INTRODUCTION:

Bio-transformations are structural modifications in a chemical compound by organisms /enzyme systems that lead to the formation of molecules with relatively greater polarity¹. This mechanism has been developed by microbes to acclimatize to environmental changes and it is useful in a wide range of biotechnological processes². The most significant aspect of bio-transformation is that it maintains the original carbon skeleton after obtaining the products³. bio-transformation, as an alternative method for structural modification, catalyzes many important reactions, especially in structurally complex natural products⁴. This method exhibited many advantages such as stereo- or region-selectivity, mild reaction conditions and avoiding complex protection, and deprotection steps over chemical synthesis⁵. Microbial bio-transformation is widely used in the transformation of various pollutants or a large variety of compounds including hydrocarbons, pharmaceutical substances, and metals.

These transformations can be congregated under the categories: oxidation, reduction, hydrolysis, isomerization, condensation, formation of new carbon bonds, and introduction of functional groups⁶. For centuries microbial biotransformation has proved to be an imperative tool in alleviating the production of various chemicals used in food, pharmaceutical, agrochemical, and other industries⁷.

The tetracyclines, which were discovered in the 1940s, are a family of antibiotics that inhibit protein synthesis by preventing the attachment of aminoacyl-tRNA to the ribosomal acceptor (A) site⁸. Tetracyclines are broad-spectrum agents, exhibiting activity against a wide range of gram-positive and gram-negative bacteria, atypical organisms such as chlamydiae, mycoplasmas, rickettsiae, and protozoan parasites. The favourable antimicrobial properties of these agents and the absence of major adverse side effects have led to their extensive use in the therapy of human and animal infections⁹. They are also used prophylactically for the prevention of malaria caused by mefloquine-resistant Plasmodium falciparum⁸. Furthermore, in some countries, including the United States, tetracyclines are added at subtherapeutic levels to animal feeds to act as growth promoters¹⁰. It is well established that tetracyclines inhibit bacterial protein synthesis by preventing the association of aminoacyl-tRNA with the bacterial ribosome. Therefore, to interact with their targets these molecules need to traverse one or more membrane systems depending on whether the susceptible organism is gram-positive or gram-negative¹¹. Hence, a discussion of the mode of action of tetracyclines requires consideration of uptake and ribosomal binding mechanisms. Also pertinent to this discussion are explanations of the joint antibacterial-antiprotozoal activity of the tetracyclines and the microbial selectivity of the class as a whole¹². Most of these issues have been considered at length in recent years, so the focus here will be on new information.

MATERIALS AND METHODS:

Procurement and Maintenance of selected Organisms:

Stenotrophomonas maltophilia DT1 and Paecilomyces Species have been reported for Tetracycline Biotransformation¹³. In this work we studied the transformation of commercially available Tetracycline (Resteclin 250mg Capsules from Abott) by using two different fungi Aspergillus niger (MTCC-548) and Candida albicans (MTCC-227) obtained from Institute of Microbial Technology, Shanthi Path, 39A, Sector 39, Chandigarh-160036.

These organisms have been maintained in our Laboratory by doing Sub culturing time to time which can provide the required nutrients to the organisms. PYG Media for fungi¹⁴.

MATERIAL AND METHODS:

General requirements:

Tetracycline (1) was obtained from market belongs to Abott Routine thin-layer chromatography was performed on Precoated TLC plates (silica gel, 20×20, 0.25mm thick PF, Merck, Germany), Recycling preparative HPLC separation was performed on a JAI LC-908W instrument, equipped with YMC L-80 (4–5 µm, 20−50mm i.d.) using MeOH-H2O as the mobile phase, with UV detection at 254nm.

Microorganism:

Fungi cultures were purchased from Microbial Type culture collection centers: (MTCC), fungi used were Aspergillus niger (MTCC 548), Candida albicans (MTCC 227),

Preparation of media:

Fungi were grown on Sabouraud dextrose agar (SDA)¹⁵, and preserved at 4°C. The constituents of a suitable medium for each fungus were dissolved in 1-liter distilled water, and the pH was then adjusted by 0.1 M NaOH, and 0.1 M HCl solutions.

