Improvement of multicatalytic properties of nitrilase from Paraburkholderia graminis for efficient biosynthesis of 2-chloronicotinic acid.

Nitrilases are promising biocatalysts to produce high-value-added carboxylic acids through hydrolysis of nitriles. However, since the enzymes always show low activity and sometimes with poor reaction specificity toward 2-chloronicotinonitrile (2-CN), very few robust nitrilases have been reported for efficient production of 2-chloronicotinic acid (2-CA) from 2-CN. Herein, a nitrilase from Paraburkholderia graminis (PgNIT) was engineered to improve its catalytic properties. We identified the beneficial residues via computational analysis and constructed the mutant library. The positive mutants were obtained and the activity of the "best" mutant F164G/I130L/N167Y/A55S/Q260C/T133I/R199Q toward 2-CN was increased from 0.14 × 10-3  to 4.22 U/mg. Its reaction specificity was improved with elimination of hydration activity. Molecular docking and molecular dynamics simulation revealed that the conformational flexibility, the nucleophilic attack distance, as well as the interaction forces between the enzyme and substrate were the main reason alternating the catalytic properties of PgNIT. With the best mutant as biocatalyst, 150 g/L 2-CN was completely converted, resulting in 2-CA accumulated to 169.7 g/L. When the substrate concentration was increased to 200 g/L, 203.1 g/L 2-CA was obtained with yield of 85.7%. The results laid the foundation for industrial production of 2-CA with the nitrilase-catalyzed route.

Biosynthesis of nicotinic acid from 3-cyanopyridine by a newly isolated Fusarium proliferatum ZJB-09150.

In this study, nitriles were used as sole sources of nitrogen in the enrichments to isolate nitrile-converting microorganisms. A novel fungus named ZJB-09150 possessing nitrile-converting enzymes was obtained with 3-cyanopyridine as sole source of nitrogen, which was identified by morphology, biology and 18S rDNA gene sequence as Fusarium proliferatum. It was found that F. proliferatum had ability to convert nitriles to corresponding acids or amides and showed wide substrate specificity to aliphatic nitriles, aromatic nitriles and ortho-substituted heterocyclic nitriles. The nitrile converting enzymes including nitrilase and nitrile hydratase in ZJB-09150 were induced by ε-caprolactam. Nitrilase obtained in this study showed high activity toward 3-cyanopyridine. It was active within pH 3.0-12.0 and temperature ranging from 25 to 65 °C with optimal at pH 9.0 and temperature 50-55 °C. The enzyme was thermostable and its half-life was 12.5 and 6 h at 45 and 55 °C, respectively. Under optimized reaction conditions, 60 mM 3-cyanopyridine was converted to nicotinic acid in 15 min, which indicated ZJB-09150 has potentials of application in large scale production of nicotinic acid.

Characterization of a newly isolated strain Rhodococcus erythropolis ZJB-09149 transforming 2-chloro-3-cyanopyridine to 2-chloronicotinic acid.

2-Chloronicotinic acid is receiving much attention for its effective applications as a key precursor in the synthesis of pesticides and medicines. In this study, a strain ZJB-09149 converting 2-chloro-3-cyanopyridine to 2-chloronicotinic acid was newly isolated and identified as Rhodococcus erythropolis, based on its physiological and biological tests, and 16S rDNA sequence analysis. In addition, the effects of inducer, carbon source and nitrogen source were examined. Maximum activity was achieved when the above parameters were set as 8 g/l ɛ-caprolactam, 7 g/l yeast extract and 5 g/l maltose. Moreover, the biotransformation pathway of 2-chloro-3-cyanopyridine to 2-chloronicotinic acid in strain ZJB-09149 was investigated as well. This study revealed that the nitrile hydratase (NHase) and amidase expressed in R. erythropolis ZJB-09149 are responsible for the conversion of 2-chloro-3-cyanopyridine. This is the first time to report on the biotransformation preparation of 2-chloronicotinic acid.

