Degradation of Acetylsalicylic Acid by a Strain of Acinetobacter Iwoffii

SUMMARY: A strain of Acinetobacter Iwoffii , isolated from a stored sample of distilled water, hydrolysed acetylsalicylic acid to salicylic and acetic acids. It grew in mineral salts medium with either of these compounds as C source and NH₄⁺ as N source. Experiments with whole cells and cell free extracts and the isolation of intermediates showed that acetylsalicylic acid was metabolized through salicylic acid, catechol, cis-cis -muconic acid, (+)-muconolactone and β-oxoadipic acid. The salicylate hydroxylase required NADH or NADPH as cofactor and 1 mole of O₂ was taken up and 1 mole of CO₂ evolved for each mole of salicylate oxidized. Catalytic quantities of flavine adenine dinucleotide (FAD) but not flavine mononucleotide (FMN) activated the enzyme. The cis-cis -muconate lactonizing enzyme was activated by Mn²⁺ and inhibited by EDTA.     

Production of 6-phenylacetylene picolinic acid from diphenylacetylene by a toluene-degrading Acinetobacter strain

Several strategies for using enzymes to catalyze reactions leading to the synthesis of relatively simple substituted picolinic acids have been described. The goal of the work described here was to synthesize a more complex molecule, 6-phenylacetylene picolinic acid [6-(2-phenylethynyl)pyridine-2-carboxylic acid], for use as a potential endcapping agent for aerospace polymers. We screened 139 toluene-degrading strains that use a variety of catabolic pathways for the ability to catalyze oxidative transformation of diphenylacetylene. Acinetobacter sp. strain F4 catalyzed the overall conversion of diphenylacetylene to a yellow metabolite, which was identified as a putative meta ring fission product (2-hydroxy-8-phenyl-6-oxoocta-2,4-dien-7-ynoic acid [RFP]). The activity could be sustained by addition of toluene at a flow rate determined empirically so that the transformations were sustained in spite of the fact that toluene is a competitive inhibitor of the enzymes. The overall rate of transformation was limited by the instability of RFP. The RFP was chemically converted to 6-phenylacetylene picolinic acid by treatment with ammonium hydroxide. The results show the potential for using the normal growth substrate to provide energy and to maintain induction of the enzymes involved in biotransformation during preliminary stages of biocatalyst development.     

Production of Oxalic Acid by a Strain of Agaricus campestris

A strain of the common mushroom Agaricus campestris was grown on a mixture of composted sawdust and CaCO₃. On incubation for 47 days, the organism produced 20.5 g of oxalic acid per 100 g of initial dry compost solids.     

Pyruvic Acid Production from 1,2-Propanediol by Thiamin-requiring Acinetobacter sp. 80-M

For the purpose of producing pyruvic acid from 1,2-propanediol (PD), three PD-utilizing and thiamin-requiring microorganisms were isolated from soil. All the isolated strains were found to be pyruvic acid producers. Among them, strain 80-M was the best producer and was identified as an Acinetobacter sp. With this bacterium, the conditions were optimized for pyruvic acid production from PD by two methods: growing cell and resting cell methods. The amount of thiamin added to the medium remarkably affected the pyruvic acid production using either method. Under optimal conditions, 14.6 and 10.0 mg/ml of pyruvate as a sodium salt were produced from 20 mg/ml of PD by the growing and resting cell methods, respectively. In the resting cell system, only PD-grown cells showed a significant pyruvate productivity from PD.     

Production of Tricarboxylic Acid Anhydrides from Fatty Acids by a Lipase-producing Strain of Pseudomonas cepacia

A lipase-producing strain of Pseudomonas cepacia isolated from a soil sample was found to produce five compounds when oleic acid was added to the culture medium as lipase inducer. The five compounds were isolated by solvent extraction, silicagel column chromatography and preparative HPLC, and their structural elucidation was performed by mass spectrometry, and infrared and nuclear magnetic resonance spectroscopies. The products were identified as dec-3-ene-1,3,4-tricarboxylic acid 3,4-anhydride (product 1 ), undec-3-ene-1,3,4-tricarboxylic acid 3,4-anhydride (product 2 ), dodec-3-ene-I,3,4-tricarboxylic acid 3,4-anhydride (product 3 ), dodec-3,8-diene-1,3,4-tricarboxylic acid 3,4-anhydride (product 4 ) and dodec-3,6-diene-I,3,4-tricarboxylic acid 3,4-anhydride (product 5 ). Accumulation of these compounds in the culture medium started after oleic acid consumption and followed a pattern similar to that found for cell growth and for lipase production. The five compounds were radioactively labeled when [U- 14 C]oleic acid was supplied to the culture medium, thus showing that they were produced by transformation of the acid. When isolated from cultures containing [1,2- 13 C]acetic acid and oleic acid as the sole sources of carbon, the compounds showed to contain the 13 C isotope only in the first five atoms of carbon of the molecule. Several long chain fatty acids also acted as precursors of these compounds, with maximal yields for chain lengths between 11 and 18 atoms of carbon. None of the five compounds acted as lipase inducer when added to the culture medium instead of oleic acid. The compounds showed moderate antibacterial and antifungal activities when tested in solid media bioassays.     

