一种产邻苯二酚菌株高通量筛选方法

目的：对产邻苯二酚菌株进行筛选。方法：采用前期筛得的产邻苯二酚菌为出发菌株,通过硫酸二乙酯诱变的方法使该菌株突变,同时建立96孔板培养和酶标仪检测方法对产邻苯二酚菌株进行高通量筛选。结果：硫酸二乙酯的终浓度为0.1%,诱变时间为8 min的条件下,突变菌致死率为84.5%,突变效果最好。筛选培养基中吸光值（495nm）和富集培养基中菌液浊度值（OD630）的加和值较大的突变菌株产邻苯二酚能力高。通过诱变和筛选得到的菌株,产邻苯二酚浓度可达0.87mg/ml,比出发菌株的提高了262.5%。经过形态学和生理生化反应,初步鉴定该菌株属于假单胞菌属（Pseudomonas sp.）。结论：硫酸二乙酯诱变和96孔板筛选的方法能以高通量方式快速筛选出产邻苯二酚菌株。     

产耐高温谷氨酰胺转胺酶菌株的快速筛选方法

目的:采用亚硝基胍(NTG)诱变结合96孔板高通量筛选方法筛选产耐高温谷氨酰胺转胺酶(MTG)的茂原链霉菌(Streptomyces mobaraensis)。方法:通过优化96孔板高通量测定MTG活性的方法、确定筛选温度和时间,建立了产耐高温MTG菌株的快速筛选方法;通过优化NTG诱变条件建立了筛选突变库;通过96孔板高通量初筛、摇瓶复筛获得了产耐高温MTG的突变株12-82,并通过摇瓶发酵对12-82所产MTG进行热稳定性分析。结果:采用2mg/ml NTG、p H8.0、60min的诱变条件获得突变株,将突变株的发酵上清液于70℃水浴7.5min,再在37℃空气浴、反应10min的条件下测定MTG活性,从5 200株突变株中筛选出5株产耐高温MTG的突变株,其中突变株12-82在50℃水浴60min以及70℃水浴1.5min的酶活残留率均比出发株高出近20%,且80℃保温2min仍有11.9%的酶活残留率。结论:利用NTG诱变结合96孔板高通量筛选的方法筛选到5株所产MTG热稳定性相对较高的突变株,其中突变株12-82在50℃、70℃和80℃的酶活残留率均有10%~20%的提高。这为高温食品加工领域所需耐高温MTG生产菌株的高效筛选提供了可行性方案。     

一株高效催化烟酸羟化反应菌株的筛选及鉴定

为开发生物法生产6-羟基烟酸,利用快速筛选方法从多个地区的土壤中筛到了一株能高效羟化烟酸为6-羟基烟酸的菌株BK-1,可以用菌体催化得到高纯度的6-羟基烟酸.通过对菌株BK-1形态观察和16S rDNA同源性分析,构建了系统发育树,BK-1菌株与Pseudomonas plecoglos-sicida形成一个类群,且有99.7%的同源性,初步判定为假单胞菌属(Pseudomonas).     

发酵液中色氨酸含量高通量快速测定

为了高通量筛选色氨酸工程菌,利用色氨酸与MAA在酸性条件下产生荧光物质(激发波长253 nm,发射波长450nm),荧光强度与色氨酸含量在一定范围内成正比的原理,建立了96孔微孔板中高通量测定发酵液色氨酸含量的方法。反应液80℃反应15 min后,测量荧光强度。线性范围为1 mg.L-1~100 mg.L-1,为大规模筛选色氨酸基因工程菌打下了基础。     

微生物高通量筛选中微孔板K_La的测定

基于微孔板高通量筛选中氧气供给与混合情况往往是微型化培养的关键,本文利用亚硫酸钠法对微孔板的K_La进行了测定。因K_La易受外界环境的影响,实验考察了摇床振幅、无菌介质、孔板装液量及种类等条件的影响,结果表明:在温度34℃、摇床振幅和转速分别为25 mm和200 r/min、以孔径为0.22μm的疏水性微孔滤膜为无菌介质、装液量为0.9 mL且加有玻璃珠的48孔板的条件下,微孔板的K_La值能达到102.00 h~(-1),在同样条件下摇床上摇瓶的对照结果为103.48 h~(-1)。就供氧能力而言,微孔板完全可以达到常规摇瓶的效果,能够保证高通量培养时高好氧微生物的需氧要求。     

