Purification,Enzymatic Properties And Active Site Of Membrane-bind Linoleic Acid Isomerase BBI

Bifidobacterium breve isomerase（BBI）is a single enzyme catalyzed production of cis 9,trans 11-conjugated linoleic acid isomer in lactic acid bacteria.As a membrane-bound protein with a nine-dimensional transmembrane structure,the expression of BBI is extremely low in primitive host,making the research of the structure-function relationship of this enzyme difficult and limiting its further development and application.In this thesis,by comparing the differences in expression levels of BBI in the model microorganisms Escherichia coli and Pichia pastoris,we determined the applicable range of BBI in the two expression systems,and on this basis,we established the purification process of BBI,carried out enzyme kinetic studies,and also predicted and validated the active site of the enzyme,and these findings laid a solid foundation for the further development and application of the enzyme.The main findings of this thesis are as follows:（1）Optimization of the expression system and determination of the applicable range of the membrane-bound linoleic acid isomerase BBI.Achieve optimization of the expression levels of BBI in E.coli that Rosetta strain with His-tag at the C-terminal is induced with 1.0 mmol/L IPTG for 8 h.After comparison between E.coli and P.pastoris systems about BBI expression,determination of its applicability had been that the application of E.coli for function and the application of P.pastoris for purification.（2）Construction of a membrane bound linoleic acid isomerase BBI purification process.For the efficient isolation and extraction of BBI,a membrane-bound protein with nine transmembrane,the membrane fraction separation conditions were firstly optimized,and10,000 g for 1 h were determined as the conditions for membrane fraction enrichment;then 10detergents including ionic,non-ionic and amphoteric were screened,and 2%（w/v）dodecyl-β-D-maltoseide and 2 h incubation at 4°C were determined as the conditions for the extraction of BBI.The extraction conditions of BBI were determined.Finally,by applying affinity chromatography and gel separation chromatography in combination.The results indicated BBI was successfully purified to homogeneity level,and the molecular weight was about 40 k Da and a specific enzyme activity of 17.44μmol·min·^-1mg^-1 was obtained,which was purified about 669.54 times.（3）Enzymatic properties and catalytic reaction kinetics of membrane-bound linoleic acid isomerase BBI were investigated.The optimum condition for temperature and p H was 37°C and 7.5～8.5,and the half-life was 32.24 min at 37°C,and BBI could maintain high catalytic activity in the p H range of 7.5～8.5.Ni²⁺and Zn²⁺could increase the enzymatic activity of BBI to 150%～160%,while Cu²⁺,Fe²⁺and Al³⁺caused the loss of BBI activity.When linoleic acid,α-linolenic acid andγ-linolenic acid were used as substrates,BBI had the highest affinity forα-linolenic acid with a K_mvalue of 125.89 mg/L,followed byα-linolenic acid,linoleic acid andγ-linolenic acid in descending order of k_cat/K_m value.The catalytic efficiency of BBI was higher forα-linolenic acid and linolenic acid when compared toγ-linolenic acid.（4）Prediction and validation of the active site of membrane-bound linoleic acid isomerase BBI.Based on protein structure modeling,molecular docking and virtual mutation techniques,recombinant with truncated fragments and mutations were constructed respectively for validation.The results showed that truncation of the N-terminal transmembrane helix resulted in the loss of BBI activity,and the C-terminal transmembrane structure was essential for maintaining the active conformation of BBI;and truncation of the carboxyl-terminal transmembrane helix resulted in the inability to express BBI,and the carboxyl-terminal transmembrane helix was essential for the expression of membrane proteins.We further used molecular docking to simulate the energetic changes before and after the amino acid mutation to screen for potential active sites,and validated the potential active sites by using the E.coli expression system and the P.pastoris expression system.The Y205M mutant strain was also screened to increase BBI relative enzyme activity to 111%.