Production of Tricarboxylic Acid Anhydrides from Fatty Acids by a Lipase-producing Strain of Pseudomonas cepacia

A lipase-producing strain of Pseudomonas cepacia isolated from a soil sample was found to produce five compounds when oleic acid was added to the culture medium as lipase inducer. The five compounds were isolated by solvent extraction, silicagel column chromatography and preparative HPLC, and their structural elucidation was performed by mass spectrometry, and infrared and nuclear magnetic resonance spectroscopies. The products were identified as dec-3-ene-1,3,4-tricarboxylic acid 3,4-anhydride (product 1 ), undec-3-ene-1,3,4-tricarboxylic acid 3,4-anhydride (product 2 ), dodec-3-ene-I,3,4-tricarboxylic acid 3,4-anhydride (product 3 ), dodec-3,8-diene-1,3,4-tricarboxylic acid 3,4-anhydride (product 4 ) and dodec-3,6-diene-I,3,4-tricarboxylic acid 3,4-anhydride (product 5 ). Accumulation of these compounds in the culture medium started after oleic acid consumption and followed a pattern similar to that found for cell growth and for lipase production. The five compounds were radioactively labeled when [U- 14 C]oleic acid was supplied to the culture medium, thus showing that they were produced by transformation of the acid. When isolated from cultures containing [1,2- 13 C]acetic acid and oleic acid as the sole sources of carbon, the compounds showed to contain the 13 C isotope only in the first five atoms of carbon of the molecule. Several long chain fatty acids also acted as precursors of these compounds, with maximal yields for chain lengths between 11 and 18 atoms of carbon. None of the five compounds acted as lipase inducer when added to the culture medium instead of oleic acid. The compounds showed moderate antibacterial and antifungal activities when tested in solid media bioassays.     

Production of Two Chitosanases from a Chitosan-Assimilating Bacterium, Acinetobacter sp. Strain CHB101

A bacterial strain capable of utilizing chitosan as a sole carbon source was isolated from soil and was identified as a member of the genus Acinetobacter. This strain, designated CHB101, produced extracellular chitosan-degrading enzymes in the absence of chitosan. The chitosan-degrading activity in the culture fluid increased when cultures reached the early stationary phase, although the level of activity was low in the exponential growth phase. Two chitosanases, chitosanases I and II, which had molecular weights of 37,000 and 30,000, respectively, were purified from the culture fluid. Chitosanase I exhibited substrate specificity for chitosan that had a low degree of acetylation (10 to 30%), while chitosanase II degraded colloidal chitin and glycol chitin, as well as chitosan that had a degree of acetylation of 30%. Rapid decreases in the viscosities of chitosan solutions suggested that both chitosanases catalyzed an endo type of cleavage reaction; however, chitosan oligomers (molecules smaller than pentamers) were not produced after a prolonged reaction.     

Lactic Acid Production from Pretreated Hydrolysates of Corn Stover by a Newly Developed Bacillus coagulans Strain

An inhibitor-tolerance strain, Bacillus coagulans GKN316, was developed through atmospheric and room temperature plasma (ARTP) mutation and evolution experiment in condensed dilute-acid hydrolysate (CDH) of corn stover. The fermentabilities of other hydrolysates with B. coagulans GKN316 and the parental strain B. coagulans NL01 were assessed. When using condensed acid-catalyzed steam-exploded hydrolysate (CASEH), condensed acid-catalyzed liquid hot water hydrolysate (CALH) and condensed acid-catalyzed sulfite hydrolysate (CASH) as substrates, the concentration of lactic acid reached 45.39, 16.83, and 18.71 g/L by B. coagulans GKN316, respectively. But for B. coagulans NL01, only CASEH could be directly fermented to produce 15.47 g/L lactic acid. The individual inhibitory effect of furfural, 5-hydroxymethylfurfural (HMF), vanillin, syringaldehyde and p-hydroxybenzaldehyde (pHBal) on xylose utilization by B. coagulans GKN316 was also studied. The strain B. coagulans GKN316 could effectively convert these toxic inhibitors to the less toxic corresponding alcohols in situ. These results suggested that B. coagulans GKN316 was well suited to production of lactic acid from undetoxified lignocellulosic hydrolysates.     

