高活力山梨醇脱氢酶氧化葡萄糖酸杆菌选育及生物催化合成米格列醇的研究
摘要
米格列醇是1-脱氧野尻霉素的N-羟乙基衍生物,是α-葡萄糖苷酶抑制剂。临床上已作为治疗2型糖尿病的首选药物。目前,其主要通过以1-脱氧野尻霉素为底物纯化学合成和以N-羟乙基葡萄糖胺为底物化学生物组合法合成,前者反应步骤较多、生产成本高,其底物1-脱氧野尻霉素无论从植物中提取或化学合成或发酵生产,工艺均较复杂；后者采用产山梨醇脱氢酶的氧化葡萄糖酸杆菌(Gluconobacter oxydans)全细胞作为生物催化剂,催化N-羟乙基葡萄糖胺反应用化学生物组合法合成米格列醇的工艺具有反应步骤少、生产成本低的优势,但通过发酵获得的全细胞,其细胞膜山梨醇脱氢酶总活力和储藏稳定性均较低,严重制约了此工艺的进一步开发和应用。本论文的目的是获得具有高活力山梨醇脱氢酶的G. oxydans菌株,以其全细胞作为生物催化剂,对化学生物组合法合成米格列醇的工艺进一步改进,进一步提高米格列醇的产量。
通过对G. oxydans A1的培养和其细胞膜山梨醇脱氢酶活力的研究表明,碳源浓度较高时,抑制细胞生长。山梨醇脱氢酶催化生物转化反应时,反应液中溶氧量越大,反应需求的细胞浓度越大,反应速率越大；当反应液中的溶氧量一定,细胞浓度达到临界值时,反应速率最大；山梨醇脱氢酶的最适作用温度为28℃,最适pH值为5.5。
以G. oxydans A1为出发菌株,通过紫外线诱变结合梯度平板半理性筛选获得一株高山梨醇脱氢酶活力突变株G. oxydans Gouv2007,其单位生物量的山梨醇脱氢酶活力与原始菌株持平,而生物量比原始菌株提高了11.2%,培养时间缩短了6小时。研究二者的发酵动力学,结果表明均符合底物抑制动力学模型,其最大比生长速率分别为0.2165h-1和0.2507h-1,Ki分别为1.5069g／L和3.9663g／L,突变株部分地解除了底物抑制现象。
单因素优化实验表明,碳、氮源分别为山梨醇、酵母浸粉,无机盐为硫酸镁和磷酸氢二钾,摇床转速为200r／min,装液量为20%,培养温度为28℃,培养基初始pH自然时,突变株的生物量和山梨醇脱氢酶的活力最高。采用中心组合设计和响应面分析,获得了细胞生长的最优培养条件：山梨醇18.0g／L,酵母浸粉10.0g／L,磷酸氢二钾3.0g／L其最终生物量达到1.333g／L,比未优化前提高了34.6%；在2L发酵罐中放大培养,生物量达到了5.580g／L,单位生物量的山梨醇脱氢酶活力为1.1U／mg干细胞。
对突变菌株细胞膜山梨醇脱氢酶性质作初步研究,最佳酶反应条件为：pH5.5,28℃。单批次操作稳定性较高,在转化反应的整个过程中基本没有失活；当底物耗尽时,连续进行两次补加底物,酶活力降低较小；多批次操作稳定性较差,首次重复使用时,酶活力有较小的降低,二次重复使用时,酶活力丧失40%。
在山梨醇培养基中梯度增大甘油浓度诱导提高山梨醇脱氢酶活力,甘油浓度为20.0g／L时,该酶的单位生物量活力达到1.9U／mg干细胞,比未诱导的提高了63%。总活力达到2.2U／mL发酵液,比未诱导的提高了30.5%。其储藏稳定性也相应提高,菌体静息细胞储存一个月时,酶活力保留75%,未经诱导的只保留30%。酶动力学显示,其Ks由诱导前的51mmol/L减小到38mmol/L,对底物的亲和力增加了1.34倍。由此,建立了甘油诱导高活力山梨醇脱氢酶的生产工艺。
以葡萄糖和乙醇胺为原料,合成N-羟乙基葡萄糖胺,葡萄糖转化率为89%；在通气搅拌下,用高活力的山梨醇脱氢酶催化N-羟乙基葡萄糖胺反应生成6-脱氧-6-氨基(N-羟乙基)-α-L-呋喃山梨糖；再加氢还原为米格列醇,分别利用TLC和离子交换层析分离检测到从N-羟乙基葡萄糖胺到生成米格列醇两步反应的总底物转化率为77.3%和73.6%,产物得率为62.7%。对合成的米格列醇经提纯后得到白色固体粉末,测得熔程为142-147℃,经红外光谱和核磁共振氢谱分析,该产物与米格列醇标准品谱图吻合良好。
Miglitol, a kind of N-hydroxyethyl ramification of 1-Deoxynojirimycin can strongly inhibit a-glucosidases. It has been first chosen as therapeutic drugs for treatment of type 2 diabetes mellitus. Up to day, there are chemical and combined biotechnological-chemical two methods to synthesize miglitol.The former is more complicated than the latter, and synthetic cost higher. In the latter method, for membrane-bound sorbitol dehydrogenase of Gluconobacter oxydans catalyticly oxidizing N-(2-hydroxyethyl)-glucamine to 6-(2-droxyethyl)amino-6-deoxy-a-L-sorbofiuanos is the key step. Sorbitol dehydrogenase total activity and storage stability of the cells produced by feimentation is small. In this study a high sorbitol dehydrogenase activity strain was obtainied. To raise the productivity of miglitol the combined biotechnological-chemical technology was improved with the high sorbitol dehydrogenase activity whole cells being catalyst.
