Method for improving vanillin yield
CN117904216A
2024-01-23 Method for improving vanillin yield
Current Assignee Tianjin University Advanced
Research Institute Of Synthetic Biology
Abstract
The
invention relates to the technical field of biology, in particular to a
method for improving the yield of vanillin produced by yeast. According
to the method, engineering saccharomyces cerevisiae is used as a
biocatalyst, ferulic acid and/or lignin are used as substrates to
produce vanillin, amino groups in chitosan molecules and aldehyde
groups of the vanillin are used for carrying out Schiff base reaction,
and the vanillin is removed from a reaction system, so that the
toxicity to cells is effectively reduced, the titer of the vanillin is
finally obviously improved, and the method has a good application
prospect.
Description
Method for improving vanillin yield
Technical Field
The invention relates to the technical field of biology, in particular to a method for improving the yield of vanillin.
Background
Vanillin
(vanillin), also known as vanillin, is known under the chemical name
4-hydroxy-3-methoxybenzaldehyde, which is present in vanilla beans, is
one of the most widely used aromatic compounds. The perfume composition
is widely used in the fields of foods, beverages, perfumes, cosmetics,
intermediates in agriculture, chemical industry, pharmaceutical
industry and the like. It was predicted that in 2025, the market demand
for vanillin would exceed 59000 tons. The vanillin sold in the market
at present is mainly produced by a chemical method, and the vanillin is
synthesized by eugenol, guaiacol and the like. However, in the age of
climate change and reduction of fossil resources, it is becoming
increasingly important to realize sustainable green production of
target products using renewable resources and environmental protection
methods. The biotechnological process offers a promising alternative
due to the inherently mild reaction conditions and the use of renewable
raw materials. In addition, according to the united states and european
food regulations, biologically produced vanillin is known as natural
vanillin. The price of natural vanillin is far higher than that of the
chemical synthesis method. However, the yield of vanillin is still low
due to the strict regulation of aromatic synthesis and metabolism by
Saccharomyces cerevisiae and the metabolic bottlenecks in the synthetic
pathway. In addition, vanillin has toxic effects on cells and causes
strains to break down vanillin into less harmful compounds such as
vanillic acid and vanillyl alcohol.
Disclosure of Invention
In
view of this, the present invention provides a method for increasing
the yield of vanillin. According to the method, the vanillin is removed
from the reaction system through the chitosan membrane, so that the
toxicity of the vanillin to cells is effectively reduced, and finally,
the titer of the vanillin is obviously improved.
In order to achieve the above object, the present invention provides the following technical solutions:
application of chitosan in improving vanillin biosynthesis yield
The substrate for vanillin biosynthesis comprises at least one of p-coumaric acid, ferulic acid or lignin.
In
the application of the invention, the microorganism adopted in the
biosynthesis is engineering bacteria with a vanillin synthesis path,
and the engineering bacteria comprise at least one of yeast,
escherichia coli, pseudomonas and streptomyces; the yeast includes
Saccharomyces cerevisiae.
The invention also provides a method for improving the yield of vanillin, comprising:
preparing a chitosan film;
mixing chitosan film, substrate for synthesizing vanillin and biocatalyst, and reacting;
acid
washing and filtering the chitosan membrane after reaction, and
carrying out acid hydrolysis and filtering on the chitosan membrane
after reaction; extracting the filtrate with ethyl acetate, evaporating
to remove an organic phase to obtain a crude product containing
vanillin;
the biocatalyst is a microorganism having a vanillin synthesis pathway.
The invention provides a method for preparing a chitosan film, which comprises the following steps of:
adding chitosan into the mixed solution of glycerol and acetic acid solution, and stirring to obtain chitosan solution;
Uniformly
spreading the chitosan solution on a substrate, drying, soaking in
alkali liquor, peeling off the film, washing with water to neutrality,
and naturally airing to obtain the chitosan film.
In some
embodiments, in the preparation method of the chitosan film, the volume
ratio of the glycerol to the acetic acid solution is 1:150; the acetic
acid solution contains 2% of acetic acid by volume.
In the
preparation method of the chitosan film, the alkali liquor is sodium
hydroxide solution, and the soaking time is 4 hours; in some
embodiments, the concentration of sodium hydroxide solution is 20 to
30g/L. The drying is carried out for 18-24 hours at 40 ℃;
The stirring is mechanical stirring at 450rpm for 5-8 hours at room temperature.
In
some embodiments, the invention is not particularly limited to the type
of substrate, any substrate commonly known in the art capable of
synthesizing vanillin, such as ferulic acid, lignin and/or p-coumaric
acid, or an extract or derivative of ferulic acid, an extract or
derivative of lignin, or an extract or derivative of p-coumaric acid.
