biotransformation of polysialogangliosides. contents 1. biotransformation 2. ganglioside 3. gm1 4....

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Biotransformation of polysialogangliosides

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Page 1: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

Biotransformation of polysialogangliosides

Page 2: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

Contents

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

Page 3: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

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

Page 4: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

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.

Page 5: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

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.

Page 6: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

1.2 Types of transformation

Chemical transformation

Biotransformation

Page 7: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

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.

Page 8: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

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.

Page 9: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

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.

Page 10: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

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.

Page 11: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

The structures of some gangliosides

Page 12: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

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.

Page 13: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

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 H2SO4 also results in the removal of sialic acids from GM1, producing asialo GM1.

Page 14: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

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.

Page 15: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

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

Page 16: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

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.

Page 17: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

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 C18 reverse-phased chromatography, followed by DEAE-Sephadex A25 anion-exchange chromatography.

Page 18: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

Methods and results

<1> 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.

Page 19: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

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.

Page 20: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

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.

Page 21: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

<2> 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.

Page 22: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

<3> Purification and characterization of GM1 from the culture of strain YF-2.

Using anion exchange chromatograph and analyzing by HPLC and MS.

Page 23: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

<4> 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

Page 24: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

4.2 Soil Bacteria

4.2.1 Methods

4.2.2 Results and discuss

Page 25: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

4.2.1 Methods

<1> Extract the ganglioside <2> Separate the ganglioside <3> Conversion <4> Purification of GM1 from culture broth

Page 26: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

<1> Extract the ganglioside

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

Page 27: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

<2> 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.

Page 28: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

<3> 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.

Page 29: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

<4> 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.

Page 30: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

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.℃

Page 31: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

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.

Page 32: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

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%.

Page 33: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

Figure1

Page 34: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion
Page 35: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

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.

Page 36: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

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

Page 37: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion
Page 38: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

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.

Page 39: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion
Page 40: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

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%)

Page 41: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

There have been many reports of sialidase-producing microoganisms.

Page 42: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

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

Page 43: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

Other examples

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

Page 44: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion

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

Page 45: Biotransformation of polysialogangliosides. Contents 1. Biotransformation 2. Ganglioside 3. GM1 4. Examples 5. Conclusion