CN101463369A - Culture medium for producing benzoylformic acid by fermentation method - Google Patents

Culture medium for producing benzoylformic acid by fermentation method Download PDF

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Publication number
CN101463369A
CN101463369A CNA2007101726400A CN200710172640A CN101463369A CN 101463369 A CN101463369 A CN 101463369A CN A2007101726400 A CNA2007101726400 A CN A2007101726400A CN 200710172640 A CN200710172640 A CN 200710172640A CN 101463369 A CN101463369 A CN 101463369A
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substratum
culture medium
yeast extract
peptone
phenylglycine
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Chinese (zh)
Inventor
胡海峰
徐梅
张琴
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Shanghai Institute of Pharmaceutical Industry
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Shanghai Institute of Pharmaceutical Industry
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Abstract

The invention discloses a culture medium for producing benzoylformic acid by fermentation method. The culture medium comprises calcium ion and D, L-phenylglycine and is characterized in that the initial pH value of the culture medium ranges from pH more than 7 to pH equal to 10. By adopting the culture medium to produce the benzoylformic acid, the substrate inventory rating can increase to 50g/L; the conversion rate can reach 36.8%, the response-specific is high, almost no byproduct exists, simple extraction is enough, and the operation is very convenient.

Description

A kind of substratum of producing benzoylformic acid by fermentation method
Technical field
The invention belongs to the microbial fermentation engineering field, specifically, the present invention relates to a kind of substratum of producing benzoylformic acid by fermentation method.
Background technology
Benzoylformic acid (phenylglyoxilic acid) has another name called benzoyl formic acid (benzoylformic acid), is the important intermediate of synthetic some agricultural chemicals and pharmaceutical prod, but synthetic herbicide metamitron, pharmaceutical prod robinul etc.R (-)-amygdalic acid is extremely important chiral drug intermediate, is widely used in the asymmetric synthesis of optically pure amino acid, angiotensin converting enzyme inhibitor, coenzyme A.Domestic existing research at present is substrate with the benzoylformic acid, asymmetric synthesis R (-)-amygdalic acid.But the The Synthesis of Benzoylformic Acid method mainly contains benzoyl cyanide hydrolysis method, two kinds of methods of styrene oxidation method at present, the former relates to the use of the sodium cyanide of severe toxicity in the preparation of benzoyl cyanide, the latter then yield is very low, so cause environmental influence big, cost is high.Therefore press for and look for new more cheap substrate raw material.
People such as Hiroshi Kanzaki are published in Agric Biol.Chem., 1990, the paper that is entitled as " Production of Benzoylformic Acid from Phenylglycine bySaccharomycopsis lipolytica " of 2101-2105 has reported that the employing Saccharomycopsis carries out microbial transformation and prepares benzoylformic acid, its method is: cultured Saccharomycopsis lipolytica seed liquor is inserted fermention medium (fructose 0.2wt% with 8% inoculum size, peptone 0.7wt%, (NH 4) 2SO 40.1wt%, KH 2PO 40.4wt%, the 2ml liquid microelement, the 1ml vitamin solution, pH7.0, the 100ml liquid microelement comprises 5g MgCl 22H 2O, 0.47g MnCl 24H 2O, FeCl 2H 2O, 0.2gZnCl 2, 0.1gCaCl 2, 0.02g CoCl 2H 2O, 0.02g CuCl 22H 2O, 0.01g NaMoO 4H 2O and 0.01g Na 2B 4O 7The 100ml vitamin solution comprises 1mg VITMAIN B1 HCl, 2mg riboflavin, 2mg calcium pantothenate, 2mg vitamin B6 HCl, 0.1mg vitamin H, 1mg ρ-M-nitro benzoic acid and 2mg niacin), the aerobic cultivation of the same terms 6d, when concentration of substrate 40g/L, the benzoylformic acid transformation efficiency is 36.25%.The shortcoming of this method is that biomass is low in the conversion culturing process; Initial pH is 7.0 o'clock, and the pH value maintains near the iso-electric point of phenylglycine in the conversion process, and the molecular conformation of substrate solubleness and substrate amino group is all impacted; Calcium ion concn 0.0002wt% is then on the low side in the former conversion substratum, does not have best action effect, therefore, causes concentration of substrate and transformation efficiency can't continue to improve.
