生产菌株的筛选及腈水合酶催化反应条件优化

1、高酶活烟酰胺生产菌株的筛选及腈水合酶催化反应条件优化摘要烟酰胺作为B族维生素的重要成员，参与了机体内两种重要辅酶的形成。烟酰胺作为重要的营养性添加剂被广泛应用于应用于饲料生产行业，其在医药、食品、化妆品行业的应用前景也非常广阔。在工业生产方面，人们主要利用化学法和微生物法进行烟酰胺生产。烟酰胺的微生物生产法有着化学法无法比拟的优越性，此方法对环境几乎不造成污染，且生产成本低，工艺简便。在本实验中，主要以下四个方面进行了实验研究并取得了一定的成果：(1) 以实验室现有菌种HD1220为出发菌株，分别使用ARTP诱变、微波诱变、EMS诱变三种单因子诱变以及在各因子最佳诱变条件下的复合诱变，确定了复合诱变的最佳条件为：ARTP诱变时间240 s；微波诱变强度60 %，微波照射时间90 s，；EMS浓度0.8 %。诱变完成后，筛选出一株能够稳定遗传且酶活高达1764U的高腈水合酶活性菌株，与出发菌株相比，诱变后菌株酶活提高了33.13 %，并将该菌株暂时命名为NHD-1。 (2)利用HPLC对烟酰胺进行检测，通过对HPLC检测的紫外检测波长、流动相流量、流动相比例三个条件进行的单因素优化实验，

2、最终确定HPLC检测烟酰胺的各个最优条件为：紫外检测波长为220 nm，流动相的流量为0.4 mL/min，流动相中甲醇：水=1：3。在以上条件下，烟酰胺出峰时间为5.306 min，与条件优化前相比，烟酰胺出峰时间明显缩短。(3) 对腈水合酶参与烟酰胺生产过程中酶催化反应条件进行单因素优化实验，我们得出各因素的最佳条件为：菌体浓度1.8 %，菌龄48 h，底物浓度100 g/L，温度29.5 ，PH 7.4，反应时间30 min，在实际发酵后期应该对产物烟酰胺进行及时分离。通过Plackett-Burman实验设计我们分析出底物浓度、温度和PH这三个因素对腈水合酶的酶活有及其显著的影响；通过对这三个因素进行响应面优化设计，我们得出了这三个因素的最佳条件组合为底物浓度98.88 g/L，反应温度29.59 ，PH值7.46。在此条件下酶活响应值为1784 U。对SAS软件统计分析结果进行三次验证实验，得到腈水合酶的实际酶活为1775 U，与多元回归分析所得理论值吻合，说明利用响应面法能够很好的优化酶催化反应条件。ABSTRACTNicotinamide is a kind of Vit

3、amin B and its very important. In the body, Its one part of the formation of two kinds of coenzyme. Nicotinamide is mainly used in feed industry as nutrition additives, it also has a very broad application prospect in medical industry, food industry and cosmetics industry. In the industrial production of nicotinamide ,we mainly use chemical method and biological method. The biological method has a big superiority that chemical method hasnt it does little harm to the environment and has a lower p

4、roduction cost, and its very simple in process.Under the conditions of laboratory, we mainly carried out our research in several aspects and achieved some results as follows:(1) Started from the strain Norcardia sp.HD9611 we had in the laboratory, we used the method of ARTP mutagenesis, microwave mutagenesis and EMS mutagenesis to make mutagenesis of the strain. The three methods were all used alone. Then we made the composite mutagenesis under best conditions of the three method of mutagenesis.

