本文選題：異槲皮素　切入點(diǎn)：微生物轉(zhuǎn)化　出處：《河北大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：異槲皮素作為一種藥理活性極高的藥物是目前醫(yī)藥領(lǐng)域研究的熱點(diǎn),但由于該物質(zhì)在自然界中含量較低,化學(xué)提取合成步驟復(fù)雜,影響了異槲皮素的利用推廣。生物酶解法轉(zhuǎn)化蘆丁定向合成異槲皮素被公認(rèn)是規(guī)模化制備異槲皮素的最有效途徑之一。本論文通過~(12)C~(6+)重離子誘變選育高效異槲皮素產(chǎn)生菌并對(duì)其進(jìn)行轉(zhuǎn)化體系及轉(zhuǎn)化條件優(yōu)化,以期提高酶法生產(chǎn)異槲皮素的生產(chǎn)效率,降低生產(chǎn)成本。對(duì)本實(shí)驗(yàn)室篩選的能降解蘆丁生成異槲皮素的岸濱芽孢桿菌(Bacillus litoralis)C44進(jìn)行~(12)C~(6+)重離子誘變育種。初步篩選得到330株pH 9.0,32℃條件下長(zhǎng)勢(shì)良好的突變株。定量TLC篩選得到13株α-L-鼠李糖苷酶轉(zhuǎn)化效率提高的菌株,其中M5B15菌株轉(zhuǎn)化效率最高,遺傳穩(wěn)定性最好。在相同條件下,該菌株相對(duì)原始菌株C44的轉(zhuǎn)化率為171.3%,利用HPLC進(jìn)行復(fù)篩測(cè)得M5B15菌株的摩爾轉(zhuǎn)化效率為出發(fā)菌株的1.55倍。對(duì)突變菌株M5B15的培養(yǎng)基成分和發(fā)酵條件進(jìn)行了優(yōu)化。優(yōu)化前后菌體OD600值分別為2.5±0.12和16.4±0.39,菌體濃度提高了5.6倍。以本實(shí)驗(yàn)室前期優(yōu)化建立的轉(zhuǎn)化條件為基礎(chǔ)進(jìn)一步優(yōu)化轉(zhuǎn)化體系。結(jié)果表明:在pH9.0甘氨酸-氫氧化鈉轉(zhuǎn)化體系中,加入3 g/L蘆丁,濕菌體濃度在100 g/L時(shí),底物完全轉(zhuǎn)化為產(chǎn)物的最短時(shí)間為10 h,比優(yōu)化前縮短14 h,比出發(fā)菌株縮短38 h;經(jīng)HPLC分析,底物的轉(zhuǎn)化率是優(yōu)化前的2.27倍,是原始出發(fā)菌株的4.62倍,有效地提高了底物的轉(zhuǎn)化效率;在超聲功率為25 kW,超聲乳化時(shí)間30 min條件下,大豆卵磷脂能夠與濃度為10 g/L的蘆丁以摩爾比1:1產(chǎn)生良好的包合作用,并且加入大豆卵磷脂后菌株的相對(duì)轉(zhuǎn)化率提高12.6%。研究得出,1L體系的最佳反應(yīng)條件為底物濃度10 g/L,細(xì)胞濃度為350 g/L,轉(zhuǎn)化時(shí)間24 h。進(jìn)一步研究得知,在轉(zhuǎn)化體系中添加濃度為20 g/L葡萄糖和10 mmol/L Zn2+,底物轉(zhuǎn)化速率提高18.0%。本論文通過對(duì)異槲皮素產(chǎn)生菌C44進(jìn)行~(12)C~(6+)重離子誘變育種,最終篩選得到一株高活性突變株M5B15,在底物濃度3 g/L時(shí),其細(xì)胞轉(zhuǎn)化時(shí)間大大縮短,摩爾轉(zhuǎn)化效率明顯提高;對(duì)突變菌株M5B15的培養(yǎng)基成分與發(fā)酵條件的優(yōu)化有效提高了菌體單位時(shí)間的生物量,降低了發(fā)酵成本。通過對(duì)轉(zhuǎn)化體系的優(yōu)化,縮短了底物濃度3 g/L時(shí)的轉(zhuǎn)化時(shí)間,底物轉(zhuǎn)化效率顯著提高。通過對(duì)底物蘆丁的乳化包合,將底物溶解度由3 g/L提高到10 g/L,同時(shí)將轉(zhuǎn)化體系擴(kuò)大至1 L,為異槲皮素的規(guī)�；a(chǎn)奠定了基礎(chǔ)。
[Abstract]:Isoquercetin, as a drug with high pharmacological activity, is a hot topic in the field of medicine. However, because of its low content in nature, the chemical extraction and synthesis process is complex. The application of isoquercetin was affected. It was recognized that the bioenzymatic transformation of rutin into isoquercetin was one of the most effective ways to produce isoquercetin on a large scale. In this paper, high efficiency isoquercetin was selected by heavy ion mutagenesis. The transformation system and transformation conditions of dermatocyte-producing bacteria were optimized. In order to improve the production efficiency of isoquercetin by enzymatic method, In order to reduce the production cost, a strain of Bacillus litoralis)C44, which could degrade rutin to produce isoquercetin, was screened by heavy ion mutagenesis. 330 mutant strains with good growth at pH 9.032 鈩，

本文編號(hào)：1616180