Pyridine salvage and nicotinic acid conjugate synthesis in leaves of mangrove species.

The metabolic fate of [carbonyl-14C]nicotinamide was surveyed in leaf disks of seven mangrove species, Bruguiera gymnorrhiza, Rhizophora stylosa, Kandeliaobovata, Sonneratia alba, Pemphis acidula, Lumnitzera racemosa and Avicennia marina, with and without 250 mM NaCl. Uptake of [14C]nicotinamide by leaf disks was stimulated by 250 mM NaCl in K. candel, R. stylosa, A. marina and L. racemosa. [Carbonyl-14C]nicotinamide was converted to nicotinic acid and was utilised for the synthesis of nucleotides and nicotinic acid conjugates. Formation of nicotinic acid by the deaminase reaction was rapid; there was little accumulation of nicotinamide in the disks 3h after administration. Radioactivity from [carbonyl-14C]nicotinamide was incorporated into pyridine nucleotides (mainly NAD and NADP) in all mangrove leaves, and the rates varied from 2% (in L. racemosa) to 15% (S. alba) of the total radioactivity taken up. NaCl generally reduced nicotinic acid salvage for NAD and NADP. In all mangrove leaf disks, the most heavily labelled compounds (up to 70% of total radioactivity) were trigonelline (N-methylnicotinic acid) and/or nicotinic acid N-glucoside. Trigonelline was formed in all mangrove plants, but N-glucoside synthesis was found only in leaves of A. marina and K. obovata. In A. marina, incorporation of radioactivity into N-glucoside (51%) was much greater than incorporation into trigonelline (2%). In general, NaCl stimulates the synthesis of these pyridine conjugates. The rate of decarboxylation of nicotinic acid in roots of A. marina seedlings was much greater than for the corresponding reaction observed in leaves.

Application of high performance liquid chromatography to the analysis of some non-volatile coffee components

High performance liquid chromatography (HPLC) was applied to the analysis of caffeine, trigonelline, nicotinic acid and sucrose in Arabica an Robusta coffee. Green and roasted coffee samples were used in this study and the degradation of sucrose and trigonelline, with the formation of nicotinic acid, was followed during roasting. Caffeine did not undergo significant degradation with only 5.4% being lost under severe roasting. Sucrose was degraded rapidly during processing with light roasting producing a 97% loss and dark roasting degrading it completely. Loss of trigonelline was strongly dependent on the degree of roasting being higher in the Robusta coffee. Trigonelline degradation was associated with nicotinic acid formation both in the Arabica and Robusta coffees as a consequence of the roasting process. Trigonelline and sucrose were determined simultaneously by partition chromatography and detection with the mass detector. Determination of caffeine was carried out using reversed phase chromatography and nicotinic acid by ion-pair reversed phase chromatography. Detection in both cases was achieved using an ultraviolet detector at 272nm or 254nm, respectively. HPLC showed adequate precision and accuracy for routine analyses. In addition, the methods used were more rapid and simple than traditional procedures. HPLC appears to be a suitable technique for quality control in the coffee industry, and for fundamental investigation on the mechanisms involved in the roasting process (AU) s

Effect of tryptophan intake on oxidation of [7a-14C]tryptophan and urinary excretion on N1-methylnicotinamide in the rat.