By studying cultivation of G. oxydans A1 and characterization of the sorbitol dehydrogenase activity, sorbitol being carbon source restrained the microorganism from growing. When the catalytical reaction was carried out by the sorbitol dehydrogenase, the more oxygen in the reactive system, the more cells required, the react rate was faster. At the same quantity of oxygen, when the concentration of the resting cells was the critical value, the react rate was the highest. The optimum temperature was 28℃and the optimum pH was 5.5.
By UV mutation to G. oxydans A1, a high sorbitol dehydrogenase activity strain named as G. oxydans Gouv2007 which biomass was raised 11.2% was screened by grads breeding with sorbitol dehydrogenase activity per boimass being the same as G. oxydans A1 and cultivating time being shortened 6 hours. The kinetics were established for G. oxydans A1 and G. oxydans Gouv2007, and the kinetic models of the two strains were substrate restrain,μmax being 0.2165h-1 and 0.2507h-1, Ki being 1.5069g/L and 3.9663g/L, respectively. G. oxydans Gouv2007 abolished restrain in part.
By optimizing the conditions of G. oxydans Gouv2007 by single experiment, the optimum carbon source sorbitol, nitrogen source yeast extract powder, minerals MgSO4 and K2HPO4, rotating speed 200r/min, liquid volume 20%, cultivating temperature 28℃and initial pH nature were obtained. Using central composite design and response surface analysis, the optimum concentration of sorbitol, yeast extract powder and K2HPO4 was 18.0g/L, 10.0g/L and 3.0g/L respectively with the biomass (1.333g/L) being raised 34.6% and enzyme activity per boimass being the same as G. oxydans A1.G. oxydans Gouv2007 was cultivated in 2L bioreactor with the biomass being 5.580g/L, sorbitol dehydrogenase activity per boimass was 1.1 U/mg.
The enzymatic characteristics about the sorbitol dehydrogenase of G. oxydans Gouv2007 was studied. When pH of the reactive system was maintained 5.5 and the temperature was 28℃sorbitol dehydrogenase activity per biomass was the highest. At the course of reaction, the sorbitol dehydrogenase almost did not lose its activity. When sorbitol was almost consumed and feed-beatch was carried out twice, the sorbitol dehydrogenase lost its activity little each time. Being reused once, it lose activity a little, but being reused twice, it lost 40% activity.
Glycerol was added gradiently in sorbitol medium cultivating G. oxydans Gouv2007 to increase sorbitol dehydrogenase activity. The concentration of glycerol being 20.0g/L, sorbitol dehydrogenase activity per biomass was 1.9U/mg being raised 63%. The storage stability was raised, too. After 30 days storage, the enzyme activity still remained 75%, not-inducted 30%. Ks was modificated from 51mmol/L to 38 mmol/L, signalling of a 1.34-fold increase in affinity toward sorbitol. So the technology with which to produce high sorbitol dehydrogenase activity by glycerol inducting was established.
