构建组织工程细胞凝胶包衣片用于合成手性醇
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摘要
高分子材料聚乙烯醇常用作固定化细胞或组织工程中,具有良好的机械强度,但其生物相容性较差,膨胀率较高,应用时有其局限性;而丝素蛋白用于组织工程时具有好的生物相容性。此外,完整细胞作为生物催化剂,能确保主酶及其辅助因子发挥作用,但游离细胞的重复使用率低。因此,本文以聚乙烯醇/丝素蛋白复合物为原料、以啤酒酵母细胞为种子、以聚乙烯醇/有机蒙脱石纳米复合凝胶为包衣材料构建组织工程啤酒酵母细胞凝胶包衣片,考察聚乙烯醇/丝素蛋白复合支架的特性,以及这种凝胶包衣片的性质研究,同时,利用该种组织工程啤酒酵母细胞凝胶包衣片作为生物催化剂、以乙酰乙酸乙酯为原料不对称催化还原合成(S)-3-羟基丁酸乙酯((S)-EHB)。
首先,以聚乙烯醇/丝素蛋白复合物为支架材料,制备出直径为5mm、厚度为2mm的碟状三维多孔支架。通过对聚乙烯醇浓度优化以及与丝素蛋白的配比、致孔剂使用量对孔隙率影响,得出浓度为14%的聚乙烯醇溶液与9.8%的丝素蛋白溶液4∶1的比例、按照1.4g/ml致孔剂的量以饱和硼酸为交联剂制备出的碟状三维多孔支架,孔隙率达92%,其膨胀率较低,机械强度高,操作较容易,适合用作细胞培养。
其次,以该种碟状三维支架作为载体,用以接种啤酒酵母细胞进行培养,以聚乙烯醇/有机蒙脱石纳米复合凝胶作为包衣材料对生长了细胞的三维支架进行包衣,得到组织工程细胞凝胶包衣片。考察支架的细胞载量、包衣效果,以及该种凝胶包衣片作为生物催化剂在不同pH和温度下凝胶包衣片内细胞醇脱氢酶(alcohol dehydrogenase, ADH)的活性影响、凝胶包衣片细胞的储藏稳定性。实验结果表明,这种三维支架组织工程凝胶包衣片的细胞载量可达34%,对支架进行包衣,厚度100～200μm无细胞泄流细胞。该种凝胶包衣片细胞胞内醇脱氢酶(ADH)活性的最适pH范围为7.0～8.0,最佳温度为30～35℃,与游离细胞相比无论是pH还是温度,其耐受能力都得到明显提高。另外,其储藏稳定性也得到了明显的提高,在4℃储藏30天后,包衣片内细胞的醇脱氢酶(ADH)活性还保留了其最初活性的74%。
最后,以这种组织工程细胞凝胶包衣片作为生物催化剂用来生物催化不对称合成手性醇(S) -3-羟基丁酸乙酯((S)-EHB)。考察其反应体系温度、环境(磷酸缓冲液) pH值以及反应时间对其产率和对映体过量(enantiomeric excess,e.e.)的影响以及该种凝胶包衣片的重复使用性。实验结果表明用PVA/OMONT纳米复合凝胶固定化细胞催化合成(S)- 3-羟基丁酸乙酯((S)-EHB)的产率可达到96.5%,e.e.值达到97.3%。这种组织工程细胞凝胶包衣片在重复使用9次后,包衣片内细胞的醇脱氢酶(ADH)活性还保留了其最初活性的67%。
Polymer polyvinyl alcohol is used as the immobilized cells or tissue engineering, has good mechanical strength, but its poor biocompatibility, high expansion rate lead to its limitations when applied. And when silk fibroin is used to tissue engineering with good biocompatibility. In addition, the whole cell could ensure its main enzymes and cofactors played key role, when used as biological catalyst, but the free cells had low utilization rate when repeated. Therefore, in this article, we constructed disk shape scaffolds with polyvinyl alcohol (PVA) / silk fibroin complex materials and cultured with Saccharomyces cerevisiae cells in it. The loading cells scaffolds were coated with nanocomposite gel of polyvinyl alcohol (PVA) / organic montmorillonite(OMMT). The characteristics of composite scaffolds and the properties of this coated cell-scaffold were studied in this article. In this study, ethyl (S) - 3 - hydroxyl butyrate ((S) - EHB) was asymmetric synthesized from bioreduction of ethyl acetoacetate by the coated cell-scaffold as biocatalyst.
