few reports on the crystallographic study on mono-and diacylglycerol lipases.For instance,AOL is a mono-and diacylglycerol lipase from Aspergillus oryzae whereas the relationship between its structure and function has not been reported so far.In the present study,we aim at the investigation into the characteristics of AOL,and its crystal structure and site-directed mutagenesis.AOL,showing a band at 31 kDa after digested by endoglycosidase Endo Hf,was expressed in P.pastoris X-33 containing aol gene and purified by anion exchange column.Details of the study showed that the recombinant AOL has a maximum catalytic activity at 50 °C and pH 6.0 and displayed good stability at near acidic or neutral environment.The best substrates for the esterification using AOL are glycerol and capric acid.In order to obtain the protein with higher homogeneity for crystallization,aol gene was cloned into the expression vector of pFL-B13 cl,and transformed into E.coli SHuffle T7 and expressed after 18 h.In this way,relatively pure AOL without SUMO tag was obtained after one-step Nickel-column purification following the digestion by SUMO protease.AOL was further purified by gel filtration chromatography.The higher purity of AOL permits the easy growth of the crystal at 20 °C in Bis-Tris buffer(100 mM,pH 6.4)with 41% w/v of polypropylene glycol P400 and a protein concentration of 10 mg/mL.Crystal data were collected using X-ray diffraction and the crystal structure was resolved with the resolution of 1.7 ?.The structure of AOL showed typical features of ?/? hydrolase fold topology with the central ? sheet formed by 8 ?-strands which was surrounded by 11 helices and connective loops on both sides.The residues forming the catalytic triad in AOL were Ser153,Asp206 and His268.The “oxyanion hole” was constituted by backbone-NH from Ser91 and Leu154,which would stabilize the tetrahedral intermediates during hydrolysis.We further substituted V269 with seven amino acids based on the relationship between the structure and function of AOL.The obtained AOL showed that hydrophilic D,E,R and Q substitutions led to the increase of hydrolysis activity in 6.1-,3.0-,2.5-and 1.2-fold,along with the increase of esterification activity in 2.6-,1.6-,1.6-and 1.4-fold,respectively.However,hydrophobic substitutions(I and L)exhibited an opposite effect with the exception of V269 A,which led to 2.3-and 1.2-fold of increase in both activities.In order to understand possible molecular mechanism of the effect of V269 on enzyme activity,homology modeling and molecular docking were performed.