General screening protocol:

Preparation of second-generation culture:

The media (100mL) were transferred into conical flasks (250mL), and autoclaved at 121°C for 15 min. Three-day old slant fungal spores were added to the media under aseptic conditions, and incubated on a shaker (128 rpm) at 28°C until a reasonable biomass was obtained¹⁶. The mycelia thus formed were distributed into conical flasks containing 100mL autoclaved media, and incubated on a shaker at 28⁰C for a few days until a good biomass was obtained.

Addition of the substrate:

The substrate was dissolved in 12mL acetone (25 mg/mL), and added to flasks containing second stage culture, so that each flask contained about 25mg of the substrate. For every screening study a positive control containing the substrate in the media without the fungus, and a negative control containing the fungus in the media without the substrate were prepared. The flasks were incubated in a shaker (128rpm) at 28°C.

Monitoring the biotransformation:

A 10mL sample of media was taken from the flask and filtered, and extracted with ethyl acetate. It was then evaporated, reconstituted with 0.5mL of chloroform, and tested by TLC. The TLC results were compared with the positive and negative controls, which were treated in the same way. The progress of the biotransformation was monitored every two days for a maximum of two weeks. Each of the fungi was screened for its ability to biotransform the substrate (Tetracycline) under study.

Biotransformation of Tetracycline (1):

Each fungus was screened for its ability to biotransform Tetracycline (1) according to the above screening procedure¹⁷. The results of the screening tests showed that two cultures, which include Aspergillus niger and Candida albicans were suitable for the biotransformation of compound (1).

HPLC of transformed Tetracyclines:

Using HPLC To measure tetracycline, 1mL solution from each batch reactor was centrifuged at 8,000rpm at 4°C for 10 min. Mcilvaine-Na2EDTA buffer was added to the sample supernatant in an equal volume fashion to chelate metal ions¹⁸, and the mixture was filtered through 0.22µm filters prior to being preserved at -20°C². Stored samples were loaded onto a Shimadzu LC-06A system¹⁹. A C18 reversed-phase column (4.6×250mm, 5µm, Agilent Technologies) was operated at 40°C, with a 1mL min-1 mobile phase consisting of 67% (v/v) 0.1M ammonium oxalate in high purity water, 22% (v/v) acetonitrile and 11% (v/v) methanol²⁰. The injection volume was 20µL, and the column isocratic elution was monitored by a UV detector at 355nm.

Identification of the Transformation Products:

Two sets of reaction conditions were evaluated: in first Set reactions the three conditions we followed are 1) FM-T solution, containing Whole Cell Microorganisms such as fungi and (hydrolysis plus biotransformation), 2) FA-T solution and containing no bacteria and (hydrolysis), and 3) FM (no tetracycline); in second Set of reactions the above three steps followed but the only difference is instead of FM-T solution, we used IFM-T solution which means Immobilized Cells taken instead of Whole cells. Solutions from each reaction condition were collected on Day 0, 3, 5, 7, 9, 11, 13, 15 and the transformation products from the three conditions were identified using LC tandem mass spectrometry Q Extractive hybrid Quadrupole-Orbitrap mass spectrometer (Thermo scientific, Bremen, Germany). Aqueous samples were extracted using solid-phase extraction cartridges (Oasis HLB, 6cc/150mg, Waters) as described in previous studies. Separation was performed on a C18 column (4.6×250mm, 5µm, Agilent Technologies) with an injection volume of 5µL. Flow rate was set at 0.3mL min-1 at the isocratic mode for 20min. Mobile phase consisted of 0.1% formic acid, acetonitrile, and methanol at 67:22:11. Mass spectra were processed using the X calibur 11 2.1 software (Thermo Scientific). The mass accuracy accepted for the experiments was estimated to be below 5ppm. The MS analysis was performed with a heated electrospray ionization (HESI) source in positive mode with a spray voltage of 3.5kV, S-lens RF level of 50%, and a capillary temperature of 300°C. The MS acquisition was performed in full scan mode 50-1000 Da with a mass resolution of 70000. The molecular structure for tetracycline and their transformation products were tentatively proposed by the detection of predicted mass, changing in full scan ion intensity, and ring double bound equivalent number (RDB) from the reacted sample (Table 1).

