1. Biotransformation of polysialogangliosides

2. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

3. 1. Biotransformation 1.1 What is transformation and why we use it 1.2 How many kinds of transformation 1.3 What is biotransformation 1.4 Why do we use biotransformation

4. 1.1 Transformation Simply say it is altered molecular structures. Compounds derived from natural reserves have long been sources of medicines and have always made immense impact on the pharmaceutical industry through the process of drug discovery. But we all know that some compounds in the nature source is so little that make the drug quite expensive.

5. 1.1 Transformation In recent years, a second generation of drug discovery is started. It altered molecular structures and gained worldwide recognition due to their improved pharmaceutical properties such as low toxicity, improved solubility and pharmacokinetics. These are primarily natural product analogs.

6. 1.2 Types of transformation Chemical transformation Biotransformation

7. 1.3 Biotransformation Biotransformation is a biochemical reaction to modify the structure or the xenobiotics by vegetal cellular or organ, animal cellular, microorganism and its orgenelle, and isolated enzyme which is mainly enzyme- catalyzed reaction.

8. 1.4 The advantages of biotransformation 1. exquisite chemo-selectivity, regio- selectivity, stereo-selectivity. 2. less by-product 3.easy to operate 4.under mild conditions 5.others Such as lower toxicity, improved solubility.

9. 2.Ganglioside Gangliosides were found to modulate the function of various membrane proteins including enzymes, ion channels receptors, and cell adhesion molecules. They are divided into several groups according to the structure of the backbone saccharides.

10. The gangliotertraose family is a major ganglioside of mature neurons and is classified, depending on the number of linked sialic acid residues per molecule, into the following: GM1, GD1, GT1, GQ1. Recently, clinical applications of GM1 for neurological disorders such as Alzheimer’s disease, Parkinson's disease, spinal-cord injury, and stroke have been reported.

11. The structures of some gangliosides

12. 3.GM1 Monosialotertrahexosylganglioside-GM1 is usually prepared from animal brain gangliosides. However, these ganglioside preparations contain less than 30% GM1, the rest being polysialogangliosides which contain two or more sialic acid residues. The importance of its physiological functions and its potential clinical applications highlight the need for a simple procedure to obtain large quantities of high purified GM1.

13. To prepared GM1 from polysialogangliosides, sialic acids are removed by either a sialidase or an acid treatment. However, both methods are unsuitable for mass production of GM1, since the conversion rate of any of polysialogangliosides to GM1 is quite low after digestion with any of the commercially available sialidases and the use of HCl or H 2 SO 4 also results in the removal of sialic acids from GM1, producing asialo GM1.

14. Given the limitations for the production of GM1, the preparation of GM1 ganglioside using sialidase-producing bacteria as a microbial biocatalyst is available alternative for large-scale production.

15. 4.Examples 4.1 Preparation of GM1 Ganglioside with Sialidase-Producing Marine Bacteria as a Microbial Biocatalyst 4.2 Highly efficient conversion of polysialoganglioside to GM1 with Brevibacterium casei as a microbial biocatalyst

16. 4.1 Marine Bacteria This paper describes the preparation of monosialoganglioside GM1 with sialidase- producing marine bacteria as a microbial biocatalyst. A new sialidase-producing bacterium, identified tentatively as pseudomonas sp. strain YF-2, was isolated from seawater by enrichment culture with ganglioside as the sole source of carbon.

17. When YF-2 was cultured in a synthetic medium containing crude bovine brain gangliosides at 25 ℃ for 3 days, 80 to 90% of the gangliosides were converted to GM1. GM1 was then purified from the supernatant of YF-2 culture by C 18 reverse-phased chromatography, followed by DEAE- Sephadex A25 anion-exchange chromatography.

18. Methods and results Isolation and identification of sialidase- producing bacteria. Sialidase-producing bacteria have been isolated from seawater, sea sand, sea mud, marine algae, and the gills of marine fish by enrichment culture with synthetic medium A containing crude bovine brain gangliosides as the sole source of carbon.

19. This result strongly suggests that sialidase- producing bacteria are widely distributed and could contribute to the degradation of polysialogangliosides in marine environments. It should be noted that polysialogangliosides have been shown to be abundantly present in the brains of marine fish.

20. Among the 28 strains isolated as sialidase- producing bacteria, YF-2 was found to have the highest productivity of sialidase acting on gangliosides. Strain YF-2, isolated from seawater, is a short rod-shaped bacterium with a long polar flagellum. Optimum growth of YF-2 was observed in medium containing 2% NaCl.