The microbial yeast is a yeast with a vanillin synthesis pathway; the
yeast includes Saccharomyces cerevisiae. In some embodiments, the
substrate is ferulic acid, lignin, and/or p-coumaric acid at a
concentration of 0.8-1.2g/L.
In some embodiments, the acid employed
for the pickling is hydrochloric acid; the volume percentage
concentration of the hydrochloric acid is preferably 6%; the pickling
time is 12 hours.
According to the invention, engineering
saccharomyces cerevisiae is used as a biocatalyst, ferulic acid and/or
lignin are used as substrates to produce vanillin, amino groups in
chitosan molecules are utilized to react with aldehyde groups of
vanillin to generate Schiff base, vanillin is removed from a reaction
system, so that the toxicity to cells is effectively reduced, and
finally, the titer of vanillin is obviously improved, and experimental
results show that when the ferulic acid is used as a substrate, the
process of the invention is used for producing vanillin, the titer of
vanillin is 12.8mM (2.0 g/L) when the reaction system is 10 ml, the
yield is 80% and is 2.2 times that of the reaction system without
chitosan, the yield is increased by 6.9mM, and the titer of vanillin is
15.3mM (2328 mg/L) when the reaction system is 100ml, and the yield is
71%. The biological conversion process of the invention produces
vanillin with a vanillin titer of 21.1mM (3.2 g/L) (100 ml reaction)
using lignin extract as substrate, corresponding to 1.4mg vanillin
produced from 1g wheat bran biomass. Therefore, the process provided by
the invention obviously improves the yield of vanillin biosynthesis and
has excellent application prospect.
Drawings
FIG. 1 shows a mechanical diagram of the reaction of vanillin with chitosan;
FIG. 2 shows the production of vanillin using the method of the invention with ferulic acid as a substrate;
FIG. 3 shows the production of vanillin using the method of the invention with lignin as a substrate.
Detailed Description
The
present invention provides a method for increasing the yield of
vanillin. Those skilled in the art can, with the benefit of this
disclosure, suitably modify the process parameters to achieve this. It
is expressly noted that all such similar substitutions and
modifications will be apparent to those skilled in the art, and are
deemed to be included in the present invention. While the methods and
applications of this invention have been described in terms of
preferred embodiments, it will be apparent to those skilled in the
relevant art that the invention can be practiced and practiced with
modification and alteration and combination of the methods and
applications herein without departing from the spirit and scope of the
invention.
Unless otherwise specified, the test materials adopted in
the invention are all common commercial products and can be purchased
in the market.
The invention is further illustrated by the following examples:
EXAMPLE 1 preparation of the chitosan film of the present invention and method for synthesizing (producing) vanillin by chitosan
1ML
of glycerin was added to 150mL of 2% (v/v) acetic acid solution and
mixed well, then 3g of chitosan powder was added, and the mixture was
mechanically stirred at 450rpm for 5-8 hours at room temperature until
the chitosan was completely dissolved. 12mL of chitosan solution was
placed on a disposable plastic microorganism culture dish and slowly
shaken to fill the bottom of the dish. The petri dish containing the
chitosan solution was then dried in an oven at 40 ℃ for 18-24 hours to
remove moisture. Adding 20-30 g/L NaOH solution into the dried chitosan
culture, soaking for 4 hours, and slowly stripping the chitosan film by
forceps. The chitosan film was repeatedly washed with distilled water
until the washing solution was neutral, and then the film was naturally
dried on gauze, and then stored in a dryer until use.
Chitosan film
(0.1 g), ferulic acid (or lignin extract) and glucose were added to
10mL 100mM PBS buffer (containing yeast catalyst), wherein the initial
final concentration of glucose was 5-10g/L and the initial final
concentration of ferulic acid was 0.8-1.2 g/L. After the reaction was
completed, the chitosan film was immersed in a 6% v/v hydrochloric acid
solution and hydrolyzed for 12 hours. The above hydrochloric acid
solution was filtered, and the filtrate was analyzed by HPLC.
Test case
1. Culture medium:
YPD
medium configuration: 20g of glucose, 10g of yeast powder and 20g of
peptone, and sterilizing with purified water to a volume of 1L at 115 ℃
under high temperature and high pressure for 15min.
SC medium
configuration: 20g of glucose, 6.7g of yeast nitrogen source and 2g of
amino acid mixed powder, leucine (100 mg), uracil (20 mg) and histidine
(20 mg) corresponding to the required defects are added, the volume is
fixed to 1L by purified water, the pH is adjusted to 6 by 2MNaOH, and
the mixture is sterilized at the temperature of 115 ℃ for 15min under
high temperature and high pressure.
