A Study On Preparation And Enzymatic Characteristics Of Three No-lid Domain Lipases

Microbial lipases are one of the most important group of biocatalysts which have been widely applied in food,pharmaceutical,pulp and energy industry.Novel lipases with outstanding features are highly desired for biotechnological applications.Unlike the common lipase,no-lid domain lipases do not conatin an alpha helix domain covering the active site,which make the active site inaccessible to the substrate.It is still unkonw whether this unusual struture feature is connected with their unique properties.In the ptesent work,lipases without lid domains from Bacillus subtilis（Bslip）,Streptomyces exfoliates（SeLip）and Pseudomonas mendocina（PmLip）were obtained,and expressed in a heterologous expression system.The recombinant enzymes were purified followed by characteristied.Furthermore,The substrate selectivity and stability of no-lid domain enzyme were studied by using Tunnel analysis and Molecular Dynamincs.This work can shed some light on understanding the relationships between the struture of the no-lid domain lipase,and also provide guidance for their industrial applications.The research contents and results are summaized as follows:（1）Cloning,expression and purification of three no-lid domain lipasesThe gene sequences encoding three no-lid domain lipases（Bslip、PmLip and Se Lip）were synthetized and cloned into the E.coin expression vector pET22b to genernate the plasmids pET22b-Bslip,pET22b-PmLip and pET22b-SeLip.Three lipases were successfully expressed in E.coin BL21-CodonPlus（DE3）,BL21 Rosetta-gami B（DE3）and BL21-CodonPlus（DE3）separately.Then the recombined lipases were purified by colume affinity chromatography and anino exchange chromatography.Using tributyrin as the substrate,the activity of Bslip,PmLip and SeLip were 108 U/mg、343 U/mg and 21 U/mg respectively.（2）Biocharacterization ofthe three no-lid domain lipasesAll three enzymes showed their highest activity around pH 7.The optimum temperature for the reaction catalyzed by BsLip and Se Lip was 35℃,while that was 45℃for Pm Lip.The half-life of PmLip at 45℃was 1.5 h,while the enzyme activity of Bslip and SeLip decreased to 30%of original enzyme activity after 0.5 h at 45℃.Three no-lid domain lipases prefered substrate as short and medium-chain triglycerides,and they showed the lower hydrolysis activity towards olive oil consisting of long-chain triglycerides.Heavy metal ions such as Zn²⁺and Cu²⁺had a significant influence on the activity of three lipases and could inhibit the enzyme activity,whereas other divalent ions such as Mg²⁺,Mn²⁺and Ca²⁺hardly influenced the enzyme activity.BsLip and SeLip were poorly tolerant to polar organic solvent,while PmLip showed good organic solvent resistance,so polar and no-polar organic solvents and surfactants inhibited hardly the enzyme activity of PmLip.（3）Molecular simulation and structural analysis of three no-lid domain lipasesThe Tunnel configurations of no-lid domain lipase,esterase,and cutinase were analyzed by using the Mole2.ui software in this study.The Tunnels of no-lid domain lipases were wide and shallow,which can be explained their preference for hydrolysis of short-chain triglycerides.As contrast,the Tunnels of esterases were narrow and deep,which may allow small molecules of fatty acid esters accece into the active site,The tunnels of cutinases were exposured to the surface,which can accommodate substrate with large size such as keratin and wax.The overall conformational change of PmLip and SeLip in methanol/water（1:1,v/v）and ethanol/water（1:1,v/v）solutions were showed by Molecular Dynamics Simulations.Through analysising the RMSDs of two lipases in polar organic solvents,it was found that Pm Lip was stable in polar organic solvents methanol/water（1:1,v/v）and ethanol/water（1:1,v/v）,and SeLip was very unstable in polar organic solvents of methanol/water（1:1,v/v）and ethanol/water（1:1,v/v）.By strucatral analysis,PmLip had an additional disulfide bond forming by Cys34 with Cys96 and a salt bridge forming by Glu35 with Arg83,whereas SeLip did not possess such bonds in the corresponding region.This may be the reason why PmLip exhibits good thermal stability and organic solvent resistance.