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.     

重组大肠杆菌生物转化甘油生产3-羟基丙酸

目的:以甘油为底物构建高效的3-羟基丙酸生产菌株。方法:以自身携带乙醛脱氢酶的E.coli BL21(DE3)plysS作为宿主,异源表达源自Klebsiella pneumoniae的甘油脱水酶基因dhaB。结果:重组菌E.coli HP获得的甘油脱水酶比活力在1.0mmol/L IPTG的诱导下达到了77.2 U/mg,摇瓶条件下,3-HP的最大产量为5.44 g/L,摩尔转化率为53%,该产量比目前报道的最高水平(4.4 g/L)提高了23.6%。结论:重组菌株E.coli HP实现了甘油向3-羟基丙酸(3-HP)的高效生物转化。     

Characterization of Cellulose Production by a Gluconacetobacter xylinus Strain from Kombucha

The aims of this work were to characterize and improve cellulose production by a Gluconoacetobacter xylinus strain isolated from Kombucha and determine the purity and some structural features of the cellulose from this strain. Cellulose yield in tea medium with both black tea and green tea and in Hestrin and Schramm (HS) medium under both static and agitated cultures was compared. In the tea medium, the highest cellulose yield was obtained with green tea (~0.20 g/L) rather than black tea (~0.14 g/L). Yield in HS was higher (~0.28 g/L) but did not differ between static and agitated incubation. ¹H-NMR and ¹³C-NMR spectroscopy indicated that the cellulose is pure (free of acetan) and has high crystallinity, respectively. Cellulose yield was improved by changing the type and level of carbon and nitrogen source in the HS medium. A high yield of ~2.64 g/L was obtained with mannitol at 20 g/L and corn steep liquor at 40 g/L in combination. In the tea medium, tea at a level of 3 g/L gave the highest cellulose yield and the addition of 3 g/L of tea to the HS medium increased cellulose yield to 3.34 g/L. In conclusion, the G. xylinus strain from Kombucha had different cellulose-producing characteristics than previous strains isolated from fruit. Cellulose was produced in a pure form and showed high potential applicability. Our studies extensively characterized cellulose production from a G. xylinus strain from Kombucha for the first time, indicating both similarities and differences to strains from different sources.     

Production of 10-Ketostearic Acid from Oleic Acid by Flavobacterium sp. Strain DS5 (NRRL B-14859)

A microbial isolate, Flavobacterium sp. strain DS5, produces 10-ketostearic acid (10-KSA) from oleic acid in 85% yield. This is the first report on this type of reaction catalyzed by a Flavobacterium strain. The product was purified to give white, plate-like crystals melting at 79.2°C. The optimum time, pH, and temperature for the production of 10-KSA are 36 h, 7.5, and 30°C, respectively. A small amount of 10-hydroxystearic acid (10-HSA) (about 10% of the amount of the main product, 10-KSA) is also produced during the bioconversion. 10-KSA is not further metabolized by strain DS5 and accumulates in the medium. In contrast to growing cells, a resting-cell suspension of strain DS5 produces 10-HSA and 10-KSA in a ratio of 1:3. The crude cell extract obtained from ultrasonic disruption of the cells converts oleic acid mainly to 10-HSA (10-HSA/10-KSA ratio, 97:3). This result strongly suggested that oleic acid is converted to 10-KSA via 10-HSA. Enzymes catalyzing the hydration and secondary alcohol dehydrogenation are cell associated. Product 10-HSA from strain DS5 is 66% enantiomeric excess in the 10(R) form. The oleic acid conversion enzyme(s) reacts with unsaturated fatty acids in the order oleic acid > palmitoleic acid > linoleic acid > linolenic acid > γ-linolenic acid > myristoleic acid.     

Some Factors Influencing Acid Production by an Oxytetracycline-Resistant Strain of Streptococcus lactis

Induction of oxytetracycline resistance in a strain of Streptococcus lactis caused this organism to display reduced acid production, salt tolerance, pyruvate synthesis, growth at alkaline pH, and a loss in ability to produce ammonia from arginine. α-Ketoglutaric and oxaloacetic acids were found to accumulate in the growth medium of resistant cells, in contrast to none in the medium of susceptible cells. No free arginine could be detected in the intracellular fraction of resistant cells, but arginine was present in the intracellular fraction of susceptible cells and decreased in concentration upon the addition of oxytetracycline to the growth medium. Depressed acid production in milk by the oxytetracycline resistant strain is evidently a consequence of the inability of this organism to metabolize arginine effectively.     

