Transcript 幻灯片 1 - nenu.edu.cn

Biotransformation of
polysialogangliosides
Contents
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1. Biotransformation
2. Ganglioside
3. GM1
4. Examples
5. Conclusion
1. Biotransformation
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1.1 What is transformation and why we use it
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1.2 How many kinds of transformation
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1.3 What is biotransformation
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1.4 Why do we use biotransformation
1.1 Transformation
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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.
1.1 Transformation
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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.
1.2 Types of transformation
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Chemical transformation
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Biotransformation
1.3 Biotransformation
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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 enzymecatalyzed reaction.
1.4 The advantages of biotransformation
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1. exquisite chemo-selectivity, regioselectivity, stereo-selectivity.
2. less by-product
3.easy to operate
4.under mild conditions
5.others
Such as lower toxicity, improved solubility.
2.Ganglioside
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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.
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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.
The structures of some gangliosides
3.GM1
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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.
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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.
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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.
4.Examples
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4.1 Preparation of GM1 Ganglioside with
Sialidase-Producing Marine Bacteria as a
Microbial Biocatalyst
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4.2 Highly efficient conversion of
polysialoganglioside to GM1 with
Brevibacterium casei as a microbial
biocatalyst
4.1 Marine Bacteria
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This paper describes the preparation of
monosialoganglioside GM1 with sialidaseproducing 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.
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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 DEAESephadex A25 anion-exchange
chromatography.
Methods and results
<1> Isolation and identification of sialidaseproducing 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.
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This result strongly suggests that sialidaseproducing 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.
Among the 28 strains isolated as sialidaseproducing 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.
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<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.
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<3> Purification and characterization of
GM1 from the culture of strain YF-2.
Using anion exchange chromatograph and
analyzing by HPLC and MS.
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<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
4.2 Soil Bacteria
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4.2.1 Methods
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4.2.2 Results and discuss
4.2.1 Methods
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<1> Extract the ganglioside
<2> Separate the ganglioside
<3> Conversion
<4> Purification of GM1 from culture broth
<1> Extract the ganglioside
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Gangliosides were extracted from pig brain
and purified by the adsorbent
chromatography method.
<2> Separate the ganglioside
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GM1 was isolated by silica gel
chromatography. So GM1 was collected as
product.
And the other gangliosides components were
collected as substrates for biotransformation.
<3> Conversion
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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.
<4> Purification of GM1 from culture
broth
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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.
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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.
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The crude GM1 was dissolved in methanolwater(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.
4.2.2 Results and discuss
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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%.
Figure1
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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.
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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
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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.
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GM1 in the culture broth was separated and
purified by X-5 adsorption resin
chromatography followed by Sephadex LH20 chromatography. Finally, 0.7g of purified
GM1 was obtained from 1.5g free GM1
gangliosides (with a purity of about 90%)
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There have been many reports of sialidaseproducing microoganisms.
Other examples
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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
Other examples
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Both the two examples used a bioreacter
about 30 liter to scale-up the bioprocess of
conversion from polysialogangliosides to
GM1.
5. Conclusion
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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