Summary of the invention
The technical problem that will solve of the present invention is further to improve the transformation efficiency that microbial enzyme method prepares benzoylformic acid.
We discover with by microbial enzyme method, are substrate with the phenylglycine of cheapness, the preparation benzoylformic acid, and crude product can be directly used in asymmetric synthesis R (-)-amygdalic acid, can save cost greatly, for its process of industrialization provides more wide space.
For this reason, the invention provides a kind of substratum of producing benzoylformic acid by fermentation method, described substratum comprises fructose, peptone, (NH 4) 2SO 4, KH 2PO 4, yeast extract, trace element, VITAMIN and D, L-phenylglycine etc. is characterized in that the initial pH value of described substratum is pH〉7 to pH=10.
The available NaOH of described pH value regulates.
The concentration of fructose can be 0-1.5wt% in the substratum of the present invention, and the concentration of peptone can be 0.3-1.5wt%, (NH 4) 2SO 4Concentration can be 0-0.4wt%, KH 2PO 4Concentration can be 0.2-1.0wt%.Described fructose, peptone, (NH 4) 2SO 4, KH 2PO 4The paper that also can deliver with reference to people such as HiroshiKanzaki of concentration described in substratum select.
The inventor finds that when people such as Hiroshi Kanzaki were 7.0 with initial pH regulator, the pH value maintained near the iso-electric point of phenylglycine in the conversion process, and this molecular conformation to substrate solubleness and substrate amino group all impacts.Therefore the inventor has done a series of tests to the pH value of substratum, the result as shown in Figure 3, can know from accompanying drawing 3 and to find out, the pH value is obvious ascendant trend greater than the relative transformation efficiency of 7 o'clock benzoylformic acids, the relative transformation efficiency that equals 9 o'clock benzoylformic acids to the pH value reaches the peak, downtrending occurring and the pH value is the relative transformation efficiency of 10 o'clock benzoylformic acids, is 7 o'clock relative transformation efficiency but still be slightly larger than pH value in the prior art.And when the pH value more than or equal to 11 the time, because of exceeding the pH subject range of yeast growth, thalli growth seriously is obstructed.Therefore, the pH value with substratum of the present invention is controlled at greater than 7 to 10.The optimal ph of substratum of the present invention is 9.
The inventor further finds, adds the transformation efficiency that yeast extract can further improve benzoylformic acid in this substratum.By accompanying drawing 1 as seen, after adding yeast extract, the relative transformation efficiency of benzoylformic acid also is obvious ascendant trend, the relative transformation efficiency of benzoylformic acid reaches the peak when the concentration of yeast extract is 1.2wt%, when the concentration of yeast extract is 2.4wt%, the relative transformation efficiency of benzoylformic acid when promoter action is 1.2wt% a little less than the concentration of yeast extract is found obviously to suppress benzoylformic acid and is transformed phenomenon.Therefore,, the interpolation concentration of the yeast extract in the substratum is controlled at 0.3～2.4wt%, preferred 1.2wt% from the cost angle.
The inventor also finds, calcium ion concn 0.0002wt% in the prior art in the trace element of conversion substratum is on the low side, when calcium ion concn in the substratum during greater than 0.0002wt%, the relative transformation efficiency of benzoylformic acid also is obvious ascendant trend, the relative transformation efficiency of benzoylformic acid reaches the peak when the concentration of calcium ion is 0.075wt%, and when the concentration of yeast extract is 0.125wt%, the relative transformation efficiency of benzoylformic acid will be lower than prior art.Therefore, the concentration with the calcium ion in the substratum is controlled at Ca 2+0.0002wt% to Ca 2+=0.015wt%, the optimum concn of described calcium ion is 0.0075wt%.
