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环氧琥珀酸水解酶工程菌的构建及在L-(+)-酒石酸生产中的应用
Alternative TitleConstruction of a cis-epoxysuccinate hydrolase engineering bacterium and its application in the efficient production of L-(+)-tartaic acid
王自强
Subtype博士
Thesis Advisor苏志国
2013-05-01
Degree Grantor中国科学院研究生院
Degree Discipline生物化工
Keyword顺式环氧琥珀酸水解酶   l-(+)-酒石酸   基因克隆与表达   非均相酶促转化   金属螯合亲和固定化
Abstract本文研究了顺式环氧琥珀酸水解酶(ESH)的发酵、纯化及生化特性,构建了表达可溶性重组ESH的E.coli工程菌,开发了基于该工程菌的L-(+)-酒石酸的酶促生产工艺。 研究了野生菌(N. tartaricans)发酵生产ESH的过程,从中发现葡萄糖对ESH的诱导表达具有一定的阻遏作用。采用补料分批发酵策略控制基质浓度可以提高ESH的发酵单位,并放大到100 L发酵罐,发酵周期约44 h,菌体浓度达48.4 g/L,ESH比酶活力达208.45 U/g,比批次发酵提高了38.9 %。 对ESH进行了分离纯化,粗酶液经硫酸铵分级沉淀以及DEAE Sepharose Fast Flow等层析获得电泳纯的ESH,比酶活力为9.24×102 μmol/(min?mg),收率为18.8 %。Edman测序显示,N-末端氨基酸序列为MQLNNANDNTQF。研究发现,ESH是一个单亚基蛋白质,相对分子质量为28,139 Da,其最适反应温度和pH分别为37℃和8.0。Fe3+、Fe2+、Cu2+、Ag+及表面活性剂显著抑制ESH的活性。此外,ESH的催化活性不依赖于二价金属离子,但其活性中心可能存在某种金属离子并在酶构象稳定方面起到一定的积极作用。 构建了温度诱导表达的、具有噬菌体抗性的E.coli Trans1-T1/pBV220-ESH工程菌,优化了其培养条件。2000 L发酵结果显示,该工程菌的菌体浓度可达62.45 g/L,重组ESH酶活力为380.17 U/mg,发酵单位为1.25×106 U/(h?L),是野生菌株ESH的150倍,高于已报道的该酶的发酵水平,而且单位酶活的生产成本降低了85 %。 研究了E.coli工程菌细胞催化生产L-(+)-酒石酸的过程工艺。ESH酶用量为240 U/100 mL时,E.coli 工程菌细胞的催化效率为8.99×10-3 g/(U?h),L-(+)-酒石酸产率为21.59 g/(L?h)。设计了Na-Ca双底物非均相L-(+)-酒石酸酶促转化工艺并完成了中试,在30 m3反应釜中,重组ESH的催化效率为7.92 ± 1.34 ×10-3 g/(U?h),L-(+)-酒石酸的产率提高了近60 %,该工艺已在企业生产中获得应用。 研究了通过组氨酸标签固定化ESH的催化过程。采用金属螯合层析介质直接吸附带His-tag的ESH,固定化ESH的酶活收率为94.59 %。最佳催化条件下,ESH的催化效率为1.04×10-2 g/(U?h),L-(+)-酒石酸的产率为1.08×103 g/(L?h),高于E.coli细胞的催化效率。
Other AbstractIn this paper, the fermentation, purification and characterization of cis-epoxysuccinate hydrolase (ESH) were studied. An E.coli engineering bacterium was constructed to express recombinant ESH as a soluble form. Based on the E.coli cell conversion process, a heterogeneous L-(+)-tartaric acid production technology was developed. The fermentation process of N. tartaricans ESH was studied. It was found that glucose could repress the expression of ESH at a certain degree. The fed-batch fermentation strategy was adopted to control the substrate concentration and improve the ESH fermentation efficiency. The 100-L fermentation results showed that the wet cell concentration reached 48.4 g/L, and the specific ESH activity was 208.45 U/g, with a significant improvement of 38.9 %, compared to the batch fermentation process. The purification of N. tartaricans ESH was investigated. ESH could be purified to electrophoretic homogeneity by ammonium sulfate precipitation and chromatographic methods. The specific activity of purified ESH was 9.24×102 μmol/(min?mg), with a yield of 18.8 %. Edman degradation analysis showed that the N-terminal amino acid sequence of ESH was MQLNNANDNTQF. It was found that ESH had a monomeric molecular weight of 28,139 Da, and its optimum reaction conditions were 37℃ and pH 8.0. ESH was strongly inhibited by Fe3+, Fe2+, Cu2+, Ag+ and surfactants. In addition, ESH did not require divalent metal ions to keep its activity, but there might be a metal ion at the active center of enzyme to play a role in the enzyme-substrate reaction or keep the stability of enzyme. An E.coli Trans 1-T1/pBV220-ESH engineering bacterium was constructed which had a phage resistant. After fermentation process optimization, the scale-up experiment was carried out in a 2000-L fermentor. The results showed that the wet cell concentration reached 62.45 g/L, and the specific ESH activity was 380.17 U/mg. The ESH fermentation efficiency reached 1.25×106 U/(h?L), about 150 times increase compared to N. tartaricans ESH, which was higher than the reported results. Moreover, the production cost of unit ESH activity decreased by 85 %. The E.coli whole-cell production of L-(+)-tartaric acid was investigated. At an ESH dosage of 240 U per 100 mL, the E.coli cell catalytic efficiency reached 8.99×10-3 g/(U?h), and the L-(+)-tartaric acid productivity was 21.59 g/(L?h). A Na-Ca double substrates heterogeneous L-(+)-tartaric acid production technology was developed. In the 30 m3 reactor, the catalytic efficiency of recombinant ESH was 7.92 ± 1.34 ×10-3 g/(U?h), with a significant improvement of 60 %. The heterogeneous production technology has been applied in industrial production of L-(+)-tartaric acid. The immobilized ESH conversion process was also investigated. The metal chelating chromatography medium was directly used to adsorb his-tagged ESH by affinity interaction, with an activity yield of 94.59 %. At the optimum reaction conditions, the catalytic efficiency of immobilized ESH was 1.04×10-2 g/(U?h), with an L-(+)-tartaric acid productivity of 1.08×103 g/(L?h), which was higher than that of free or immobilized E.coli cell.
Pages139
Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/8294
Collection研究所(批量导入)
Recommended Citation
GB/T 7714
王自强. 环氧琥珀酸水解酶工程菌的构建及在L-(+)-酒石酸生产中的应用[D]. 中国科学院研究生院,2013.
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