Oxidation of tryptophan and urinary excretion of niacin metabolites were studied in weanling rats fed ad libitum for 12 to 16 days niacin-free and nicotinamide-supplemented (20 mg/kg of diet) diets containing 15% of crystalline amino acids and from 0.04 to 1.0% of L-tryptophan. N1-methylnicotinamide was measured in urine collected during the last 3 days of the feeding period. After the feeding period, rats were placed in metabolic cages, and each rat was given 4 g (dry weight) of diet containing a tracer dose of DL-[7a-14C]tryptophan. Expired CO2 was collected hourly for 10 hours. In both the niacin-free and nicotinamide-supplemented groups, weight gains increased with increasing increments of tryptophan up to about 0.16% L-tryptophan. With more than 0.16% of L-tryptophan, the amount of N1-methylnicotinamide excreted per day by both groups of rats increased linearly with increasing tryptophan intake, but proportionately less N1-methylnicotinamide was excreted when the dietary tryptophan level was 1.0%. The amount of tryptophan oxidized per 10 hours increased linearly with increasing dietary tryptophan levels between 0.16 and 1.0%. Therefore, the decline in excretion of N1-methylnicotinamide with the 1.0% tryptophan level could not be accounted for by increased oxidation. These results suggest that after the tryptophan requirement for growth is met, the amount of tryptophan oxidized and converted to nicotinamide is directly proportional to the level of tryptophan intake up to about 3 times the requirement for growth. The tryptophan requirement of the growing rat fed niacin-free and nicotinamide-supplemented diets estimated from the inflection points of N1-methylnicotinamide excretion curves was in good agreement with the requirement of about 100 mumoles/day determined from the growth curve.

Effect of nicotinamide intake on urinary excretion of N1-methylnicotinamide and oxidation of [7a-14C]tryptophan in the rat.

Oxidation of tryptophan and urinary excretion of N1-methylnicotinamide were studied in weanling rats fed ad libitum for 10 days 12% casein diets containing from 0 to 500 mg of nicotinamide per kilogram of diet. N1-methylnicotinamide was measured in urine collected during the last 3 days of the experiment. After the feeding period, rats fed the niacin-free and 500 mg nicotinamide diets were placed in metabolic cages, and each rat was given 4 g (dry weight) of diet containing a tracer dose of DL-[7a-14C]tryptophan. Expired CO2 was collected hourly for 10 hours. From rats consuming levels of nicotinamide ranging between 20 and 150 mg/kg of diet, N1-methylnicotinamide excretion increased linearly with increasing nicotinamide intake. From these results and others from this laboratory (4), it was calculated that an additional 10--11 mg of dietary tryptophan above the approximately 20 mg/day essential for growth are needed by the growing rat for the formation of 1 mg of nicotinamide. The addition of 500 mg of nicotinamide per kilogram of diet to a niacin-free diet had no effect on the oxidation of DL-[7a-14C]tryptophan.

Acrodermatitis enteropathica and the relation to pellagra.

A patient with a variant form of acrodermatitis enteropathica (AE) without hypozincemia is presented who showed a rise in plasma zinc and partial improvement on a pancreatic enzyme preparation, apparently because of its content of picolinic acid (PA). Complete recovery occurred on 60 mg zinc (1). This patient has now been treated with zinc PA (equal to only 5 mg zinc) and subsequently with PA. Both maintained elevated plasma zinc levels. Because of the similarity of AE with pellagra and the common origin of PA and nicotinic acid from tryptophan, a hypothesis is presented which suggests that skin manifestations in the two disorders are due to PA deficiency since picolinic carboxylase forms NAD preferentially when there is competition for the common precursor.

Excretion of tryptophan-niacin metabolites by young men: effects of tryptophan, leucine, and vitamin B6 intakes.

Three human metabolic studies, each 35 days in length, were performed to investigate the relationship between tryptophan intake and the proportion of dietary tryptophan converted to niacin and the effect of supplements of L-leucine and vitamin B6 on this conversion. Nine college men consumed a basal diet that provided 8 mg of niacin, 1 mg of vitamin B6, and either 245, 548, or 845 mg of tryptophan from proteins per day. During each 35-day study, for one 15-day period basal diet alone was consumed, for another 15-day period basal diet plus 10 g of L-leucine per day was consumed, and for the last 5-day period, 20 mg of vitamin B6 per day was added to the diets of both groups. N1-methylnicotinamide, N1-methyl-2-pyridone-5-carboxamide, and quinolinic acid were measured in 24-hr urine samples. There were no significant or consistent effects of L-leucine or vitamin B6 supplements on the excretin of any of the metabolites measured. The proportion of tryptophan converted to niacin tended to increase as tryptophan consumption increased; however, this change was small and was probably not significant over the range of tryptophan intakes studied. The average conversion ration of tryptophan to niacin was approximately 72:1 in these subjects.