First of all, the disk shape three-dimensional porous scaffolds(5mm diameter, 2mm thickness) were prepared with PVA/SF composites by crosslinking with saturated boric acid, and its porosity reached 92%. After screening concentration of PVA, the ratio of PVA and SF and quantity of porogen, optimization result is that the ratio of gel beads of 14% PVA and 9.8% SF is 4∶1 and amount of porogen is 1.4g/ml. The porous scaffolds prepared according above conditions possess high mechanical strength, low-expansion ratio and preparation of them also were easily operated. It was suitable for cells culture.
Secondly, this kind of disc scaffolds as a carrier for seeding of Saccharomyces cerevisiae were cultured. Then the cell-scaffold composites were coated with about 200μm thickness PVA/OMONT nanocomposite gel. We investigated embedding situation, cells load of the coated cell-scaffold, effects of pH and temperature on intracellular ADH activity of the coated cell-scaffold and storage stability of it. The experimental results indicate that the cells load capacity of the coated cell-scaffold reach to 34% and it has not cells leakage after coating. In addition, the optimum pH and temperature on ADH activity of the coated cell-scaffold at pH7.0-8.0 and 35℃and the intracellular ADH in the coated cell-scaffold retained over 74% of it original activity after one month of storage. It can be seen that the coated cell-scaffold had good storage stability.
Finally, we investigated that pH and temperature affect on the yield and the enantiomeric excess (e.e.) of the (S)-ethyl-3-hydroxy butyrate when using the coated cell-scaffold as biocatalyst. The result showed that the yield reached 96.5% and e.e reached 97.3% at pH7.0-8.0 and 35℃. Furthermore, the coated cell-scaffold also showed good reusability. The intracellular alcohol dehydrogenase (ADH) in the coated cell-scaffold retained 67% of its initial activity after 9 cycles.
首先,以聚乙烯醇/丝素蛋白复合物为支架材料,制备出直径为5mm、厚度为2mm的碟状三维多孔支架。通过对聚乙烯醇浓度优化以及与丝素蛋白的配比、致孔剂使用量对孔隙率影响,得出浓度为14%的聚乙烯醇溶液与9.8%的丝素蛋白溶液4∶1的比例、按照1.4g/ml致孔剂的量以饱和硼酸为交联剂制备出的碟状三维多孔支架,孔隙率达92%,其膨胀率较低,机械强度高,操作较容易,适合用作细胞培养。
其次,以该种碟状三维支架作为载体,用以接种啤酒酵母细胞进行培养,以聚乙烯醇/有机蒙脱石纳米复合凝胶作为包衣材料对生长了细胞的三维支架进行包衣,得到组织工程细胞凝胶包衣片。考察支架的细胞载量、包衣效果,以及该种凝胶包衣片作为生物催化剂在不同pH和温度下凝胶包衣片内细胞醇脱氢酶(alcohol dehydrogenase, ADH)的活性影响、凝胶包衣片细胞的储藏稳定性。实验结果表明,这种三维支架组织工程凝胶包衣片的细胞载量可达34%,对支架进行包衣,厚度100～200μm无细胞泄流细胞。该种凝胶包衣片细胞胞内醇脱氢酶(ADH)活性的最适pH范围为7.0～8.0,最佳温度为30～35℃,与游离细胞相比无论是pH还是温度,其耐受能力都得到明显提高。另外,其储藏稳定性也得到了明显的提高,在4℃储藏30天后,包衣片内细胞的醇脱氢酶(ADH)活性还保留了其最初活性的74%。
最后,以这种组织工程细胞凝胶包衣片作为生物催化剂用来生物催化不对称合成手性醇(S) -3-羟基丁酸乙酯((S)-EHB)。考察其反应体系温度、环境(磷酸缓冲液) pH值以及反应时间对其产率和对映体过量(enantiomeric excess,e.e.)的影响以及该种凝胶包衣片的重复使用性。实验结果表明用PVA/OMONT纳米复合凝胶固定化细胞催化合成(S)- 3-羟基丁酸乙酯((S)-EHB)的产率可达到96.5%,e.e.值达到97.3%。这种组织工程细胞凝胶包衣片在重复使用9次后,包衣片内细胞的醇脱氢酶(ADH)活性还保留了其最初活性的67%。
Polymer polyvinyl alcohol is used as the immobilized cells or tissue engineering, has good mechanical strength, but its poor biocompatibility, high expansion rate lead to its limitations when applied. And when silk fibroin is used to tissue engineering with good biocompatibility. In addition, the whole cell could ensure its main enzymes and cofactors played key role, when used as biological catalyst, but the free cells had low utilization rate when repeated. Therefore, in this article, we constructed disk shape scaffolds with polyvinyl alcohol (PVA) / silk fibroin complex materials and cultured with Saccharomyces cerevisiae cells in it. The loading cells scaffolds were coated with nanocomposite gel of polyvinyl alcohol (PVA) / organic montmorillonite(OMMT). The characteristics of composite scaffolds and the properties of this coated cell-scaffold were studied in this article. In this study, ethyl (S) - 3 - hydroxyl butyrate ((S) - EHB) was asymmetric synthesized from bioreduction of ethyl acetoacetate by the coated cell-scaffold as biocatalyst.
