Bacillus litoralis C44 α-L-鼠李糖苷酶基因的克
发布时间：2018-10-21 18:51
【摘要】：以芦丁为底物,利用α-L-鼠李糖苷酶生物转化法生产异槲皮素成为当今研究的热点。但迄今为止发现的α-L-鼠李糖苷酶产生菌都有β-D-葡萄糖苷酶与(或)芸香糖苷酶活性,致使其专一性很差,产物中常含大量槲皮素等成分,给分离纯化造成困难的同时,也降低了异槲皮素的收率。本研究以本实验室前期筛选得到的能特异高效水解芦丁为异槲皮素的岸滨芽孢杆菌(Bacillus litoralis)C44为实验对象,通过全基因组测序来发掘其潜在的α-L-鼠李糖苷酶基因、β-D-葡萄糖苷酶基因和芸香糖苷酶基因,实现这些基因的克隆及原核表达。通过对重组蛋白进行分离纯化、活性检测、酶学性质测定等方法来阐明C44菌株专一转化芦丁为异槲皮素的作用机制,从而为促进该酶在生物转化法生产异槲皮素中应用,降低异槲皮素生产成本奠定基础。分析C44菌株基因组测序结果,得到4个潜在编码α-L-鼠李糖苷酶的基因:rha1、rha2、rha3和rha4,其大小分别为2859 bp、2910 bp、2712 bp、1572 bp。同时发现C44编码3个β-D-葡萄糖苷酶基因,不编码芸香糖苷酶基因。Bacillus litoralis C44的3个β-D-葡萄糖苷酶基因克隆、表达后,经测定没有特异或非特异水解芦丁的能力。分别将4个α-L-鼠李糖苷酶基因(rha)进行T克隆并构建重组表达载体pET-28a(+)-rha,经诱导,各基因在E.coli BL21(DE3)中编码的酶蛋白(RHA)大小分别为:112.468 KD,110.67 KD,106.764 KD,64.595 KD。当菌液OD600为0.6时,加入0.2 mmol/L的IPTG,37℃下诱导5 h,则各重组蛋白表达量占总蛋白的10.13%,18.016%,19.36%,35.258%。在含6%甲醇(V/V),pH 7.0的0.1 mol/L PBS缓冲液中,分别用含有重组蛋白的破碎全菌液转化终浓度为3 mg/mL的芦丁,经TLC检测,发现仅rha4编码蛋白(RHA4)具有活性。对其进行Ni-NTA亲和纯化,回收浓度为0.335mg/mL,收率为15.59%。RHA4在50℃,pH 8.0的Atkins-Pantin缓冲液中水解芦丁效果最好;在此条件下,20 mg的破碎菌体,3 h可转化约1.8 mg底物且没有副产物的产生。以pNP-α-L-鼠李糖苷为底物测定重组酶酶学性质,结果是:重组酶最适温度为60℃,于20-55℃使用时稳定;最适pH值是6.0,且仅在p H6.0附近保持稳定;在60℃,p H 6的Mcilvaine’s buffer中,测得纯酶酶活力为372.76 U/mL,比活力为1112.7 U/mg,Vm值是0.31 mmol/L·h,Km值为0.6933 mmol/L;10 mmol/L的Mn2+和Cd2+都可以促进RHA4酶活性。
[Abstract]:The production of isoquercetin by 伪-L-rhamnosidase biotransformation with rutin as substrate has become a hot topic. However, the 尾 -D-glucosidase and / or rutosidase activities of 伪 -L-rhamnosidase producing bacteria have been found so far, the specificity of 伪 -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 伪 -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 伪 -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 伪 -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，
本文编号：2286034
[Abstract]:The production of isoquercetin by 伪-L-rhamnosidase biotransformation with rutin as substrate has become a hot topic. However, the 尾 -D-glucosidase and / or rutosidase activities of 伪 -L-rhamnosidase producing bacteria have been found so far, the specificity of 伪 -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 伪 -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 伪 -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 伪 -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，
本文编号：2286034
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