Substrate D in the substratum of the present invention, the concentration range of L-phenylglycine can be 2～5wt%, preferred 2wt%.When concentration of substrate during less than 2wt%, then uneconomical from the cost angle, then because substrate is too much, and solubleness is little greater than 5wt%, and causes fermented liquid too dense, influence dissolved oxygen, is unfavorable for thalli growth, and transformation efficiency reduces, thereby causes substrate to waste.
Therefore, the most preferred substratum of the present invention is made up of following ingredients:
Fructose 0.2wt%
Peptone 0.7wt%
(NH 4) 2SO 4 0.1wt％
KH 2PO 4 0.4wt％
Yeast extract 1.2wt%
Calcium chloride 0.0075wt%
D, L-phenylglycine 2wt%
And the initial pH value of this substratum is 9.0.
The HPLC detection method is: transform and finish back taking-up 5ml fermented liquid, concentrated hydrochloric acid is acidified to about pH1.0, adds the ethyl acetate of 3ml.Get 50 μ l supernatant liquor vacuum and drain, carry out HPLC after the methyl alcohol of adding 1ml fully dissolves and analyze.Chromatographic column: Waters C 18Post (4.6mm * 250mm, 5 μ m); Sample size: 20 μ l; Detect wavelength: 254nm; Moving phase: phosphate buffered saline buffer: methyl alcohol=9:1; Flow velocity: 0.8ml/min.The mensuration of transformation efficiency: the sample solution preparation is measured with HPLC, and reference substance solution is the methanol solution of 0.6g/L standard substance.The calculating of transformation efficiency: the transformation efficiency of sample=substrate conversion concentration/substrate concentration * 100% that feeds intake.
Advantage of the present invention is: optimizing under the culture medium condition, the substrate charging capacity can be brought up to 50g/L, cultivates 6d, and transformation efficiency reaches 36.8%, the atopic height, and almost no coupling product only needs simple extraction to get final product, and operation is very easy.
Description of drawings
Add behind the different concns yeast extract influence (transformation efficiency with Comparative Examples is reference) in the curve display substratum of Fig. 1 to the relative transformation efficiency of benzoylformic acid;
Add behind the different concns calcium ion influence (transformation efficiency with Comparative Examples is reference) in the curve display substratum of Fig. 2 to the relative transformation efficiency of benzoylformic acid;
The substratum of the different initial pH of the curve display of Fig. 3 is to the influence (transformation efficiency with Comparative Examples is reference) of the relative transformation efficiency of benzoylformic acid.
Embodiment
Embodiment 1:
With Candida lipolytica SIPI0201 bacterial classification inoculation in the 25ml seed culture medium (glucose 3wt%, yeast extract 0.3wt%,, peptone 0.3wt%, K 2HPO 43H 2O 0.1wt%, MgSO 47H 2O 0.05wt%, KCl 0.05wt%, natural pH), 30 ℃, the aerobic cultivation of 230r/min 20h obtain seed liquor, insert 25ml fermention medium (fructose 0.2wt%, peptone 0.7wt%, (NH with 8% inoculum size then 4) 2SO 40.1wt%, KH 2PO 40.4wt%, yeast extract 0.3wt%, calcium chloride 0.0003wt%, D, L-phenylglycine 2wt% is adjusted to 8.0 with NaOH with the pH value), the aerobic cultivation of the same terms 6d, it is 35.9% that HPLC analyzes the benzoylformic acid yield.
Embodiment 2:
With Candida lipolytica SIPI0201 bacterial classification inoculation in the 25ml seed culture medium (glucose 3wt%, yeast extract 0.3wt%,, peptone 0.3wt%, K 2HPO 43H 2O 0.1wt%, MgSO 47H 2O 0.05wt%, KCl 0.05wt%, natural pH), 30 ℃, the aerobic cultivation of 230r/min 20h obtain seed liquor, insert 25ml fermention medium (fructose 0.2wt%, peptone 0.7wt%, (NH with 8% inoculum size then 4) 2SO 40.1wt%, KH 2PO 40.4wt%, yeast extract 1.2wt%, calcium chloride 0.005wt%, D, L-phenylglycine 2wt% is adjusted to 7.5 with NaOH with the pH value), the aerobic cultivation of the same terms 6d, it is 39.5% that HPLC analyzes the benzoylformic acid yield.
