高产甘露醇明串珠菌中甘露醇脱氢酶的特性分析

发布时间：2018-06-28 15:00

  本文选题：甘露醇 + 甘露醇脱氢酶　； 参考：《延边大学》2015年硕士论文

【摘要】：甘露醇,学名己六醇,可作为甜味剂在饲料中广泛应用。生产甘露醇的方法主要有提取法、化学合成法、酶转化法、微生物发酵法等。微生物发酵法因其可以提高甘露醇的产率并且能避免山梨醇等副产物的产生成为甘露醇生产的主要趋势。目前已经报道了多种微生物具有产生甘露醇的能力,包括霉菌、酵母菌和细菌,其中异型发酵乳酸菌为主要产生菌。明串珠菌作为异型发酵乳酸菌的一种,其合成甘露醇的代谢途径是在甘露醇脱氢酶的作用下催化果糖生成甘露醇。为了研究明串珠菌中甘露醇脱氢酶的特性,使其更好地应用于甘露醇的生产,本实验以假肠膜明串珠菌为研究菌株,从假肠膜明串珠菌中获得甘露醇脱氢酶基因,并在大肠杆菌中获得表达。结果显示：以假肠膜明串珠菌基因组DNA为模板,经PCR扩增出大量甘露醇脱氢酶目的基因,PCR扩增产物为1017bp。将重组质粒pETDuet-1-mdh转入大肠杆菌BL21(DE3)进行转化并且通过SDS-PAGE分析重组蛋白是否成功表达,结果表明,蛋白表达的分子量约为36.007 kDa,与期望的分子量大小一致,说明重组蛋白成功表达,且表达量较高。.为了优化IPTG诱导甘露醇脱氢酶基因表达的条件,试验以IPTG不同浓度、不同诱导时间和不同诱导温度对重组甘露醇脱氢酶基因进行诱导表达。结果显示：IPTG诱导甘露醇脱氢酶表达的最佳浓度为0.5 mM、最佳诱导时间为16 h、最佳诱导温度为30℃。为了进一步了解甘露醇脱氢酶的特性,实验分别对甘露醇脱氢酶最适反应温度、pH,热稳定性以及化学抑制剂进行了测定。结果显示：甘露醇脱氢酶最适反应温度为30℃；最适反应pH值为7；加入Ca2+、尿素会促进酶反应,提高酶活性,但差别不大；加入Fe2+、Ba2+、Zn2+、Cu2+、Na+以及SDS酶活性受抑制,、其中,Na+、SDS对酶活性的抑制作用较强；对酶在30℃-80℃热处理10 min,其中,30℃、40℃处理后,酶活性损失25%左右,50℃酶活性损失32%,60℃酶活性损失47%、70℃处理后酶活性损失较严重,酶活性损失60%左右；80℃处理10 min后酶活性全部损失,残余酶活为0。本实验得到结果为明串珠菌应用于甘露醇的生产提供理论依据。
[Abstract]:Mannitol, the scientific name of hexanol, can be widely used as a sweetener in feed. The main methods of producing mannitol include extraction, chemical synthesis, enzymatic transformation, microbial fermentation and so on. Microbial fermentation can improve the yield of mannitol and avoid the production of sorbitol and other by-products become the main trend of mannitol production. A variety of microbes have been reported to have the ability to produce mannitol, including mold, yeast and bacteria. As a kind of lactic acid bacteria, the metabolic pathway for the synthesis of mannitol is to catalyze the production of mannitol from fructose by mannitol dehydrogenase. In order to study the properties of mannitol dehydrogenase in Streptococcus spp. And to make it more suitable for the production of mannitol, the gene of mannitol dehydrogenase was obtained from Candida spp. And expressed in E. coli. The results showed that a large amount of mannitol dehydrogenase target gene was amplified by PCR using the genomic DNA of Streptococcus pseudointestinal membrane as template and the PCR product was 1017bp. The recombinant plasmid pETDuet-1-mdh was transformed into E. coli BL21 (DE3) and the recombinant protein was successfully expressed by SDS-PAGE. The results showed that the molecular weight of the recombinant protein was about 36.007 kDa, which was consistent with the expected molecular weight, indicating that the recombinant protein was successfully expressed. And the expression amount is higher. In order to optimize the expression conditions of mannitol dehydrogenase gene induced by IPTG, the recombinant mannitol dehydrogenase gene was induced by IPTG with different concentration, time and temperature. The results showed that the optimal concentration, time and temperature for inducing the expression of mannitol dehydrogenase were 0.5 mm, 16 h and 30 鈩，

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