5、 The best conditions of composite mutagenesis were: the time of ARTP mutagenesis was 240 s; the time and intensity of microwave were 90 s and 60 %; the concentration of EMS mutagenesis was 0.8 %. By the mutagenesis we screened one stain whose nitrile hydratase had a very high enzyme activity of 1764 U and the stain had stable heredity. Compared to the stain Norcardia sp.HD9611, the enzyme activity increased by 33.13 %, and we named the stain as NHD-1.(2)In the detection of Nicotinamide, we used

6、the HPLC method. By optimizing the wavelength of UV detection, the flow rate of mobile phase and the proportion of mobile phase in the HPLC assay, we got the best conditions of HPLC method : the wavelength of UV detection was 220 nm; the flow rate of mobile phase was methanol: water=1:3; the proportion of mobile phase was 0.4 mL/min. under these conditions, the peak time of niacinamide was 5.306 min. Compared to the method before optimization we used, the peak time significantly shortened. (3) B

7、y optimizing the conditions of catalytic reaction of the nitrile hydratase, we got the best conditions: the concentration of bacteria producing niacinamide was 1.8 %; the fungus age was 48 h; the concentration of substrate was 100 g/L; the temperature was 29.5 ; the PH was 7.4; the reaction time is 30 min, and we should separate nicotinamide late in the actual fermentation. By Plackett-Burman experiment we confirmed that conditions of concentration of substrate, temperature, PH had significant i

8、nfluences on the enzyme activity of nitrile hydratase. By response surface test we optimized these three factors: the concentration of substrate was 98.88 g/L; the temperature was 29.59 ; the PH was 7.46. Under these conditions, we analyzed the enzyme activity is 1784 U. Through verification test, we confirmed the enzyme activity of nitrile hydratase was 1775 U. It was very close to the theoretical value. It showed that the response surface test was a very effective method in optimization of enz

9、yme catalysis.Key word: mutagenes nitrile hydratase enzyme activity HPLC optimization 1前言1.1烟酰胺的概述 1.1.1烟酰胺简介烟酰胺（Nicotinamide）化学名称为3吡啶甲酰胺，又维生素B3英文简称VB3、NSA1。烟酰胺最早从动物肝脏中提取，市场上销售产品多为化学合成品2。烟酰胺的分子式如图1-1：图1-1 NSA分子式 Fig.1-1 The structure of NSA1.1.2烟酰胺化学性质 烟酰胺为白色针状结晶或结晶性粉末。熔点为128 131 ，沸点为150 160 。烟酰胺极易溶于水，在一般极性有机溶剂溶解，在苯和乙醚中无溶解性3。烟酰胺在空气中对光、热等条件稳定，干燥状态50 以下极其稳定。1.1.3烟酸简介烟酸又称尼克酸，分子式为C6H5NO2，其整体性质与烟酰胺极其相似，但由于羧基的存在导致在某些方面不用与含有酰胺基的烟酰胺。两者通称维生素PP（二者混合物），在实际应用中，两者可相互等量替代4。在加热条件下有无机酸和碱存在条件下，烟酰胺发生水解反应转化为烟酸。烟酸分子式如图2图1-2 烟酸分子式Fig.1-1 The structure of niacin1.1.4烟酰胺用途烟酰胺参与生物体内糖原分解、脂类代谢及各种物质的氧化作用5。烟酰胺有促进细胞新陈代谢等等作用6。当烟酰胺缺乏时会出现细胞正常代谢不能正常进行，糖原分解受阻等状况而引发糙皮病。在市场上烟酰胺共分为三种：医药级、食品级和饲料。烟酰胺最主要作为营养添加剂在饲料广泛应用，在食品和医药行业主要以维生素形式为人们所使用，烟酰胺在电镀及生化方面也有一定的应用7。医学上常用来治疗糙皮病、口炎、舌炎、肝脏疾病及日光性皮炎的药物中均含有烟酰胺，其也被用来降低人体胆固醇和甘油三酯含量8。烟酰胺应用最广泛的应用是在饲料工业，其作为重要的饲料添加剂而被大量应用9。1.1.5国外和国内烟酰胺生产及消费状况 在20世纪50年代，国外烟酰胺的生产已经实现工业大批量生产。目前

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