1，3-甘油二酯对2型糖尿病的影响及选择性水解甘油三酯sn-2位酯键酶基因的克隆与表达
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摘要
2型糖尿病(T2DM)是一种世界性的流行性疾病,其患病率日益升高,且日趋年轻化。研究表明,T2DM与膳食脂肪摄入之间存在相关性。1,3-甘油二脂(1,3-DAG)是天然油脂的微量组成成分,以1,3-DAG替换膳食中的甘油三酯(TAG),可以降低T2DM老鼠血清胰岛素和瘦素的浓度,改善T2DM人群的血脂代谢。但1,3-甘油二酯的摄入是否可以改善T2DM患者葡萄糖代谢相关指标,目前尚无大规模人体实验证实。天然油脂中1,3-DAG的含量极低,需通过对现有油脂资源进行改造而制备。目前在市场上销售的富含1,3-DAG的食用油是采用化学法制备而成,不易为消费者所接受,而利用脂肪酶制备1,3-DAG受到越来越多的关注。南极假丝酵母脂肪酶A(CAL-A)具有倾向性水解TAG sn-2位酯键的特性,可以利用其制备1,3-DAG。但该酶在原始菌株中产量极低。本学位论文拟调查1,3-DAG对T2DM的影响,并在外源蛋白表达系统中对编码CAL-A的基因进行表达,利用表达的重组脂肪酶水解TAG而制备1,3-DAG。
本学位论文主要内容分述如下:
采用双盲的、平行对照的膳食干预研究,评价1,3-DAG对T2DM的影响。共招募杭州地区127名40～65岁的T2DM受试者,随机分成两组,分别摄入等量DAG(1,3-DAG占64%)或TAG 120天。分别在第0、60和120天测定受试者生理参数并采集血液样品。采用商业化试剂盒对受试者的肝肾功能指标、血脂代谢指标、血糖浓度、血清胰岛素和瘦素浓度进行检测;采用气相色谱法检测受试者血清磷脂脂肪酸组成;胰岛素抵抗指数(HOMA-IR)由以下公式进行计算:HOMA-IR=空腹胰岛素浓度×空腹血糖浓度/22.5。共有112名受试者完成本次实验,其中DAG组60人(36名女性,24名男性,平均年龄54.1±6.7岁),TAG组52人(29名女性,23名男性,平均年龄53.9±6.0岁)。结果发现,DAG组和TAG组受试者的体重均显著下降(p＜0.05),但DAG组的下降幅度(1.2 kg)显著大于TAG组的幅度(0.5kg)(p＜0.05)。DAG组受试者的腰围和臀围显著下降(p＜0.05),TAG组的腰围和臀围未出现显著变化。DAG组受试者的血清葡萄糖浓度由第0天的7.4 mmol/L下降至第120天的7.06mmol/L,而TAG组则由第0天的7.81 mmol/L上升至第120天的7.88 mmol/L,均未达到显著水平。DAG组受试者的血清胰岛素和瘦素浓度均明显下降,但未达到显著水平,而TAG组胰岛素和瘦素浓度均显著升高(p＜0.05)。DAG组受试者的胰岛素抵抗指数显著下降(p＜0.05),而TAG组的胰岛素抵抗指数明显升高,但未达到显著水平。两组之间肝肾功能相关指标及血清磷脂必需脂肪酸组成无显著差异。
以南极假丝酵母基因组为模板,对编码CAL-A的基因序列进行扩增,然后与原核表达载体pET-28a连接构建成重组载体,并将其转化入大肠杆菌DH5α中。通过抗生素平板和PCR扩增,筛选和鉴定重组子,并对其进行诱导表达。聚丙烯酰胺凝胶电泳(SDS-PAGE)显示,重组脂肪酶以包涵体的形式存在。采用尿素处理后,未恢复其活性。
将编码CAL-A的PCR扩增产物与酵母分泌型载体pPIC9K连接构建成重组载体,并将其电转化入毕赤酵母GS115中。通过最小葡萄糖培养基和最小甲醇培养基及PCR扩增,筛选和鉴定重组子。重组酵母经192 h培养后,发酵液上清酶活达到17.4 U/mL。SDS-PAGE显示,重组脂肪酶分子量约为50 kD。粗酶液经中空纤维素膜超滤浓缩和离子柱交换层析后,得到经SDS-PAGE分析表明为单一条带的重组脂肪酶。该酶最适反应温度为75℃;热稳定性高,在70℃及以下温度保持30 min后仍具90%以上的酶活性;最适反应pH值为7.0,在pH 6.0～8.0的范围内具有较好的稳定性;Co~(2+)和EDTA溶液对重组脂肪酶的活性具有促进作用,Hg~(2+)对脂肪酶活性具有抑制作用,Ca~(2+)、Zn~(2+)、Ag~+等离子对脂肪酶的活性无显著影响。
将三油酸甘油酯与水以3:1(w:w)的比例混合,并加入占三油酸甘油酯重量2%的重组脂肪酶,60℃200 rpm反应35 h后,反应体系中的1,3-DAG含量约为14%。采用短程蒸馏法除去反应体系中的水、甘油、自由脂肪酸和甘油单酯后,最终产物中1,3-DAG的含量可以达到43%。
本学位论文检验了1,3-DAG对T2DM的影响。与TAG相比,1,3-DAG的摄入可显著降低T2DM患者体重、体重指数、腰围、臀围、胰岛素抵抗、血清胰岛素和瘦素浓度等指标:1,3-DAG可提供与TAG相同的必需脂肪酸,且对患者肝肾功能无毒副作用。将CAL-A基因序列与酵母分泌型载体pPIC9K连接构建重组载体,并将其转化进入组氨酸缺陷型酵母GS115中,成功进行了表达。重组脂肪酶分子量约为50kD;粗酶液经纯化后,得到电泳纯的重组脂肪酶。该重组脂肪酶具有很好的热稳定性,且对金属离子的毒害作用具有一定的抵抗性。利用重组CAL-A倾向性水解TAGsn-2位酰基,成功制备了1,3-DAG,1,3-DAG的含量可达43%。
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. Co~(2+) and EDTA solution promoted the activity of CAL-A, Hg~(2+) decreased the activity of lipase, Ca~(2+)、Zn~(2+)、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.
