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。     

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%。     

Effect of 6059-S,a novel oxacephem,on cross-linking reaction of peptidoglycan biosynthesis in Escherichia coli,Pseudomonas aeruginosa and Serratia marcescens

The effect of 6059-S, a novel 1-oxacephem, on peptidoglycan synthesis was investigated using ether-treated cells of Escherichia coli K 12, Pseudomonas aeruginosa KM 338 and Serratia marcescens IFO 12648. The cross-linking reaction of peptidoglycan synthesis in these organisms was inhibited by markedly low concentration of 6059-S.Non-standard abbreviations PBP penicillin binding protein - MIC minimum inhibitory concentration - ETB ether treated bacterial cells - SDS sodium dodecylsulfate     

Nitrile Hydratase-Catalyzed Production of Nicotinamide from 3-Cyanopyridine in Rhodococcus rhodochrous J1

Nitrile hydratase, which occurs abundantly in cells of Rhodococcus rhodochrous J1 isolated from soil samples, catalyzes the hydration of 3-cyanopyridine to nicotinamide. By using resting cells, the reaction conditions for nicotinamide production were optimized. Under the optimum conditions, 100% of the added 12 M 3-cyanopyridine was converted to nicotinamide without the formation of nicotinic acid, and the highest yield achieved was 1,465 g of nicotinamide per liter of reaction mixture containing resting cells (1.48 g as dry cell weight) in 9 h. The nicotinamide produced was crystallized and then identified physicochemically. The further conversion of the nicotinamide to nicotinic acid was due to the low activity of nicotinamide as a substrate for the amidase(s) present in this organism.     

A Combined Hydroxylation of 3‐Cyanopyridine to 3‐Cyano‐6‐hydroxypyridine and 6‐Hydroxynicotinic Acid by Resting Cells of Comamonas testosteroni JA1 Grown on Nicotinic Acid

A strain of Comamonas testosteroni JA1 known for its capacity to hydroxylate 3‐cyanopyridine to 3‐cyano‐6‐hydroxypyridine was found to be also capable to hydroxylate nicotinic acid at a higher rate. In the course of the induced cultivation the forming 6‐hydroxynicotinic acid was degraded either slightly, in the presence of nicotinic acid in the medium, or faster, in the absence of nicotinic acid. In a combined process of hydroxylation of nicotinic acid by growing culture and hydroxylation of 3‐cyanopyridine by resting cells of Comamonas testosteroni JA1, not only an additional amount of 50.38 g of solid 6‐hydroxynicotinic acid was produced from 1 L of cultivation broth with a 99.97 % molar conversion yield, but also the yield of 3‐cyano‐6‐hydroxypyridine produced was more than doubled. This can be compared to that of the resting cells from the induced cultivation broth where within 8 h an amount of 5.77 g of solid 3‐cyano‐6‐hydroxypyridine was produced by resting cells from 1 L of the cultivation broth. This also was superior to 4.39 g/L of cultivation broth of resting cells reported in the literature.     

New synthesis of the 4- and 6-pyridones of 1-methylnicotinamide and 1-methylnicotinic acid (trigonelline).

New routes are described for preparation of the 4- and 6-pyridones of 1-methylnicotinamide and 1-methylnicotinic acid (trigonelline). For the 4-pyridone series, a carboxyl group is introduced into 4-hydroxypyridine at C-3 by high-pressure carbonation and the resulting 4-hydroxynicotinic acid converted to the amide through ammonolysis of the methyl ester. Both the acid and the amide are then N¹-alkylated with methyl iodide. For the 6-pyridone series, commercially available 6-hydroxynicotinic acid is N¹-alkylated with methyl iodide and the resulting 6-pyridone of 1-methylnicotinic acid converted to the 6-pyridone of 1-methylnicotinamide by ammonolysis of the acid chloride. The above alkylations do not occur under neutral conditions but do proceed smoothly in the presence of alkali. Spectrophotometric data are given for the compounds of major interest, and a chromatographic separation is described for the naturally occurring pyridones and other urinary metabolites of nicotinamide and nicotinic acid.     