Interactions of thiamin, riboflavin, and other B-vitamins.

Interactions of the B-complex vitamins are essential in the performance of metabolic and catabolic reactions in the body. Even vitamin C and the fat-soluble vitamins may be involved in these interactions. Clinical and biochemical aberrations associated with various disease states can often be explained on the basis of these vitamin interrelationships. Health and nutritional well-being are dependent upon the maintenance and proper functioning of these vitamin-dependent metabolic pathways.

Three simple tests as an adjunct to the niacin test for the small mycobacteriology laboratory.

With the change in the management of tuberculosis, many bacteriology laboratories should be prepared to examine sputum for the presence of acid-fast bacilli. The niacin test is the most reliable test that can be performed in any mycobacteriology laboratory to differentiate Mycobacterium tuberculosis from the other acid-fast bacilli, but it is not perfect. Other tests can be used to supplement it are cord formation, growth at 24 C, and the catalase test at 68 C. Incompletely identified strains should be submitted to a reference laboratory.

[Mass-spectrometric study of the mechanism of microbial oxidation of 3-pyridinaldehyde]. / Mass-spektrometricheskoe izuchenie mekhanizma mikrobiologicheskogo okisleniia 3-piridinal'degida

In order to clarify mechanisms of nicotinic acid synthesis during microbial transformation of 3-methylpyridine, microbial and spontaneous air oxygen oxidation of 3-pyridinaldehyde to nicotinic acid in the H218O environment was studied. It was shown that during spontaneous oxidation the label was incorporated into one atom of oxygen in the carboxylic group of nicotinic acid. During the microbial oxidation the label was equally incorporated into both atoms of oxygen in the carboxylic group of nicotinic acid. It is concluded that the mechanisms of spontaneous and microbial oxidation of 3-pyridinaldehyde are different. It is suggested that the possible precursor of nicotinic acid during microbial oxidation may be hydroxy-3-pyridinaldehyde.

Studies on niacin biosynthesis from 3-hydroxyanthranilic acid in streptozotocin diabetic rats in vivo and in vitro.

Biosynthesis of niacin from 3-hydroxyanthranilic acid (3-OHAA) in normal and streptozotocin diabetic rats was studied in vivo and in vitro. Streptozotocin (SZ) diabetic rats were found to excrete lesser quantities of 3-OHAA, quinolinic acid, niacin and N1-methylnicotinamide (nmn) in their urines following 3-OHAA administration than corresponding normal rats. In vitro studies indicated that SZ diabetic livers form less quinolinic acid from 3-OHAA than normal livers. It appears that this may be due to elevated picolinic carboxylase activity in the livers of SZ diabetic rats.

[Vitamin biosynthesis by germinating seeds at low temperature and in overmoistened soil]. / Biosintez vitaminov prorastaiushchimi semenami v usloviiakh nizkoi temperatury i pereuvlazhneniia pochvy

The distribution of farnesene and farnesene hydroperoxide in apples of different varieties was studied. The relationship between the content of these compounds and the pathological browning of the apple surface, scald was followed. It was shown that 1) farnesene and farnesene hydroperoxide were concentrated in the cuticle; 2) there was no distinct correlation between farnesene concentration and scald, 3) there was a direct correlation between the concentration of farnese hydroperoxide and scald development; 4) the factors that inhibited farnesene oxidation (antioxidants, low oxygen concentration, mineral oils--farnesene absorbers) slowed down the scald development during apple storage.