First of all, the disk shape three-dimensional porous scaffolds(5mm diameter, 2mm thickness) were prepared with PVA/SF composites by crosslinking with saturated boric acid, and its porosity reached 92%. After screening concentration of PVA, the ratio of PVA and SF and quantity of porogen, optimization result is that the ratio of gel beads of 14% PVA and 9.8% SF is 4∶1 and amount of porogen is 1.4g/ml. The porous scaffolds prepared according above conditions possess high mechanical strength, low-expansion ratio and preparation of them also were easily operated. It was suitable for cells culture.
Secondly, this kind of disc scaffolds as a carrier for seeding of Saccharomyces cerevisiae were cultured. Then the cell-scaffold composites were coated with about 200μm thickness PVA/OMONT nanocomposite gel. We investigated embedding situation, cells load of the coated cell-scaffold, effects of pH and temperature on intracellular ADH activity of the coated cell-scaffold and storage stability of it. The experimental results indicate that the cells load capacity of the coated cell-scaffold reach to 34% and it has not cells leakage after coating. In addition, the optimum pH and temperature on ADH activity of the coated cell-scaffold at pH7.0-8.0 and 35℃and the intracellular ADH in the coated cell-scaffold retained over 74% of it original activity after one month of storage. It can be seen that the coated cell-scaffold had good storage stability.
Finally, we investigated that pH and temperature affect on the yield and the enantiomeric excess (e.e.) of the (S)-ethyl-3-hydroxy butyrate when using the coated cell-scaffold as biocatalyst. The result showed that the yield reached 96.5% and e.e reached 97.3% at pH7.0-8.0 and 35℃. Furthermore, the coated cell-scaffold also showed good reusability. The intracellular alcohol dehydrogenase (ADH) in the coated cell-scaffold retained 67% of its initial activity after 9 cycles.
引文
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[20] Bezbradica D, Obradovic B, Leskosek-Cukalovic I, et al. Immobilization of yeast cells in PVA particles for beer fermentation[J]. Proc Biochem. 2007; 42: 1348- 1351.
[21] Kubá? D, ?ejkováA, Masák J, et al. Biotransformation of nitriles by Rhodococcus equi A4 immobilized in LentiKats?[J]. Journal of Molecular Catalysis B: Enzymatic. 2006;39(1-4):59-61.
[22] Gong G H, Hou Y, Yu M-A*,et al. A new approach for the immobilization of permeabilized brewer’s yeast cells in a modified composite polyvinyl alcohol lens-shaped capsule containing montmorillonite and dimethyldioctadecylammonium bromide for use as a biocatalyst[J]. Process Biochemistry, 2010,45:1445-1449.学博士论文,2005年.
[17]敬科举,徐志南,林建平等.重组大肠杆菌细胞不对称还原4-氯乙酰乙酸乙酯合成(R)- (+)-4-氯-3-羟基丁酸乙酯[J].催化学报, 2005;26(11):993-998.
[18] Gowda LR, Bachhawat N and Bhat SG. Permeabilization of bakers’yeast by cetyltrimethylammonium bromide for intracellular enzyme catalysis[J]. Enzyme Microb Technol,1991;13(2):154-157.
[19] Yu M-A, Wei YM, Jiang L, et al. Bioconversion of ethyl 4-chloro-3-oxobutanoate by permeabilized fresh brewer's yeast cells in the presence of allyl bromide[J]. J Ind Microbiol Biotechnol, 2007;34(2):151-156.
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