Production of a Novel Extracellular Polysaccharide by a Bacillus Strain Isolated from Soil

A bacterium that was isolated from soil and identified as Bacillus circulans was found to produce a highly viscous extracellular polysaccharide when it was grown aerobically in a medium containing glucose as a sole source of carbon. The product was characterized by TLC and GC analyses as a novel heteropolysaccharide consisted of rhamnose, mannose, galactose, and mannuronic acid as sugar components. A maximal yield of polysaccharide reached about 2 g/liter by jar-fermentor culture at 30°C for 48 hr with a medium containing 1% glucose, 0.05% asparagine, 0.005% yeast extract, and small amounts of inorganic salts. Some culture conditions for the production of polysaccharide were investigated with flask culture; an optimal production was attained with a medium containing 0.1–1 % glucose and 0.01–0.05% asparagine, pH 7–8, at 30°C under aerobic conditions.     

Propionic Acid Production by a Propionic Acid-Tolerant Strain of Propionibacterium acidipropionici in Batch and Semicontinuous Fermentation

A propionic acid-tolerant derivative of Propionibacterium acidipropionici P9 was obtained by serially transferring strain P9 through broth that contained increasing amounts of propionic acid. After 1 year of repeated transfers, a strain (designed P200910) capable of growth at higher propionic acid concentrations than P9 was obtained. An increase in the proportion of cellular straight-chain fatty acids and uncoupling of propionic acid production from growth were observed for strain P200910. Growth rate, sugar utilization, and acid production were monitored during batch and semicontinuous fermentations of semidefined medium and during batch fermentation of whey permeate for both strain P200910 and strain P9. The highest propionic acid concentration (47 g/liter) was produced by P200910 in a semicontinuous fermentation. Strain P200910 produced a higher ratio of propionic acid to acetic acid, utilized sugar more efficiently, and produced more propionic acid per gram of biomass than did its parent in all fermentations.     

Production of 6-Aminopenicillanic Acid by Kluyvera citrophila KY 3641

Kluyvera citrophila KY 3641 cultivated aerobically more than 48 hr produced penicillin acylase. Sucrose, monosodium glutamate and aspartic acid stimulated the growth of the cells, whereas glucose, fructose, maltose and lactose inhibited the growth and acylase production. Enzymic reaction was carried out with whole broth, too, instead of using separated intact cells. The cells maintained its acylase activity more than one month and could be used repeatedly. Acetone-dried or freeze-dried cell was also available for enzymic reaction. Identification of 6-APA was also described.     

Molecular Characterization of a Toluene-Degrading Methanogenic Consortium

A toluene-degrading methanogenic consortium enriched from creosote-contaminated aquifer material was maintained on toluene as the sole carbon and energy source for 10 years. The species in the consortium were characterized by using a molecular approach. Total genomic DNA was isolated, and 16S rRNA genes were amplified by using PCR performed with kingdom-specific primers that were specific for 16S rRNA genes from either members of the kingdom Bacteria or members of the kingdom Archaea. A total of 90 eubacterial clones and 75 archaeal clones were grouped by performing a restriction fragment length polymorphism (RFLP) analysis. Six eubacterial sequences and two archaeal sequences were found in the greatest abundance (in six or more clones) based on the RFLP analysis. The relative abundance of each putative species was estimated by using fluorescent in situ hybridization (FISH), and the presence of putative species was determined qualitatively by performing slot blot hybridization with consortium DNA. Both archaeal species and two of the six eubacterial species were detected in the DNA and FISH hybridization experiments. A phylogenetic analysis of these four dominant organisms suggested that the two archaeal species are related to the genera Methanosaeta and Methanospirillum. One of the eubacterial species is related to the genus Desulfotomaculum, while the other is not related to any previously described genus. By elimination, we propose that the last organism probably initiates the attack on toluene.     

Pectin Lyase Production by a Penicillium italicum Strain

Growth and concomitant production of an extracellular pectin lyase (PL) [poly(methoxylgalactosiduronate) endolyase; EC 4.2.2.10] were investigated in a group of 16 fungi grown in liquid medium containing pectin as a supplementary carbon source. Culture filtrates of both Penicillium italicum (CECT 2294) and P. expansum (CECT 2275) showed the highest PL activity and contained polygalacturonase but not pectinesterase activity. The effect of the inoculum size, the carbon source (sucrose and glucose syrup), and the presence of pectin on the production of PL by P. italicum was studied. The presence of 2.6 mM glycerophosphate in the culture medium enhanced the appearance of PL but was not inhibitory for the in vitro activity. However, glycerol inhibited the enzyme nearly 50% at such a concentration.