Amino acid mixed powder
configuration: respectively weighing 5g of adenine, 20g of alanine, 20g
of arginine, 20g of aspartic acid, 20g of asparagine, 20g of proline,
20g of glutamic acid, 20g of glutamine, 20g of glycine, 20g of
isoleucine, 20g of lysine, 20g of methionine, 2g of para-aminobenzoic
acid, 20g of phenylalanine, 20g of cysteine, 20g of serine, 20g of
threonine, 20g of serine, 20g of tyrosine, 20g of valine and 20g of
inositol. The above powder was placed in a dry blue cap bottle and
mixed overnight in a shaker until homogeneous for use.
The inorganic
salt culture medium comprises the following components: 20g/L glucose,
7.5g/L ammonium sulfate, 14.4g/L potassium dihydrogen phosphate, 0.5g/L
magnesium sulfate heptahydrate, 2mL/L trace elements and 1mL/L vitamin
solution.
The trace element components were 3.0g/L ferrous sulfate
heptahydrate, 4.5g/L zinc sulfate heptahydrate, 4.5g/L calcium chloride
dihydrate, 0.84g/L manganese chloride dihydrate, 0.3g/L cobalt chloride
hexahydrate, 0.3g/L copper sulfate pentahydrate, 0.4g/L sodium
molybdate dihydrate, 1.0g/L boric acid, 0.1g/L potassium iodide, and
19.0g/L disodium ethylenediamine tetraacetate dihydrate.
The vitamin
solution had a composition of 0.05 g/LD-biotin, 1.0 g/LD-calcium
pantothenate, 1.0g/L thiamine hydrochloride, 1.0g g/L pyridoxine
hydrochloride, 1.0g/L niacin, 0.2g/L para-aminobenzoic acid, and
25.0g/L inositol.
The optimized formula is to add 5g/L peptone on the basis of inorganic salt culture medium.
2. Full cell bioconversion of vanillin without chitosan
1) Primary seed culture:
Inoculating
Saccharomyces cerevisiae yVAN glycerol bacterial liquid stored in a
refrigerator at-80deg.C into shaking tube containing 5mLYPD liquid
culture medium, and shake culturing at 30deg.C and 220rpm for 16-20 hr
to middle and late stage of index as primary seed liquid.
2) Secondary seed culture:
The
primary seed solution was transferred to shaking culture medium
containing 5mL of liquid medium at 30℃and 220rpm for 16-20 hours to
medium and late exponential phase at OD600 of about 5-6 in SC culture
and OD600 of about 8-10 in YPD medium according to OD 600=0.2.
3) Fermentation culture:
The
secondary seeds are inoculated into a fermentation medium (YPD medium
or inorganic salt medium or medium plus 5g/L peptone) for culture until
the secondary seeds are in the middle and later periods of the index.
4) Whole cell bioconversion:
the
cultured fermentation broth was centrifuged at 5000rpm at 4℃to collect
cells, which were washed twice with 100mM PBS. The cells were
resuspended to the desired OD for the experiment with 100mM PBS,
glucose, ferulic acid (or p-coumaric acid or lignin extract) were
added, and the whole cell bioconversion reaction was performed at
30℃and 220 rpm. Wherein, the initial concentration of the ferulic acid
is 0.8-1.2g/L, and the glucose is 10 g/L.
3. Preparation of the
chitosan film of the present invention and in situ product removal by
chitosan synthesis (production) of vanillin
1ML of glycerin was
added to 150mL of 2% (v/v) acetic acid solution and mixed well, then 3g
of chitosan powder was added, and the mixture was mechanically stirred
at 450rpm for 5-8 hours at room temperature until the chitosan was
completely dissolved. 12mL of chitosan solution was placed on a
disposable plastic microorganism culture dish and slowly shaken to fill
the bottom of the dish. The petri dish containing the chitosan solution
was then dried in an oven at 40 ℃ for 18-24 hours to remove moisture.
To the dried chitosan culture, a 3% v/v NaOH solution was added, and
after soaking for 4 hours, the chitosan film was slowly peeled off with
tweezers. The chitosan film was repeatedly washed with distilled water
until the washing solution was neutral, and then the film was naturally
dried on gauze, and then stored in a dryer until use.
Adding
chitosan film (0.1 g), ferulic acid and glucose into 10mL 100mM PBS
buffer solution (containing yeast yVAN/L which is resuspended in step
4), wherein the initial final concentration of glucose is 5-10g/L, the
initial concentration of ferulic acid is 0.8-1.2g/L, the ferulic acid
feed is added in real time according to the liquid phase detection
concentration, and the final concentration of each addition is
0.6-1.2g/L. After the reaction was completed, the chitosan film was
immersed in a 6% v/v hydrochloric acid solution and hydrolyzed for 12
hours. The above hydrochloric acid solution was filtered, and the
filtrate was analyzed by HPLC.
