本文選題：甘露醇 + 甘露醇脫氫酶�。� 參考：《延邊大學(xué)》2015年碩士論文

【摘要】：甘露醇,學(xué)名己六醇,可作為甜味劑在飼料中廣泛應(yīng)用。生產(chǎn)甘露醇的方法主要有提取法、化學(xué)合成法、酶轉(zhuǎn)化法、微生物發(fā)酵法等。微生物發(fā)酵法因其可以提高甘露醇的產(chǎn)率并且能避免山梨醇等副產(chǎn)物的產(chǎn)生成為甘露醇生產(chǎn)的主要趨勢。目前已經(jīng)報道了多種微生物具有產(chǎn)生甘露醇的能力,包括霉菌、酵母菌和細菌,其中異型發(fā)酵乳酸菌為主要產(chǎn)生菌。明串珠菌作為異型發(fā)酵乳酸菌的一種,其合成甘露醇的代謝途徑是在甘露醇脫氫酶的作用下催化果糖生成甘露醇。為了研究明串珠菌中甘露醇脫氫酶的特性,使其更好地應(yīng)用于甘露醇的生產(chǎn),本實驗以假腸膜明串珠菌為研究菌株,從假腸膜明串珠菌中獲得甘露醇脫氫酶基因,并在大腸桿菌中獲得表達。結(jié)果顯示：以假腸膜明串珠菌基因組DNA為模板,經(jīng)PCR擴增出大量甘露醇脫氫酶目的基因,PCR擴增產(chǎn)物為1017bp。將重組質(zhì)粒pETDuet-1-mdh轉(zhuǎn)入大腸桿菌BL21(DE3)進行轉(zhuǎn)化并且通過SDS-PAGE分析重組蛋白是否成功表達,結(jié)果表明,蛋白表達的分子量約為36.007 kDa,與期望的分子量大小一致,說明重組蛋白成功表達,且表達量較高。.為了優(yōu)化IPTG誘導(dǎo)甘露醇脫氫酶基因表達的條件,試驗以IPTG不同濃度、不同誘導(dǎo)時間和不同誘導(dǎo)溫度對重組甘露醇脫氫酶基因進行誘導(dǎo)表達。結(jié)果顯示：IPTG誘導(dǎo)甘露醇脫氫酶表達的最佳濃度為0.5 mM、最佳誘導(dǎo)時間為16 h、最佳誘導(dǎo)溫度為30℃。為了進一步了解甘露醇脫氫酶的特性,實驗分別對甘露醇脫氫酶最適反應(yīng)溫度、pH,熱穩(wěn)定性以及化學(xué)抑制劑進行了測定。結(jié)果顯示：甘露醇脫氫酶最適反應(yīng)溫度為30℃；最適反應(yīng)pH值為7；加入Ca2+、尿素會促進酶反應(yīng),提高酶活性,但差別不大；加入Fe2+、Ba2+、Zn2+、Cu2+、Na+以及SDS酶活性受抑制,、其中,Na+、SDS對酶活性的抑制作用較強；對酶在30℃-80℃熱處理10 min,其中,30℃、40℃處理后,酶活性損失25%左右,50℃酶活性損失32%,60℃酶活性損失47%、70℃處理后酶活性損失較嚴重,酶活性損失60%左右；80℃處理10 min后酶活性全部損失,殘余酶活為0。本實驗得到結(jié)果為明串珠菌應(yīng)用于甘露醇的生產(chǎn)提供理論依據(jù)。
[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，

本文編號：2078362