A New Exploration of Extracting and Purifying the Coenzyme F420 from Natural Sludge

Coenzyme F420 is one kind of special flavin cofactors which exist in some Archaea and bacteria, it is a low potential electron transfer carrier in methanogens. In this study, we designed three specific primers of F420-dependent glucose-6-phos-phate dehydrogenase gene to determine which environment contains the more abundant coenzyme F420, and methods for separating, extracting and purifying the coenzyme F420 from this environmental samples were carried out, the pure coenzyme F420 was obtained by some optimizing methods from the natural sludge , and the purity and concentration of coenzyme F420 we got is just as good as the FMN standard sample which confirmed by their fluorescence emission spectrum. These optimizing methods would save more energy and time to get the good quality coenzyme F420.


Introduction
5-deazaflavin coenyme (F 420 ) was first discovered in methanogenic archaea 1972 by Chessman [1], and it was deemed only exist in the methanogenic bacteria and as a low potential electron carrier, emitting fluorescence in 420nm and 480nm [2]. Recently, however, coenzyme F 420 has been found in some Methanogenbacterium sp. and Actinomycetes [3], and mostly Streptomyces sp. with ability to produce a wide array of bioactive secondary metabolites. The metabolites include many medically-important compounds with antibacterial, antifungal, antiviral, anti-parasitic, anticancer and immunosuppressive activities [4].
Researchs showed coenzyme F 420 plays an irreplaceable role in the synthesis of new active substances. Essentially, coenzyme F 420 acts as a 5-deazaflavin analog that mediates exclusively two electron transfer reactions, and has an acidic 8-hydroxy substituent that can modulate reactivity in response to its protonation state [1]. F 420 is a flavin-like redox-active coenzyme commonly used by archaea and some eubacteria in a variety of biochemical reactions in methanogenesis, the formation of secondary metabolites, the degradation of nitroaromatic compounds, activation of nitroimidazofurans, and F 420 -dependent photolysis in DNA repair [5].
Coenzyme F 420 has structural features common to flavin cofactors, but more similar to nicotinamide adenine dinucleotide phosphate when participate in reactions [6]. More and more studies focused on the mechanism of the catalytic reaction, the conformation of this enzyme, the selection of special conformation protein and substrate for coenzyme F 420 . Based on the phylogenetic analysis of large numbers of F 420 genes, it was found that many of the F 420 biosynthetic proteins in actinomycetes were F 420 dependent enzymes [7].
Overall, F 420 may confer an advantage to mycobacteria in anaerobic environments because it has a lower redox potential than NADP [8]. In nature most of enzymes exploit small molecules such as metals and cofactors, to enhance their catalytic properties,and these molecules can aid catalysis and expand the range of available enzymatic functions [9]. The auxiliary factor of these important and widely used, including nicotinamide adenine dinucleotide coenzyme A, pyridoxal phosphate, flavin adenine nucleotide, biotin, 5 '-deoxyadenosyl cobalamin, thiamine pyrophosphate, folic acid and coenzyme F 420 [10]. Coenzyme F 420 and coenzyme FMN are similar in structure (Fig 1), and they have the same effect in the reaction are used as carriers for electron transfer [ 11]. Coenzymes F 420 can have two to seven glutamate residues ligated to the coenzyme ribitol side chain through a phospholactate. The reduction of F 420 at the C 5 atom (labelled) gives F 420 -H 2 (Fig.2) [12]. At present, the direction of researches on coenzyme F 420 dependent enzymes is becoming increasingly diverse, and it has been studied in different application fields. For example in the field of Medicine. Each year more than 10 million people are affected by TB and nearly 1/3 of the patients to death by this disease. There are many resistance and side effects of drugs are now being used, and the study on this issue of coenzyme F 420 dependent enzymes in the process, drugs aimed at F 420 -related targets would act by mechanisms completely different from those of the more widely used drugs [13]. On the other hand, the application of F 420 in the field of chemical electrodes has also made great progress, and the advantages of coenzyme F 420 as an electron transfer vector are not negligible. Using the elementary step thermodynamic parameter of coenzyme F 420 mode releasing hydride ions in acetonitrile. The information disclosed in this work can not only fill the blank of chemical thermodynamics of coenzyme F 420 models as a kind of important organic source electron, hydride ions, hydrogen atoms and protons, but also strongly promote the fast development of the chemistry and applications of coenzyme F 420 [6].
However, the most popular research field of coenzyme F 420 is the special role which plays in the synthesis of new compounds. Some steps in tetracycline [14] and lincomycin [15]  Before the experiment, we make the verification of the existence of FGD gene in the bacteria, and then carried out the extraction experiment.
The coenzyme F 420 is extracted from some anaerobic Three pairs of primers were designed based on the F 420 -dependent glucose-6-phosphate dehydrogenase(FGD) gene in this study [13]. The corresponding pairs of primers as listed in Table.1. We selected samples from methane digesters, ponds and rivers, and extracted the genomic DNA using the PowerSoil DNA kit of Sigma , and then amplified the gene by PCR with different primers.