Biotransformation of Tetracycline:

Two different fungi were studied for their ability to transform Tetracycline The screening results showed that two fungi A. niger and C. albicans were able to biotransform 1. Large-scale fermentation experiments were carried out in order to isolate metabolites 2-5, produced as a result of whole-cell biocatalysis of the enzymatic systems of fungi Aspergillus niger and Candida albicans.

Compounds 2-5, are characterized based on detailed spectral analysis.

Aspergillus niger and Candida albicans were selected for further analysis due to their ability to bio transform tetracycline. They were grown on SDA media. They could grow on CM-T agar plate which contained 20mg L-1 tetracycline. Growth was observed over a temperature range of 20-40°C, a pH range of 6.0–10.0, and a salinity range of 0-5% NaCl²¹. They showed negative results in the methyl red test, in the catalase/ oxidase/urease test, in indole production and nitrate reduction.

Insights of Tetracycline Biotransformation:

Because the biotransformation experiments were conducted in submerged fermentation, hydrolysis was an intrinsic part of the experiment. Hence, biotransformation cannot be one and only method to alternate the Tetracycline Concentrations in Medium. Hence, to estimate the biotransformation, we conducted different experiments that contained strains individually are Aspergillus niger and Candida albicans. whole cells and Immobilized cells and a control experiment that contained no bacterial cells. The differences in the tetracycline profiles between the experiments were attributed to biotransformation. Both the overall transformation and the hydrolysis followed first order reaction kinetics. The fastest overall transformation appeared at 30°C, where 20mg L -1 tetracycline was transformed in 14 days. The changes of tetracycline concentrations due to hydrolysis and due to combined hydrolysis and biotransformation plotted in Scheme. 1,2,3. The initial biotransformation rate was the highest at 30°C with a value of 10.13mg L-1 d -7. it is difficult to estimate fungal biomass. Because the fungal strains are very possible to form the biofilm on the inner wall of the fermentation vessels. The hydrolysis and the biotransformation of tetracycline were also affected by solution PH. The maximum hydrolysis occurred when the initial pH was 4. The hydrolysis rate will be increases with the increasing initial pH. The Maximum initial biotransformation rate was at pH 6. At the end of the transformation experiment, the final pH of all two sets was close to 6. If the initial p^His <4, it leads to lagging of biotransformation of Tetracyclines.

Tetracycliine Transformed Products:

From the fermentation vessels, where both hydrolysis and biotransformation occurred, the initial concentration of the tetracycline reduced from 20.00mg L-1 to approximately 6.23mg L-1 by Day 14²². In comparison, in the control reactors where only hydrolysis occurred, residual tetracycline dropped from 20.00mg L-1 to 11.93 mg L-1 by Day 14. Both organisms are involved in the transformation of Tetracycline, the residual concentrations are nearer to the above values. T A peak corresponding to either an epimer or isomer of tetracycline was identified (Fig:1 and Table 1) in Day 3 of the experiment. Tetracycline eluted at 7.40min with an m/z value of 445.1601 for its protonated form, while the peak for either 2-epi-tetracycline (ETC) or is tetracycline (ISO-TC) 3, had the same m/z value but an earlier retention time (i.e., 6.49min). Previous work in aqueous systems at near neutral pH suggests that formation of ISO-TC is unusual. 4 possible biotransformation products were also identified (Fig. 1 to 3 and Table 1.) and the mass spectra of the parent compound and proposed transformation products are shown in Fig. 1 to 3. A putative biotransformation pathway of tetracycline by A.niger and C.albicans was proposed in Fig 1 to 3. Formation of compound A.n TTC could occur when the carbonylation occurs. Formation of C.a TTC may occur via acetylation.