Cloning of a Novel 6-Chloronicotinic Acid Chlorohydrolase from the Newly Isolated 6-Chloronicotinic Acid Mineralizing Bradyrhizobiaceae Strain SG-6C

A 6-chloronicotinic acid mineralizing bacterium was isolated from enrichment cultures originating from imidacloprid-contaminated soil samples. This Bradyrhizobiaceae, designated strain SG-6C, hydrolytically dechlorinated 6-chloronicotinic acid to 6-hydroxynicotinic acid, which was then further metabolised via the nicotinic acid pathway. This metabolic pathway was confirmed by growth and resting cell assays using HPLC and LC-MS studies. A candidate for the gene encoding the initial dechlorination step, named cch2 (for 6-chloronicotinic acid chlorohydrolase), was identified using genome sequencing and its function was confirmed using resting cell assays on E. coli heterologously expressing this gene. The 464 amino acid enzyme was found to be a member of the metal dependent hydrolase superfamily with similarities to the TRZ/ATZ family of chlorohydrolases. We also provide evidence that cch2 was mobilized into this bacterium by an Integrative and Conjugative Element (ICE) that feeds 6-hydroxynicotinic acid into the existing nicotinic acid mineralization pathway.     

Nitrilase-Catalyzed Production of Nicotinic Acid from 3-Cyanopyridine in Rhodococcus rhodochrous J1

The nitrilase which occurs abundantly in cells of Rhodococcus rhodochrous J1 catalyzes the direct hydrolysis of 3-cyanopyridine to nicotinic acid without forming nicotinamide. By using resting cells, the reaction conditions for nicotinic acid production were optimized. Under the optimum conditions, 100% of the added 3-cyanopyridine could be converted to nicotinic acid, the highest yield achieved being 172 mg of nicotinic acid per 1.0 ml of reaction mixture containing 2.89 mg (dry weight) of cells in 26 h.     

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.     

Production of protease using wastewater from the manufacture of shochu

The production of protease using wastewater from a shochu distillery was investigated in order to devise a process for the treatment of shochu distillery wastewater. Aspergillus usami mut. shirousami IFO 6082 was selected from among eight strains for production of protease. Production of 240 U/ml of proteolytic activity was achieved after 72 h in a jar-fermentor culture under the following conditions: initial pH, 5; 30°C; aeration, 1 vvm; and agitation, 600 rpm. The protease was purified by column chromatography on Sephadex G-150 and isoelectrofocusing. The molecular weight of the purified enzyme determined by SDS-PAGE was 56 kDa, and the isoelectric point was pH 4.0. The optimum pH for the reaction was about 4.0, so the enzyme is therefore an acid protease. The optimum temperature for proteolysis ranged between 55 and 60°C, while the enzyme was unstable at temperatures above 60°C.     

Product induction of purine hydroxylase II in Asperigillus nidulans.

Summary By the use of a mutation, hxB-20, that eliminates purine hydroxylase II activity but retains cross-reacting material and an ancillary NADH dehydrogenase activity, it has been established that 6-hydroxynicotinic acid rather than nicotinic acid, is the true inducer of purine hydroxylase II.     

Mutually exclusive occurrence and metabolism of trigonelline and nicotinic acid arabinoside in plant cell cultures

In 50 cell suspension cultures of wide taxonomic origin, formation of trigonelline and nicotinic acid N-α-l-arabinoside from nicotinate was strictly alternative. The arabinoside was only found in cell cultures of the subclass Asteridae and in the higher orders of the subclasses Rosidae and Dilleniidae. Degradation of nicotinic acid could only be observed in cell cultures producing the arabinoside. Nicotinic acid degradation does not involve free 6-hydroxynicotinic acid. Cross feeding experiments with both conjugates and measurements of a nicotinic acid N-arabinoside: UDP-arabinosyltransferase support the hypothesis that metabolism of these two derivatives in cell cultures may be of chemosystematic value. Finally various discrepancies between plants and cell cultures with respect to nicotinate metabolism and to the natural occurrence of the two conjugates are discussed.     