Glucose and ethanolamines were catalytically hydrogenated to N-(2-hydroxyethyl)-glucamine with a transformation rate of 89%. N-(2-hydroxyethyl)-glucamine was then dehydrogenated to 6-(2-droxyethyl)amino-6-deoxy-a-L-sorbofiuanos by the high activity sorbitol dehydrogenase of G. oxydans Gouv2007 through aeration. At last 6-(2-droxyethyl)-amino-6-deoxy-a-L-sorbofiuanose was hydrogenated catalytically to miglitol. From the transformation of N-(2-hydroxyethyl)-glucamine to miglitol, the total transformation rate was 77.3% and 73.6% respectively by separating and identifying with TLC and ion exchange chromatography and the product rate was 62.7%. After purifying the sample of miglitol, it was white powder and its melting point was 142～147℃. At last, it was confirmed with IR and 1HNMR.
通过对G. oxydans A1的培养和其细胞膜山梨醇脱氢酶活力的研究表明,碳源浓度较高时,抑制细胞生长。山梨醇脱氢酶催化生物转化反应时,反应液中溶氧量越大,反应需求的细胞浓度越大,反应速率越大；当反应液中的溶氧量一定,细胞浓度达到临界值时,反应速率最大；山梨醇脱氢酶的最适作用温度为28℃,最适pH值为5.5。
以G. oxydans A1为出发菌株,通过紫外线诱变结合梯度平板半理性筛选获得一株高山梨醇脱氢酶活力突变株G. oxydans Gouv2007,其单位生物量的山梨醇脱氢酶活力与原始菌株持平,而生物量比原始菌株提高了11.2%,培养时间缩短了6小时。研究二者的发酵动力学,结果表明均符合底物抑制动力学模型,其最大比生长速率分别为0.2165h-1和0.2507h-1,Ki分别为1.5069g／L和3.9663g／L,突变株部分地解除了底物抑制现象。
单因素优化实验表明,碳、氮源分别为山梨醇、酵母浸粉,无机盐为硫酸镁和磷酸氢二钾,摇床转速为200r／min,装液量为20%,培养温度为28℃,培养基初始pH自然时,突变株的生物量和山梨醇脱氢酶的活力最高。采用中心组合设计和响应面分析,获得了细胞生长的最优培养条件：山梨醇18.0g／L,酵母浸粉10.0g／L,磷酸氢二钾3.0g／L其最终生物量达到1.333g／L,比未优化前提高了34.6%；在2L发酵罐中放大培养,生物量达到了5.580g／L,单位生物量的山梨醇脱氢酶活力为1.1U／mg干细胞。
对突变菌株细胞膜山梨醇脱氢酶性质作初步研究,最佳酶反应条件为：pH5.5,28℃。单批次操作稳定性较高,在转化反应的整个过程中基本没有失活；当底物耗尽时,连续进行两次补加底物,酶活力降低较小；多批次操作稳定性较差,首次重复使用时,酶活力有较小的降低,二次重复使用时,酶活力丧失40%。
在山梨醇培养基中梯度增大甘油浓度诱导提高山梨醇脱氢酶活力,甘油浓度为20.0g／L时,该酶的单位生物量活力达到1.9U／mg干细胞,比未诱导的提高了63%。总活力达到2.2U／mL发酵液,比未诱导的提高了30.5%。其储藏稳定性也相应提高,菌体静息细胞储存一个月时,酶活力保留75%,未经诱导的只保留30%。酶动力学显示,其Ks由诱导前的51mmol/L减小到38mmol/L,对底物的亲和力增加了1.34倍。由此,建立了甘油诱导高活力山梨醇脱氢酶的生产工艺。
以葡萄糖和乙醇胺为原料,合成N-羟乙基葡萄糖胺,葡萄糖转化率为89%；在通气搅拌下,用高活力的山梨醇脱氢酶催化N-羟乙基葡萄糖胺反应生成6-脱氧-6-氨基(N-羟乙基)-α-L-呋喃山梨糖；再加氢还原为米格列醇,分别利用TLC和离子交换层析分离检测到从N-羟乙基葡萄糖胺到生成米格列醇两步反应的总底物转化率为77.3%和73.6%,产物得率为62.7%。对合成的米格列醇经提纯后得到白色固体粉末,测得熔程为142-147℃,经红外光谱和核磁共振氢谱分析,该产物与米格列醇标准品谱图吻合良好。
Miglitol, a kind of N-hydroxyethyl ramification of 1-Deoxynojirimycin can strongly inhibit a-glucosidases. It has been first chosen as therapeutic drugs for treatment of type 2 diabetes mellitus. Up to day, there are chemical and combined biotechnological-chemical two methods to synthesize miglitol.The former is more complicated than the latter, and synthetic cost higher. In the latter method, for membrane-bound sorbitol dehydrogenase of Gluconobacter oxydans catalyticly oxidizing N-(2-hydroxyethyl)-glucamine to 6-(2-droxyethyl)amino-6-deoxy-a-L-sorbofiuanos is the key step. Sorbitol dehydrogenase total activity and storage stability of the cells produced by feimentation is small. In this study a high sorbitol dehydrogenase activity strain was obtainied. To raise the productivity of miglitol the combined biotechnological-chemical technology was improved with the high sorbitol dehydrogenase activity whole cells being catalyst.