RESULTS AND DISCUSSION:

Different from sulphonamide compounds, whose degradation by pure fungal cultures. and microbially mediated abiotic processes have been reported, little is known about how microbial processes may transform tetracycline compounds. Several Stenotrophomonas strains have demonstrated the ability to degrade organic pollutants, such as pesticide, insecticide, BTEX, PAH, and steroid hormones. These strains can transform toxic pollutants by opening their loop structure or by cutting functional groups. For example, Stenotrophomonas Sp. THZ-XP can bio transform the pesticide acetamiprid by cutting it cyano substituent. We noticed that although strains of Aspergillus and Candida species could form colonies on SDA-T agar plates, they could not grow in SD-T liquid medium. This suggests that strains may not be able to use tetracycline as sole carbon and energy source. The growth of strains on SD-T agar plates might be due to the utilization of the trace number of organic matters in agar. The biotransformation of tetracycline by fungal strains might be attributed to the detoxification mechanism, which relies on the flavin-dependent monooxygenases. Temperature and pH can influence the hydrolysis of tetracycline. Similar to the findings from a previous study, this study demonstrated that high temperatures accelerated tetracycline hydrolysis. According to the collision theory, higher temperatures lead to higher reaction rates by causing more collisions between particles. High pH also accelerated the hydrolysis of tetracycline. Tetracycline is an amphoteric molecule with three dissociation constants (pKa=3.3, 7.7, 9.7). As pH increased from 4 to 6, different reactive species with various degrees of ionization might have dominated the solution and led to different hydrolysis rates. For the temperature and pH tested (except pH <4), biotransformation increased in the first four days before levelling off. Despite the buffer system in the solution, strains changed the final pH of the solution to ~6 at the end of the experiment regardless the initial pH values. Fungal cells can break down peptone in the reaction medium and release alkaline compounds such as ammonia and amines, which can cause increase in pH. Previous studies showed that the tetracycline transformation products from photolysis had higher toxicity than the parent compound. In contrast, the tetracycline transformation products from fungal lactase had lower toxicity than the parent compound. Hence, microbial transformation of tetracycline may be more desirable from an environmental perspective than physicochemical processes as it reduces the biological activities of the antibiotic. During hydrolysis, under the action of hydroxide ion from water the hydroxyl group on C6 may attack the carbonyl group on C12 and form iso-tetracycline (ISO-TC) irreversibly in alkaline pH range. Also, tetracycline can undergo reversible epimerization on C6 under certain acidic conditions and form the corresponding 6-epi-tetracyline (ETC) with the participation of hydrogen ions from water at acidic pH conditions. Moreover, tetracycline can turn into ISO-TC or ETC at neutral or weak alkaline conditions (pH =6.5–9), causing decrease in tetracycline concentrations during hydrolysis [fig.1]. The intensity of subsequent transformation products C.a TTC1 and C.aTTC2 were nearly an order of magnitude lower than A.nTTC2 (Table 1). suggesting the pathway through ISO-TC was not the dominant pathway.

Figure 1: Hydrolysis of Tetracycline in the presence of Aspergillus niger and Candida albicans

Figure: 2 Carbonylation of Tetracycline in the presence of Aspergillus niger

Figure: 3 Acetylation of Tetracycline in the presence of Candida albicans

CONCLUSION:

Chemical synthesis is challenging to accomplish these highly selective reactions, especially under moderate circumstances. The investigation's findings enable a more accurate prediction of the fate and delivery of antibiotics to changed or transformed forms, which will have more advantages than those of present forms.

Recovery of Biotransformation products has a lot of potential to develop novel Tetracycline compounds that might overcome bacterial resistance in the not too distant future.

ACKNOWLGEMENT:

I am thankful to Dr. Bodla R.B for helping me through the various analysis stages, and for providing helpful criticism and feedback throughout the work and writing process.

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Received on 21.05.2023 Modified on 18.09.2023

Research J. Pharm. and Tech 2024; 17(5):2201-2205.

DOI: 10.52711/0974-360X.2024.00346

Retention time	Compound	Ion	Predicted mass m/z	Measured mass m/z	Elemental composition	Double bond equivalents (RDB)	Intensity
6.49	ETC/ISO-TC	[M+H]⁺	445.1601	445.1580	C₂₂H₂₅O₈N2	11.5	2.75E+07
7.40	TC*	[M+H]⁺	445.1601	445.14801	C₂₂H₂₅O₈N₂	11.5	4.79+05
7.80	A.nTTC	[M+H+CO]⁺	473.1445	472.8327	C₂₃H₂₃O9N₂	12.5	5.24E+05
9.84	C.aTTC	[M+H+COCH₃]⁺]⁺	488.1131	488.0016	C₂₄H28O9N₂	12.5	6.57E+06