本学位论文主要内容分述如下:
采用双盲的、平行对照的膳食干预研究,评价1,3-DAG对T2DM的影响。共招募杭州地区127名40～65岁的T2DM受试者,随机分成两组,分别摄入等量DAG(1,3-DAG占64%)或TAG 120天。分别在第0、60和120天测定受试者生理参数并采集血液样品。采用商业化试剂盒对受试者的肝肾功能指标、血脂代谢指标、血糖浓度、血清胰岛素和瘦素浓度进行检测;采用气相色谱法检测受试者血清磷脂脂肪酸组成;胰岛素抵抗指数(HOMA-IR)由以下公式进行计算:HOMA-IR=空腹胰岛素浓度×空腹血糖浓度/22.5。共有112名受试者完成本次实验,其中DAG组60人(36名女性,24名男性,平均年龄54.1±6.7岁),TAG组52人(29名女性,23名男性,平均年龄53.9±6.0岁)。结果发现,DAG组和TAG组受试者的体重均显著下降(p＜0.05),但DAG组的下降幅度(1.2 kg)显著大于TAG组的幅度(0.5kg)(p＜0.05)。DAG组受试者的腰围和臀围显著下降(p＜0.05),TAG组的腰围和臀围未出现显著变化。DAG组受试者的血清葡萄糖浓度由第0天的7.4 mmol/L下降至第120天的7.06mmol/L,而TAG组则由第0天的7.81 mmol/L上升至第120天的7.88 mmol/L,均未达到显著水平。DAG组受试者的血清胰岛素和瘦素浓度均明显下降,但未达到显著水平,而TAG组胰岛素和瘦素浓度均显著升高(p＜0.05)。DAG组受试者的胰岛素抵抗指数显著下降(p＜0.05),而TAG组的胰岛素抵抗指数明显升高,但未达到显著水平。两组之间肝肾功能相关指标及血清磷脂必需脂肪酸组成无显著差异。
以南极假丝酵母基因组为模板,对编码CAL-A的基因序列进行扩增,然后与原核表达载体pET-28a连接构建成重组载体,并将其转化入大肠杆菌DH5α中。通过抗生素平板和PCR扩增,筛选和鉴定重组子,并对其进行诱导表达。聚丙烯酰胺凝胶电泳(SDS-PAGE)显示,重组脂肪酶以包涵体的形式存在。采用尿素处理后,未恢复其活性。
将编码CAL-A的PCR扩增产物与酵母分泌型载体pPIC9K连接构建成重组载体,并将其电转化入毕赤酵母GS115中。通过最小葡萄糖培养基和最小甲醇培养基及PCR扩增,筛选和鉴定重组子。重组酵母经192 h培养后,发酵液上清酶活达到17.4 U/mL。SDS-PAGE显示,重组脂肪酶分子量约为50 kD。粗酶液经中空纤维素膜超滤浓缩和离子柱交换层析后,得到经SDS-PAGE分析表明为单一条带的重组脂肪酶。该酶最适反应温度为75℃;热稳定性高,在70℃及以下温度保持30 min后仍具90%以上的酶活性;最适反应pH值为7.0,在pH 6.0～8.0的范围内具有较好的稳定性;Co~(2+)和EDTA溶液对重组脂肪酶的活性具有促进作用,Hg~(2+)对脂肪酶活性具有抑制作用,Ca~(2+)、Zn~(2+)、Ag~+等离子对脂肪酶的活性无显著影响。
将三油酸甘油酯与水以3:1(w:w)的比例混合,并加入占三油酸甘油酯重量2%的重组脂肪酶,60℃200 rpm反应35 h后,反应体系中的1,3-DAG含量约为14%。采用短程蒸馏法除去反应体系中的水、甘油、自由脂肪酸和甘油单酯后,最终产物中1,3-DAG的含量可以达到43%。
本学位论文检验了1,3-DAG对T2DM的影响。与TAG相比,1,3-DAG的摄入可显著降低T2DM患者体重、体重指数、腰围、臀围、胰岛素抵抗、血清胰岛素和瘦素浓度等指标:1,3-DAG可提供与TAG相同的必需脂肪酸,且对患者肝肾功能无毒副作用。将CAL-A基因序列与酵母分泌型载体pPIC9K连接构建重组载体,并将其转化进入组氨酸缺陷型酵母GS115中,成功进行了表达。重组脂肪酶分子量约为50kD;粗酶液经纯化后,得到电泳纯的重组脂肪酶。该重组脂肪酶具有很好的热稳定性,且对金属离子的毒害作用具有一定的抵抗性。利用重组CAL-A倾向性水解TAGsn-2位酰基,成功制备了1,3-DAG,1,3-DAG的含量可达43%。
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. Co~(2+) and EDTA solution promoted the activity of CAL-A, Hg~(2+) decreased the activity of lipase, Ca~(2+)、Zn~(2+)、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.
引文
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