By studying cultivation of G. oxydans A1 and characterization of the sorbitol dehydrogenase activity, sorbitol being carbon source restrained the microorganism from growing. When the catalytical reaction was carried out by the sorbitol dehydrogenase, the more oxygen in the reactive system, the more cells required, the react rate was faster. At the same quantity of oxygen, when the concentration of the resting cells was the critical value, the react rate was the highest. The optimum temperature was 28℃and the optimum pH was 5.5.
By UV mutation to G. oxydans A1, a high sorbitol dehydrogenase activity strain named as G. oxydans Gouv2007 which biomass was raised 11.2% was screened by grads breeding with sorbitol dehydrogenase activity per boimass being the same as G. oxydans A1 and cultivating time being shortened 6 hours. The kinetics were established for G. oxydans A1 and G. oxydans Gouv2007, and the kinetic models of the two strains were substrate restrain,μmax being 0.2165h-1 and 0.2507h-1, Ki being 1.5069g/L and 3.9663g/L, respectively. G. oxydans Gouv2007 abolished restrain in part.
By optimizing the conditions of G. oxydans Gouv2007 by single experiment, the optimum carbon source sorbitol, nitrogen source yeast extract powder, minerals MgSO4 and K2HPO4, rotating speed 200r/min, liquid volume 20%, cultivating temperature 28℃and initial pH nature were obtained. Using central composite design and response surface analysis, the optimum concentration of sorbitol, yeast extract powder and K2HPO4 was 18.0g/L, 10.0g/L and 3.0g/L respectively with the biomass (1.333g/L) being raised 34.6% and enzyme activity per boimass being the same as G. oxydans A1.G. oxydans Gouv2007 was cultivated in 2L bioreactor with the biomass being 5.580g/L, sorbitol dehydrogenase activity per boimass was 1.1 U/mg.
The enzymatic characteristics about the sorbitol dehydrogenase of G. oxydans Gouv2007 was studied. When pH of the reactive system was maintained 5.5 and the temperature was 28℃sorbitol dehydrogenase activity per biomass was the highest. At the course of reaction, the sorbitol dehydrogenase almost did not lose its activity. When sorbitol was almost consumed and feed-beatch was carried out twice, the sorbitol dehydrogenase lost its activity little each time. Being reused once, it lose activity a little, but being reused twice, it lost 40% activity.
Glycerol was added gradiently in sorbitol medium cultivating G. oxydans Gouv2007 to increase sorbitol dehydrogenase activity. The concentration of glycerol being 20.0g/L, sorbitol dehydrogenase activity per biomass was 1.9U/mg being raised 63%. The storage stability was raised, too. After 30 days storage, the enzyme activity still remained 75%, not-inducted 30%. Ks was modificated from 51mmol/L to 38 mmol/L, signalling of a 1.34-fold increase in affinity toward sorbitol. So the technology with which to produce high sorbitol dehydrogenase activity by glycerol inducting was established.
Glucose and ethanolamines were catalytically hydrogenated to N-(2-hydroxyethyl)-glucamine with a transformation rate of 89%. N-(2-hydroxyethyl)-glucamine was then dehydrogenated to 6-(2-droxyethyl)amino-6-deoxy-a-L-sorbofiuanos by the high activity sorbitol dehydrogenase of G. oxydans Gouv2007 through aeration. At last 6-(2-droxyethyl)-amino-6-deoxy-a-L-sorbofiuanose was hydrogenated catalytically to miglitol. From the transformation of N-(2-hydroxyethyl)-glucamine to miglitol, the total transformation rate was 77.3% and 73.6% respectively by separating and identifying with TLC and ion exchange chromatography and the product rate was 62.7%. After purifying the sample of miglitol, it was white powder and its melting point was 142～147℃. At last, it was confirmed with IR and 1HNMR.
引文
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