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     

基于微孔板的高通量筛选6-羟基烟酸转化菌方法的建立

建立了一种基于96孔板-酶标仪的双波长紫外分光光度法高通量筛选6-羟基烟酸转化菌的方法.实验以251nm为测定波长、231nm为参比波长测定转化样品的6-羟基烟酸含量,6-羟基烟酸与△A251-231在0.5～11 μg/mL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99.11%～100.81%.利用96孔板-酶标仪,每天筛选量可达到2000～5000个反应,达到高通量筛选要求.     

Bioelectrochemical transformation of nicotinic acid into 6-hydroxynicotinic acid on Pseudomonas fluorescens TN5-immobilized column electrolytic flow system

Pseudomonas fluorescens TN5 catalyzes the hydroxylation of nicotinic acid (NA) into 6-hydroxynicotinic acid (6HNA), an important compound as a starting material for the synthesis of a new type of pesticides. Under aerobic conditions, however, 6HNA is metabolized in the P. fluorescens cells. The use of Fe(CN)₆³⁻ as an extracellular electron acceptor enhances the biotransformation of NA into 6HNA and completely suppresses the subsequent oxidation of 6HNA. The function of the P. fluorescens cell was combined with the electrode process by immobilizing the P. fluorescens cells on the carbon fiber electrode surface in the column, where Fe(CN)₆³⁻ was used as an electron transfer mediator. Continuous-flow electrolysis of NA in the presence of Fe(CN)₆³⁻ at the P. fluorescens-immobilized column electrode realized the accelerated and complete transformation of NA into 6HNA without any by-product.     

Formation and Purification of Serratia marcescens Arylsulfatase

The effects of culture conditions on arylsulfatase production by six strains of the genus Serratia were studied. Synthesis of arylsulfatases in all six strains was repressed in media with inorganic sulfate or methionine as the sole source of sulfur and derepressed by the addition of tyramine. Serratia marcescens IFO 3046 grew most rapidly and produced a high level of arylsulfatase when cultured on mannitol with inorganic sulfate and tyramine. The derepressed synthesis of arylsulfatase in S. marcescens was not subject to strong catabolite repression. The molecular weight of purified arylsulfatase was determined to be between 46,000 and 49,000. Arylsulfatase from S. marcescens differed in K_m and V_max values, substrate specificities, fluoride inhibition, and electrophoretic mobility from the enzyme from K. aerogenes, but had the same molecular weight as the latter.     

A molybdenum-containing dehydrogenase catalyzing an unusual 2-hydroxylation of nicotinic acid

An enzyme of Ralstonia/ Burkholderia strain DSM 6920 catalyzing the initial hydroxylation of 6-methylnicotinic acid at position 2 was purified to apparent homogeneity. It also catalyzed the unusual conversion of nicotinic acid to 2-hydroxynicotinic acid and was therefore designated as nicotinic acid dehydrogenase (NDH). Native NDH had a molecular mass of 280 kDa and was composed of subunits of 75, 30 and 16 kDa. It contained molybdenum, iron, acid-labile sulfur and FAD in a ratio of 1.6:7.3:8.0:0.6 mol(-1) of native enzyme. The molybdenum cofactor was characterized as molybdopterin cytosine dinucleotide. Zinc was identified as an additional metal ion in a molar ratio of 1.8 mol mol(-1) of native enzyme. Purified NDH exhibited a maximal specific activity of 22.6 micromol nitro blue tetrazoliumchloride reduced min(-1) mg(-1) of protein, using nicotinic acid as electron donor. The apparent K(m) value for nicotinic acid was determined to be 154 microM. Pyridine-3,5-dicarboxylic acid and quinoline-3-carboxylic acid were further substrates, but exhibited significantly different activity pH optima. Several artificial electron acceptors were reduced by NDH, but no activity was detected with NAD or O(2). NDH was inactivated upon incubation with cyanide, but no loss of activity was obtained in the presence of arsenite.     