4. Lignin extract preparation
Wheat
bran was ground into powder with a grinder and sieved through a 180
mesh stainless steel mesh. The crushed wheat bran was subjected to
alkaline hydrolysis with 0.5m noh solution. Hydrolysis conditions: the
solid-to-liquid ratio was 0.35g/mL and hydrolyzed in an incubator
shaker at 30℃and 220rpm for 4 hours. After the hydrolysis is completed,
solid-liquid separation is performed by centrifugation. The alkaline
hydrolysate was acidified to pH1.5 with 72% w/v H 2SO4 and then
centrifuged at 12000rpm for 30 minutes. The supernatant (called acid
liquor) and solids (called acid solids) were collected. Both the acidic
liquid and the acidic solid were extracted with ethyl acetate (EtOAc).
The organic phase was concentrated by rotary evaporation and dried in a
vacuum oven. The resulting extract was dissolved in 100mM potassium
phosphate buffer pH 7.0 and used for HPLC analysis and as a substrate
for whole cell bioconversion.
The reaction mechanism of vanillin and chitosan is shown in figure 1.
Vanillin
is synthesized (produced) by the method of step 3 with ferulic acid and
the lignin extract as substrates, respectively, wherein yeast is
yVAN005,005, and the results are shown in fig. 2-3.
As can be seen
from FIG. 2, vanillin was produced by the process of the invention
using ferulic acid as a substrate, with a vanillin titer of 12.8mM (2.0
g/L) and a yield of 80% (reaction system 10 ml). The yield was 2.2
times that without chitosan, 6.9mM was added. Vanillin was produced by
an in situ product removal whole cell bioconversion process with a
vanillin titer of 15.3mM (2.3 g/L) and a yield of 71% (100 ml reaction
system).
As can be seen from FIG. 3, vanillin was produced by the in
situ product removal whole cell bioconversion process of the invention
using lignin extract as substrate, with a vanillin titer of 21.1mM (3.2
g/L) (100 ml reaction system). This corresponds to 1.4mg of vanillin
produced with 1 gram of wheat bran biomass, as shown in figure 3.
The
foregoing is merely a preferred embodiment of the present invention and
it should be noted that modifications and adaptations to those skilled
in the art may be made without departing from the principles of the
present invention, which are intended to be comprehended within the
scope of the present invention.
Claims (10)
Hide Dependent
1. Use of chitosan for increasing the biosynthesis yield of vanillin.
2.
The use according to claim 1, wherein the substrate for vanillin
biosynthesis comprises at least one of p-coumaric acid, ferulic acid or
lignin.
3. The use according to claim 1, wherein the microorganism
used for biosynthesis is an engineering bacterium having a vanillin
synthesis pathway, the engineering bacterium comprising yeast,
escherichia coli, pseudomonas or streptomyces; the yeast includes
Saccharomyces cerevisiae.
4. A method for increasing vanillin production comprising:
preparing a chitosan film;
Mixing chitosan film, substrate for synthesizing vanillin and biocatalyst, and reacting;
washing
the chitosan film after reaction with acid and filtering; extracting
the filtrate with ethyl acetate, evaporating to remove an organic phase
to obtain a crude product containing vanillin;
the biocatalyst is a microorganism having a vanillin synthesis pathway.
5. The method according to claim 4, wherein the preparation method of the chitosan film comprises:
adding chitosan into the mixed solution of glycerol and acetic acid solution, and stirring to obtain chitosan solution;
Uniformly
spreading the chitosan solution on a substrate, drying, soaking in
alkali liquor, peeling off the film, washing with water to neutrality,
and naturally airing to obtain the chitosan film.
6. The method of
claim 5, wherein the volume ratio of glycerol to acetic acid solution
is 1:150; the acetic acid solution contains 2% of acetic acid by volume.
7. The method of claim 5, wherein the step of determining the position of the probe is performed,
The alkali liquor is sodium hydroxide solution with the concentration of 20-30 g/L, and the soaking time is 4 hours;
the drying is carried out for 18-24 hours at 40 ℃;
The stirring is mechanical stirring at 450rpm for 5-8 hours at room temperature.
8.
The method of claim 4, wherein the substrate is ferulic acid,
p-coumaric acid and/or lignin, or an extract, derivative thereof; the
microorganism comprises at least one of yeast, escherichia coli,
pseudomonas or streptomyces.
9. The method of claim 8, wherein the substrate is present at a concentration of 0.8-1.2g/L.
10.
The method of claim 4, wherein the acid used for the acid washing is
hydrochloric acid and the time for the acid washing is 12 hours.
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