恶臭假单胞菌NA-1菌体培养转化和静息细胞转化联合工艺生产6-羟基烟酸研究

现有微生物羟基化烟酸采用的是静息细胞转化工艺。但研究揭示,恶臭假单胞菌NA-1(Pseudomonas putidaNA-1)在培养过程中不降解发酵液中由诱导剂烟酸转化形成的6-羟基烟酸,这是由于烟酸的存在抑制了羟基烟酸降解酶的作用,而不是因为细胞停止生长不利用羟基烟酸的缘故。因而尝试利用菌体诱导培养过程进行烟酸转化生产,建立了一种新的生产工艺,即菌体培养转化和静息细胞转化联合工艺。该工艺在恶臭假单胞菌NA-1培养过程中持续补充烟酸以维持1%(W/V)浓度,使烟酸被生长细胞转化为羟基化烟酸并在发酵液中线性积累,而不被进一步降解;培养转化结束后,发酵液中的静息细胞依然拥有很高的羟基化酶活力,能够再次用于转化反应。该联合转化工艺与传统的静息细胞转化工艺相比,不仅节约了诱导剂烟酸,而且6-羟基烟酸的产量提高了65%。     

一株烟酸羟基化转化菌株的筛选和鉴定

从南京地区的土壤中筛选到一株高效转化烟酸为 6_羟基烟酸的菌株NA_1。形态及生理生化特征测定结果表明 ,NA_1菌株与假单胞菌属 (Pseudomonas)中的恶臭假单胞菌 (P .putida)种的特征基本一致。测定了该菌株的16SrDNA序列并根据 16SrDNA构建了系统发育树 ;在系统发育树中 ,NA_1菌株与恶臭假单胞菌形成一个类群 ,序列同源性为 99%。因此将NA_1菌株鉴定为恶臭假单胞菌     

全细胞高通量筛选α-氨基酸酯水解酶突变体的方法

【目的】建立高效敏感的高通量筛选方法,用于筛选头孢克洛合成活性提高或热稳定性提高的α-氨基酸酯水解酶。【方法】根据头孢克洛在碱性条件下水解生成的衍生物在340 nm处有特征吸收峰的原理,制作出标准曲线。采用全细胞96孔板紫外分光光度法高通量测定α-氨基酸酯水解酶突变体的头孢克洛合成活性。【结果】头孢克洛含量与△A340?405在(0.1?0.6)×10?3 mol/L浓度范围内有良好的线性关系,服从朗伯-比尔定律,平均回收率为99.8%?101.3%。一轮定点饱和突变产生的2 300个克隆经该方法的筛选,获得3株kcat提高40%以上,4株半失活温度较野生型提高5°C以上的突变体酶。【结论】该方法准确可靠,每天筛选量可达到2 000个反应,达到高通量筛选的要求。     

一种3-氰基吡啶水解酶产生菌的高通量筛选策略及应用

利用改进的羟肟酸铁分光光度比色法建立了一种简单、快速、高通量的腈水解酶筛选方法.应用该方法从土壤中筛选获得1株具有3-氰基吡啶水解酶活性的菌株CCZU10 -1,经16S rDNA序列分析,鉴定该菌为红球菌属Rhodococcus sp.;同时确定了最适反应温度、pH和金属离子添加剂分别为30℃、7.0和Ca2+ (0.1 mmol/L).在最适催化反应条件下,催化转化50 mmol/L烟腈36 h,烟酸的产率可达到93.5％.     