Bioprocess engineering to produce 10-hydroxystearic acid from oleic acid by recombinant Escherichia coli expressing the oleate hydratase gene of Stenotrophomonas maltophilia

Microbial hydroxylation of long chain fatty acids has been extensively investigated. However, biotransformation productivity remains below ca. 1.0 g/g cell dry weight (CDW)/h under process conditions. In the present study, a highly efficient microbial hydroxylation process to convert oleic acid into 10-hydroxystearic acid was developed. A recombinant Escherichia coli expressing ohyA, the gene encoding oleate hydratase of Stenotrophomonas maltophilia, was used as the biocatalyst. Investigation of the ohyA expression and biotransformation conditions (e.g., inducer concentration, gene expression period before initiating biotransformation, mixing condition of reaction medium) enabled 10-hydroxystearic acid to accumulate to a final concentration of approximately 46 g/L in the culture medium. The specific product formation rate and product yield reached approximately 2.0 g/g CDW/h (i.e., 110 U/g CDW) and 91%, respectively. The specific product formation rate was more than 3-fold higher than those of a bioprocess using wild type Stenotrophomonas sp. cells. Additionally, the product of the whole-cell biotransformation was recovered at a yield of 70.9% and a purity of 99.7% via solvent fraction crystallization at low temperature. These results will contribute to developing a biological process for hydroxylation of oleic acid.     

Oxidation of terfenadine by Streptomyces platensis: Influence of culture medium on metabolite formation

The biotransformation of terfenadine into a primary alcohol, hydroxyterfenadine, followed by its oxidation to an acid, fexofenadine, was investigated using Streptomyces platensis cells. Time-courses of metabolite formation were established, and the results underlined the modulation of the alcohol to acid formation ratio according to culture conditions. Optimization of the hydroxylation step (pH, temperature, culture medium composition) led to the preparation of hydroxyterfenadine with a good yield (51%) using cells grown in culture medium without soybean peptone. In contrast, when incubations were performed with cells cultured in a medium containing soybean peptone, the alcohol to acid formation ratio decreased. The efficiency of the conversion to fexofenadine was shown to depend on the age of the cells, thus suggesting the induction of an oxidative activity. Both the hydroxylation reaction and the following two-oxidation steps leading to the acid seemed to depend on oxygen.     

Bioprocess development for nicotinic acid hydroxamate synthesis by acyltransferase activity of Bacillus smithii strain IITR6b2

In this work, acyltransferase activity of a new bacterial isolate Bacillus smithii strain IITR6b2 was utilized for the synthesis of nicotinic acid hydroxamate (NAH), a heterocyclic class of hydroxamic acid. NAH is an important pyridine derivative and has found its role as bioligand, urease inhibitor, antityrosinase, antioxidant, antimetastatic, and vasodilating agents. Amidase having acyltransferase activity with nicotinamide is suitable for nicotinic acid hydroxamate production. However, amidase can also simultaneously hydrolyze nicotinamide and nicotinic acid hydroxamate to nicotinic acid. Nicotinic acid is an undesirable by-product and thus any biocatalytic process involving amidase for nicotinic acid hydroxamate production needs to have high ratios of acyltransferase to amide hydrolase and acyltransferase to nicotinic acid hydroxamate hydrolase activity. Isolate Bacillus smithii strain IITR6b2 was found to have 28- and 12.3-fold higher acyltransferase to amide and hydroxamic acid hydrolase activities, respectively. This higher ratio resulted in a limited undesirable by-product, nicotinic acid (NA) synthesis. The optimal substrate/co-substrate ratio, pH, temperature, incubation time, and resting cells concentration were 200/250 mM, 7, 30 °C, 40 min, and 0.7 mg_DCW ml^?1, respectively, and 94.5 % molar conversion of nicotinamide to nicotinic acid hydroxamate was achieved under these reaction conditions. To avoid substrate inhibition effect, a fed-batch process based on the optimized parameters with two feedings of substrates (200/200 mM) at 40-min intervals was developed and a molar conversion yield of 89.4 % with the productivity of 52.9 g h^?1 g _DCW ^?1 was achieved at laboratory scale. Finally, 6.4 g of powder containing 58.5 % (w/w) nicotinic acid hydroxamate was recovered after lyophilization and further purification resulted in 95 % pure product.