【摘要】：以蘆丁為底物,利用α-L-鼠李糖苷酶生物轉(zhuǎn)化法生產(chǎn)異槲皮素成為當(dāng)今研究的熱點(diǎn)。但迄今為止發(fā)現(xiàn)的α-L-鼠李糖苷酶產(chǎn)生菌都有β-D-葡萄糖苷酶與(或)蕓香糖苷酶活性,致使其專一性很差,產(chǎn)物中常含大量槲皮素等成分,給分離純化造成困難的同時(shí),也降低了異槲皮素的收率。本研究以本實(shí)驗(yàn)室前期篩選得到的能特異高效水解蘆丁為異槲皮素的岸濱芽孢桿菌(Bacillus litoralis)C44為實(shí)驗(yàn)對(duì)象,通過(guò)全基因組測(cè)序來(lái)發(fā)掘其潛在的α-L-鼠李糖苷酶基因、β-D-葡萄糖苷酶基因和蕓香糖苷酶基因,實(shí)現(xiàn)這些基因的克隆及原核表達(dá)。通過(guò)對(duì)重組蛋白進(jìn)行分離純化、活性檢測(cè)、酶學(xué)性質(zhì)測(cè)定等方法來(lái)闡明C44菌株專一轉(zhuǎn)化蘆丁為異槲皮素的作用機(jī)制,從而為促進(jìn)該酶在生物轉(zhuǎn)化法生產(chǎn)異槲皮素中應(yīng)用,降低異槲皮素生產(chǎn)成本奠定基礎(chǔ)。分析C44菌株基因組測(cè)序結(jié)果,得到4個(gè)潛在編碼α-L-鼠李糖苷酶的基因:rha1、rha2、rha3和rha4,其大小分別為2859 bp、2910 bp、2712 bp、1572 bp。同時(shí)發(fā)現(xiàn)C44編碼3個(gè)β-D-葡萄糖苷酶基因,不編碼蕓香糖苷酶基因。Bacillus litoralis C44的3個(gè)β-D-葡萄糖苷酶基因克隆、表達(dá)后,經(jīng)測(cè)定沒(méi)有特異或非特異水解蘆丁的能力。分別將4個(gè)α-L-鼠李糖苷酶基因(rha)進(jìn)行T克隆并構(gòu)建重組表達(dá)載體pET-28a(+)-rha,經(jīng)誘導(dǎo),各基因在E.coli BL21(DE3)中編碼的酶蛋白(RHA)大小分別為:112.468 KD,110.67 KD,106.764 KD,64.595 KD。當(dāng)菌液OD600為0.6時(shí),加入0.2 mmol/L的IPTG,37℃下誘導(dǎo)5 h,則各重組蛋白表達(dá)量占總蛋白的10.13%,18.016%,19.36%,35.258%。在含6%甲醇(V/V),pH 7.0的0.1 mol/L PBS緩沖液中,分別用含有重組蛋白的破碎全菌液轉(zhuǎn)化終濃度為3 mg/mL的蘆丁,經(jīng)TLC檢測(cè),發(fā)現(xiàn)僅rha4編碼蛋白(RHA4)具有活性。對(duì)其進(jìn)行Ni-NTA親和純化,回收濃度為0.335mg/mL,收率為15.59%。RHA4在50℃,pH 8.0的Atkins-Pantin緩沖液中水解蘆丁效果最好;在此條件下,20 mg的破碎菌體,3 h可轉(zhuǎn)化約1.8 mg底物且沒(méi)有副產(chǎn)物的產(chǎn)生。以pNP-α-L-鼠李糖苷為底物測(cè)定重組酶酶學(xué)性質(zhì),結(jié)果是:重組酶最適溫度為60℃,于20-55℃使用時(shí)穩(wěn)定;最適pH值是6.0,且僅在p H6.0附近保持穩(wěn)定;在60℃,p H 6的Mcilvaine’s buffer中,測(cè)得純酶酶活力為372.76 U/mL,比活力為1112.7 U/mg,Vm值是0.31 mmol/L·h,Km值為0.6933 mmol/L;10 mmol/L的Mn2+和Cd2+都可以促進(jìn)RHA4酶活性。
[Abstract]:The production of isoquercetin by 偽-L-rhamnosidase biotransformation with rutin as substrate has become a hot topic. However, the 尾 -D-glucosidase and / or rutosidase activities of 偽 -L-rhamnosidase producing bacteria have been found so far, the specificity of 偽 -L-rhamnosidase is very poor, and the products often contain a lot of quercetin, which makes it difficult to isolate and purify. The yield of isoquercetin was also decreased. In this study, (Bacillus litoralis) C44, which could hydrolyze rutin as isoquercetin, was selected as experimental object. The potential genes of 偽 -L-rhamnosidase, 尾 -D-glucosidase and rutosidase were identified by whole genome sequencing, and the cloning and prokaryotic expression of these genes were realized. The mechanism of specific transformation of Rutin to isoquercetin from C44 strain was elucidated by means of purification, activity detection and enzymatic property determination of the recombinant protein, so as to promote the application of the enzyme in the production of isoquercetin by biotransformation. To reduce the cost of isoquercetin production laid the foundation. Four genes encoding 偽 -L-rhamnosidase were obtained by sequencing the genome of C44 strain. The size of rha1,rha2,rha3 and rha4, were 2859 bp,2910 bp,2712 bp,1572 bp., respectively. At the same time, we found that C44 encodes three 尾 -D-glucosidase genes and three 尾 -D-glucosidase genes that do not encode rutasidase gene. Bacillus litoralis C44. After expression, we have determined that there is no specific or non-specific ability to hydrolyze rutin. Four 偽 -L-rhamnosidase gene (rha) were cloned by T clone and the recombinant expression vector pET-28a ()-rha, was constructed. After induction, the (RHA) size of the enzyme protein encoded by each gene in E.coli BL21 (DE3) was 112.468 KD,110.67 KD,106.764 KD,64.595 KD.. When the concentration of OD600 was 0. 6, the recombinant protein was induced at 0. 2 mmol/L IPTG,37 鈩，

本文編號(hào)：2286034