全细胞高通量筛选a-氨基酸酯水解酶突变体的方法

【目的】建立高效敏感的高通量筛选方法,用于筛选头孢克洛合成活性提高或热稳定性提高的a-氨基酸酯水解酶。【方法】根据头孢克洛在碱性条件下水解生成的衍生物在340 nm处有特征吸收峰的原理,制作出标准曲线。采用全细胞96孔板紫外分光光度法高通量测定a-氨基酸酯水解酶突变体的头孢克洛合成活性。【结果】头孢克洛含量与△A340?405在0.1?0.6×10?3 mol/L浓度范围内有良好的线性关系, 服从朗伯-比尔定律, 平均回收率为99.8%?101.3％。一轮定点饱和突变产生的2 300个克隆经该方法的筛选, 获得3株kcat提高40%以上, 4株半失活温度较野生型提高5 °C以上的突变体酶。【结论】该方法准确可靠,每天筛选量可达到2 000个反应, 达到高通量筛选要求。     

Microbial production of 6-hydroxynicotinic acid,an important building block for the synthesis of modern insecticides

Production of 6-hydroxynicotinic acid, an important starting material for the synthesis of modern pesticides through bacterial position-specific hydroxylation of nicotinic acid, was investigated. Resting cells of Serratia marcescens IFO 12648 were found to catalyze the potential hydroxylation activity of nicotinic acid to produce 6-hydroxynicotinic acid. The optimum culture conditions of S. marcescens IFO 12648 for the accumulation of 6-hydroxynicotinic acid were investigated. The addition to the culture medium of molybdenum and iron ions and of nicotinic acid as an inducer greatly enhanced the hydroxylation activity. Under the optimum conditions, 98.5% of the added 2.2 M nicotinic acid was converted to 6-hydroxynicotinic acid, and the highest yield achieved was 301 g of 6-hydroxynicotinic acid per liter of reaction mixture containing 3.98 g dry weight of resting cells during a 72-h reaction at 35°C.     

Molybdenum-dependent degradation of nicotinic acid by Bacillus sp. DSM 2923

Abstract Growth of Bacillus sp. DSM 2923 on nicotinic acid in mineral medium was dependent on the concentration of sodium molybdate added. Addition of increasing amounts of tungstate to the medium resulted in an inhibition of growth on nicotinic acid or 6-hydroxynicotinic acid as sole source of carbon and energy. Chlorate-resistant mutants were isolated which were not able to degrade nicotinic acid and 6-hydroxynicotinic acid nor to reduce nitrate. Additionally, enzyme activities of nicotinic acid dehydrogenase and 6-hydroxynicotinic acid dehydrogenase increased with increasing concentrations of molybdate (10⁻⁸ to 10⁻⁶ M) added to the medium, and decreased with increasing amounts of tungstate (10⁻⁶ to 10⁻⁵ M) in the medium.     

恶臭假单胞菌NA-1菌株烟酸羟基化酶活性的诱导和转化条件的研究

恶臭假单胞菌NA-1菌株的培养和产酶特性与已报道的产酶菌株粘质沙雷氏菌(Serratiamarcescens)IFO12648和荧光假单胞菌(Psudomonasfluorescens)TN5有所不同,主要反映在最适碳源及浓度、最适诱导剂浓度和最适培养温度等方面。最适的转化条件是温度为30℃,pH为7.0,烟酸的浓度为3%。采用初步优化后的条件和流加底物的方式进行4L上罐生产,恶臭假单胞菌NA-1菌株的6-羟基烟酸产率可达到108.39gL。     

Oxidation of Nicotinic Acid by a Bacillus Species: Purification and Properties of Nicotinic Acid and 6-Hydroxynicotinic Acid Hydroxylases

The enzymes of a Bacillus species that hydroxylate nicotinic acid to 6-hydroxynicotinic acid and 6-hydroxynicotinic acid to 2,6-dihydroxynicotinic acid were purified and characterized. The purified enzymes contained approximately two molecules of flavine and eight molecules of iron per molecule of enzyme. The enzymes were large (molecular weight, 400,000 to 450,000) and appeared to consist of subunits.     

