Effect Of 1,3-diacylglycerol On Type 2 Diabetes Mellitus And Gene Cloning And Expression Of The Lipase With A Preference For The Sn-2 Position Of Triacylglycerol

Type 2 diabetes mellitus (T2DM) is a world prevalent disease with increasing prevalence and trends to be younger. It was reported that T2DM is related with dietary fat intake. 1,3-diacylglycerol (1,3-DAG) is the minor composition of natural oil. Human and animal studies showed that 1,3-DAG could decrease the serum insulin and leptin concentration of T2DM rats and improve the lipid metabolism parameters of the T2DM patients. Whether 1,3-DAG can improve the glucose metabolism parameters of T2DM patients has not been tested in human studies.The content of 1,3-DAG is very low in the natural oil. A commercial available edible oil rich in 1,3-DAG was prepared with chemical methods. However, the utilization of chemical reagents is always a concern for consumers Preparation of 1,3-DAG with enzymatic methods attracted the interest of the scientists. Lipase A from Candida antarctica (CAL-A) was reported to be the lipase with the most sn-2 preference and it can be used to prepare 1,3-DAG by hydrolyzing TAG. However, CAL-A is scarcely available in the original strain. In the present study, we attempted to express the gene of CAL-A in expression system of E.coli and Pichia pastoris and prepare 1,3-DAG with the recombinant lipase.The main contents of this study including:The effect of 1,3-DAG on T2DM was examined in a double-blind controlled parallel study with 127 T2DM patients (aged 40 to 65) recruited in Hangzhou, China. All subjects consumed TAG oil in the washout period (14 days), then were randomly divided into two groups and consumed DAG or TAG oil respectively with similar fatty acid composition (25 g/day) for 120 days. Physiological parameters were measured and blood samples were collected on days 0, 60 and 120. Parameters of liver and kidney functions, lipid metabolism and T2DM were measured by standard methods using commercially available kits. Fatty acids composition of serum phospholipids was measured with gas chromatography. 112 subjects completed the study. Energy and diet intake did not differ significantly between two groups. Body weight decreased significantly in both DAG and TAG group (p<0.05), but the reduction in DAG group (1.2 kg) was significantly greater than that in TAG group (0.5) (p<0.05). Waist and hip circumferences decreased significantly in DAG group (p<0.05) but not in TAG group. The insulin resistance (HOMA-IR) was significantly reduced from baseline in the DAG oil group (p<0.05). However, in the TAG group, the HOMA-IR increased obviously although not significantly. Serum insulin and leptin concentrations were both increased significantly in the TAG oil group (p<0.05). But in the DAG group, their concentrations were reduced obviously although not significantly. Compared with TAG, DAG oil consumption had no significant effect on the parameters of liver and kidney functions and essential fatty acids composition in serum phospholipids.The CAL-A gene was cloned and ligated with pET-28a to construct recombinant plasmid, which was then transformed into E.coli DH5α.Recombinant E.coli was obtained by LB plate with ampecillin and confirmed by PCR. The expression product was analyzed by SDS-PAGE. Results indicated that CAL-A gene was functionally expressed but the recombinant CAL-A existed in form of inclusion body. After treated with urea, the activity of recombinant CAL-A was not recovered.The CAL-A gene was then cloned into the yeast integrative plasmid pPIC9K, which was then transformed into His4 mutant yeast GS115. Recombinant Pichia strain was obtained by minimal dextrose and minimal methanol plates and confirmed by PCR. The expression product of CAL-A gene was analyzed by SDS-PAGE. Results indicated that CAL-A gene was functionally expressed in Pichia pastoris and the recombinant lipase had a molecular mass of 50 kD. After 192 h fermentation, the lipase activity of the supernant reached 17.4 U/mL.Crude enzyme was purified with hollow fiber membrane and ion exchange chromatography and its properties were studied. The lipase showed maximum activity at 75℃and pH 7.0. Lipase was relatively stable under 70℃and at pH range 6.0-8.0. Co²⁺ and EDTA solution promoted the activity of CAL-A, Hg²⁺ decreased the activity of lipase, Ca²⁺,Zn²⁺,Ag⁺ etc had no significant effect on the recombinant CAL-A. Hydrolysis reaction was carried out in closed test tubes containing triolein and water (3:1 w:w), CAL-A (2% of triolein) was then added. After incubated at 60℃for 35 h, the product rate of 1,3-DAG reached 13%. After purified with short path distillation, the ultimate yield of 1,3-DAG could reach 43 %.In conclusions, The effect of 1,3-DAG on T2DM was examined. Compared with TAG, 1,3-DAG consumption improved biomarkers and anthropometric parameters of T2DM patients and no adverse reactions were observed. 1,3-DAG oil has an equivalent bioavailability as TAG in relation to providing essential fatty acids. The CAL-A gene was cloned into yeast integrative plasmid pPIC9K and expressed in P. pastoris GS115 successfully. Electrophoretic pure recombinant CAL-A was obtained after purification with hollow fiber membrane and ion exchange chromatography. This recombinant lipase was very thermostable and had great resistibility to the inhibition of metal ions. The CAL-A was then used to prepare 1,3-DAG successfully.