21. Conversion of polysialogangliosides to GM1 by strain YF-2. Conversion of polysialogangliosides to GM1 proceeded gradually, and after 3 days the conversion was more than 80% complete. Asialo GM1 was not detected during the cultivation period, indicating that sialic acids were removed from polysialogangliosides but not from GM1.

22. Purification and characterization of GM1 from the culture of strain YF-2. Using anion exchange chromatograph and analyzing by HPLC and MS.

23. Purification of sialidase. Sialidase was purified 33-fold with 13.3% recovery from a culture supernatant of newly isolated Pseudomonas sp. strain YF-2 by anion-exchange, gel filtration and hydroxyapatite chromatographies

24. 4.2 Soil Bacteria 4.2.1 Methods 4.2.2 Results and discuss

25. 4.2.1 Methods Extract the ganglioside Separate the ganglioside Conversion Purification of GM1 from culture broth

26. Extract the ganglioside Gangliosides were extracted from pig brain and purified by the adsorbent chromatography method.

27. Separate the ganglioside GM1 was isolated by silica gel chromatography. So GM1 was collected as product. And the other gangliosides components were collected as substrates for biotransformation.

28. Conversion Cells from agar slant culture were transferred into preculture medium containing 2% glycerol as carbon source. After this step, the seed culture were transferred into conversion medium containing 0.5% (w/v) free GM1 ganglioside as sole carbon source.

29. Purification of GM1 from culture broth Following the conversion procedure, the culture broth was harvested by centrifugation. An equal volume of methanol containing NaCl at a final concentration of 0.03M was added to the supernatant, and the supernatant applied to the column, which was washed successively with water, methanol-water (1:1, v/v) to remove the water soluble impurities.

30. The gangliosides were then eluted with methanol and the fractions containing gangliosides were collected. The combined gangliosides fractions were concentrated in a rotary evaporator under vacuum at 40 ℃. Three volumes of concentrated acetone were then added to precipitate the gangliosides, the precipitate collected by centrifugation and dried under vacuum at 20-30 ℃ as crude GM1.

31. The crude GM1 was dissolved in methanol- water(1:1, v/v) and then loaded onto a Sephadex LH-20 column that had been equilibrated with methanol-water(1:1, v/v). Fractions were assayed by High performance thin-layer chromatography (HPTLC). The fractions containing GM1 were collected and evaporated to dryness to obtain the final product.

32. 4.2.2 Results and discuss GM1 is a component of gangliosides and makes up less than 20% of the total gangliosides as shown in Figure 1. After silica chromatography separation of gangliosides, the polysialoganglioside fractions were collected for subsequent biotransformation. About 0.7 g polysialogangliosides were obtained from 1 g gangliosides, the yield was nearly 70%.

33. Figure1

35. Strain YZ-1, isolated from soil with gangliosides as sole carbon source by enrichment cultivation and spread plaiting, was a short rod-shaped bacterium. A seed culture in exponential growth phase was transferred into conversion medium, where strain YZ-1 was cultured with free GM1 gangliosides as the sole carbon source.

36. The polysialogangliosides were converted to GM1 by strain YZ-1 as biocatalyst. The time course of cells growth and concentration of GM1 is shown in Figure 2

38. The conversion procedure increased gradually paralleling the growth of the bacterium. The highest concentration of GM1 in the broth was obtained after two days of cultivation as the culture entered stationary phase. As the cultivation proceeded beyond this point the concentration of GM1 in broth decreased gradually, presumably because it was being metabolized by the bacterium as a nutrient. The optimal conversion was obtained within 48h.

40. GM1 in the culture broth was separated and purified by X-5 adsorption resin chromatography followed by Sephadex LH- 20 chromatography. Finally, 0.7g of purified GM1 was obtained from 1.5g free GM1 gangliosides (with a purity of about 90%)

41. There have been many reports of sialidase- producing microoganisms.

42. Other examples Efficient conversion from polysialogangliosides to monosialotetrahexosylganglioside using Oerskovia xanthineolytica YZ-2 Development of a large scale process for the conversion of polysialogangliosides to monosialotetrahexosylganglioside with a novel strain of Brevibacterium casei producing sialidase

43. Other examples Both the two examples used a bioreacter about 30 liter to scale-up the bioprocess of conversion from polysialogangliosides to GM1.

44. 5. Conclusion 1. Find the natural product analogs which have better activity 2. Find the way to alter the molecular structure 3. Consider the transformation methods 4. Transformation 5. Purity the final product