The Study Of Relationship Between Structure And Thermostability And Substrate Specificity Of MAS1 Lipase

Our studies resolved the first crystal structure of the member of I.7subfamily bacterial lipase,which is a thermostable enzyme coded by a gene?mas1?from marine Streptomyces sp.strain W007.According to the structure features,we explored the molecular basis for thermostability and regiospecificity toward triglyceride of MAS1 lipase.Therefore,our studies not only provide insights into the molecular basis for thermostability and regiospecificity of lipases,but also offer possibilities for engineering lipases with desired catalytic properties for industrial applications.1.Purification and Screening of crystallizing condition of MAS1 lipase: Pichia pastoris X-33 recombinant strain containing pPICZ?A-MAS1 vector was cultured in a 20 L fermentor.Under the optimal fermentation condition,vast target protein MAS1 was obtained by methanol induced.We got the 95%purified protein by Ni cheated affinity chromatography and gel exclusion.The purity of protein was higher than 95% as determined by SDS–PAGE.The crystal concentration of protein was determinate as 21mg/mL by pre-crystallizition.Two initial conditions were optimised according to shape and diffraction quality crystals were obtained from 0.2M zinc acetate,0.1M imidazole pH 6.5 and 10%?w/v?PEG8000,and 1.0 M ammonium citrate dibasic,0.1M sodium acetate trihydrate pH 4.6.2.The resolution and description of crystal structure of MAS1 lipase: Two x-ray diffraction data with 2.3 and 2.34 ? resolution were collected at the Shanghai Synchrotron Radiation Facility beamline BL17U1.Because of the present of Zn²⁺ in the first crystallization condition and the anomalous scattering signal in diffraction data,we thought some Zn²⁺ may be binding with MAS1.Despite lacking of homological structure,we can find the phase of the Zn²⁺ to resolve the overall structure of MAS1 rather than the use of molecular replacement method.Based on the heavy metal positioning method,we got the crystal structure of MAS1 lipase with high resolution.3.Study on the mechanism of thermostability of MAS1 lipase: The analysis of structure information revealed that there are two disulfide bonds at the N-and C-terminus of MAS1 lipase,respectively.They are stabilizing the long terminal loops which were located on surface of protein.To break up the disulfide bonds,the mutants C22 S,C260S and MAS1 wild-type after DTT treatment were constructed.The thermostability profiles of these proteins were investigated.Compared to the wild-type MAS1,The half-lives?t1/2,70°C?of each treated protein was decreased 4.8,3.1,And all mutants decreased their Tm values by 2.5-4oC as compared to that of MAS1wild-type.Thus,our results suggested that the disulfide bonds might play a part in stabilizing the overall conformation of the enzyme.4.Study on the mechanism of regiospecificity of MAS1 lipase toward triacylglyceride: MAS1 lipase is a non-regiospecific lipase.Through alignment of sequences and structure with same family lipase,we find that the second site of conserved pentapeptide?G-X-S-X-G?of MAS1 play a important role in regiospecificity of lipase belonged to this family.Conversion of MAS1 from a non-regiospecific lipase to sn-1,3 specific was achieved by substitution H108 W,probably by varying the distance between the second residue in the conserved pentapeptide and the catalytic serine.Our present study provides insights into the molecular basis for the thermostability and regiospecificity of MAS1 lipase which is from marine Streptomyces sp.strain W007 by biochemical tests,crystal structure analysis and mutation experiments.Therefore,our studies not only provide insights into the molecular basis for thermostability and regiospecificity of lipases,but also offer possibilities for engineering lipases with desired catalytic properties for industrial applications.