Coenzyme extraction
Samples will be taken from the biogas production, ponds and rivers, named F420-1 F420-2, F420-3 respectively. Took 10 grams of three samples each, Results and analysis

1.PCR results
By continuously improving the PCR condition, and finally using the PrimeSTAR GXL DNA Polymerase under the condition of adding GC-buffer, we obtained the corresponding size of the gene segments from the samples which collected from the biogas digester and pond (Fig.3). Therefore, we believe that these places where the bacteria can produce coenzyme F 420 .
According the result, we found the FGD genes of samples from biogas pools and ponds are much easier amplified, but there is hardly no amplified bands in the river samples, which indicates that there is more coenzyme F 420 in the digester and the pond then the river, so we can get the coenzyme F 420 much easier from the mud of digester and pond. Moreover, the amplification efficiency of primers FGD-1011 and FGD-546 was high relatively, while the other two pairs of primers did not amplify the corresponding size of the target band. Leary-UV1801 were used to measure the fluorescence emission spectra and UV-Vis absorption spectra. Figure A shows the spectra of crude extract product with isopropyl alcohol, we can find that there are many twists and turns in the fluorescence spectra through the emission spectra of excitation light at 420nm and 480nm, fluorescence emission spectrum displays purity of the sample is not high, which needs further purification. Figure B shows that fluorescence emission spectra of the crude extract product through distillation for 2 times, the purity has been greatly improved, and the fluorescence emission spectra of samples which collected from methane digesters was significantly higher than ponds and rivers samples. In the Figure C, compare fluorescent emission spectra of 0.1μm FMN standard sample with the diluted sample 1's fluorescent emission spectra which after purified, we can find that the quality of purified coenzyme F 420 is good as standard sample. Figure D is a UV-Vis absorption spectrum of coenzyme F 420 extracted from sample 1.

Conclusion and Discussion
In this study, coenzyme F 420 was extracted and purified from the natural sludge of biogas digesters, ponds and rivers. It found that the coenzyme F 420 in biogas digesters are more abundant than ponds and rivers by PCR testing for F 420 -dependent glucose-6-phosphate dehydrogenase(FGD) gene.
The results of the experiments show that the coenzyme F 420 which extracted from the sludge samples has good purity which confirmed by the fluorescence emission spectrum and UV-Vis absorption spectrum. What's more, the quality of purified coenzyme F 420 is much better then we expect, and it could be used to make other further research. Therefore, it is feasible to extract and purify coenzyme F 420 from sludge of biogas digesters in the future which could avoid spending much energy, money and time to cultivate the methanogenic bacteria in the laboratory.
In the future, more research will be made on the function of coenzyme F 420 which extracted from the sludge of biogas digesters, aim at verifying whether the coenzyme F 420 extracted from natural is just as good as the product extracted from anaerobic bacteria which cultivated in the lab.