Metabolism of nicotinic acid in plant cell suspension cultures, IV: Occurrence and metabolism of nicotinic acid N-alpha-arabinoside (author's transl)]

Application of nicotinic acid to cell suspension cultures of Petroselinum hortense Hoffm., Daucus carota, Nicotiana tabacum and Nicotiana glauca leads to the formation of the recently isolated[2] nicotinic acid N-alpha-L-arabinoside. In these cell cultures the arabinoside is a metabolically active compound; the nicotinic acid moiety is used for NAD synthesis and nicotinic acid degradation involving decarboxylation and ring fission. N-Methylnicotinic acid (trigonelline) and nicotinic acid N-alpha-L-arabinoside occur alternatively in plant cell suspension cultures, but seem to fulfil the same function as a reserve form for nicotinic acid. Catabolism of nicotinic acid in parsley cell suspension cultures does not involve 6-hydroxynicotinic acid as an intermediate.     

A visible wavelength spectrophotometric assay suitable for high-throughput screening of 3-hydroxy-3-methylglutaryl-CoA synthase

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase catalyzes the first physiologically irreversible step in biosynthesis of isoprenoids and sterols from acetyl-CoA. Inhibition of enzyme activity by β-lactone-containing natural products correlates with substantial diminution of sterol synthesis, identifying HMG-CoA synthase as a potential drug target and suggesting that identification of effective inhibitors would be valuable. A visible wavelength spectrophotometric assay for HMG-CoA synthase has been developed. The assay uses dithiobisnitrobenzoic acid (DTNB) to detect coenzyme A (CoASH) release on acetylation of enzyme by the substrate acetyl-CoA, which precedes condensation with acetoacetyl-CoA to form the HMG-CoA product. The assay method takes advantage of the stability of recombinant enzyme in the absence of a reducing agent. It can be scaled down to a 60 μl volume to allow the use of 384-well microplates, facilitating high-throughput screening of compound libraries. Enzyme activity measured in the microplate assay is comparable to values measured by using conventional scale spectrophotometric assays with the DTNB method (412 nm) for CoASH production or by monitoring the use of a second substrate, acetoacetyl-CoA (300 nm). The high-throughput assay method has been successfully used to screen a library of more than 100,000 drug-like compounds and has identified both reversible and irreversible inhibitors of the human enzyme.     

Isolation of new 6-methylnicotinic-acid-degrading bacteria, one of which catalyses the regioselective hydroxylation of nicotinic acid at position C2

2-Hydroxynicotinic acid is an important building block for herbicides and pharmaceuticals. Enrichment strategies to increase the chances of finding microorganisms capable of hydroxylating at the C2 position and to avoid the degradation of nicotinic acid via the usual intermediate, 6-hydroxynicotinic acid, were used. Three bacterial strains (Mena 23/3–3c, Mena 25/4–1, and Mena 25/ 4–3) were isolated from enrichment cultures with 6-methylnicotinic acid as the sole source of carbon and energy. Partial characterization of these strains indicated that they represent new bacterial species. All three strains completely degraded 6-methylnicotinic acid, and evidence is presented that the first step in the degradation pathway of strain Mena 23/3–3c is hydroxylation at the C2 position. Resting cells of this strain grown on 6-methylnicotinic acid also hydroxylated nicotinic acid at the C2 position, but did not further degrade the product. Strain Mena 23/ 3–3c showed the highest degree of 16S rRNA sequence similarity to members of the genera Ralstonia and Burkholderia. Received: 4 April 1997 / Accepted: 10 June 1997     

Microplate enzyme assay for screening lipoxygenases to degrade wood extractives

Lipoxygenases are non-heme iron-containing dioxygenases, capable of catalyzing the oxidation of unsaturated fatty acids. The enzyme has the potential to degrade problematic wood extractives in the paper-making process. However, commercially available lipoxygenase is currently too expensive for this application. A 96-well UV microplate assay was developed to screen enzymes from fungal sources for a more cost-effective alternative lipoxygenase. The substrate used for this assay was linoleic acid, a predominant fatty acid in wood. The enzyme activity and reaction kinetics determined by this microplate assay were compared to those obtained from a conventional bench scale assay. A number of hydrolytic enzymes and other oxidases were also tested using this protocol, to examine the specificity of the assay. The results show that the microplate assay developed can provide an inexpensive method for accelerated screening of a large number of enzymes to identify potential oxidative enzymes with specific action in degrading wood extractives.