Embodiment 3:
With Candida lipolytica SIPI0201 bacterial classification inoculation in the 25ml seed culture medium (glucose 3wt%, yeast extract 0.3wt%,, peptone 0.3wt%, K 2HPO 43H 2O 0.1wt%, MgSO 47H 2O 0.05wt%, KCl 0.05wt%, natural pH), 30 ℃, the aerobic cultivation of 230r/min 20h obtain seed liquor, insert 25ml fermention medium (fructose 0.2wt%, peptone 0.7wt%, (NH with 8% inoculum size then 4) 2SO 40.1wt%, KH 2PO 40.4wt%, yeast extract 2.4wt%, calcium chloride 0.005wt%, D, L-phenylglycine 2wt% is adjusted to 8.0 with NaOH with the pH value), the aerobic cultivation of the same terms 6d, it is 37.7% that HPLC analyzes the benzoylformic acid yield.
Embodiment 4:
With Candida lipolytica SIPI0201 bacterial classification inoculation in the 25ml seed culture medium (glucose 3wt%, yeast extract 0.3wt%,, peptone 0.3wt%, K 2HPO 43H 2O 0.1wt%, MgSO 47H 2O 0.05wt%, KCl 0.05wt%, natural pH), 30 ℃, the aerobic cultivation of 230r/min 20h obtain seed liquor, insert 25ml fermention medium (fructose 0.2wt%, peptone 0.7wt%, (NH with 8% inoculum size then 4) 2SO 40.1wt%, KH 2PO 40.4wt%, calcium chloride 0.0075wt%, D, L-phenylglycine 2wt% is adjusted to 8.0 with NaOH with the pH value), the aerobic cultivation of the same terms 6d, it is 41.1% that HPLC analyzes transformation efficiency.
Embodiment 5:
With Candida lipolytica SIPI0201 bacterial classification inoculation in the 25ml seed culture medium (glucose 3wt%, yeast extract 0.3wt%,, peptone 0.3wt%, K 2HPO 43H 2O 0.1wt%, MgSO 47H 2O 0.05wt%, KCl 0.05wt%, natural pH), 30 ℃, the aerobic cultivation of 230r/min 20h obtain seed liquor, insert 25ml fermention medium (fructose 0.2wt%, peptone 0.7wt%, (NH with 8% inoculum size then 4) 2SO 40.1wt%, KH 2PO 40.4wt%, calcium chloride 0.015wt%, D, L-phenylglycine 2wt% is adjusted to 8.5 with NaOH with the pH value), the aerobic cultivation of the same terms 6d, it is 37.3% that HPLC analyzes transformation efficiency.
Embodiment 6:
With Candida lipolytica SIPI0201 bacterial classification inoculation in the 25ml seed culture medium (glucose 3wt%, yeast extract 0.3wt%,, peptone 0.3wt%, K 2HPO 43H 2O 0.1wt%, MgSO 47H 2O 0.05wt%, KCl 0.05wt%, natural pH), 30 ℃, the aerobic cultivation of 230r/min 20h obtain seed liquor, insert 25ml fermention medium (fructose 0.2wt%, peptone 0.7wt%, (NH with 8% inoculum size then 4) 2SO 40.1wt%, KH 2PO 40.4wt%, calcium chloride 0.0075wt%, D, L-phenylglycine 2wt% is adjusted to 8.0 with NaOH with the pH value), the aerobic cultivation of the same terms 6d, it is 39.7% that HPLC analyzes transformation efficiency.
Embodiment 7:
With Candida lipolytica SIPI0201 bacterial classification inoculation in the 25ml seed culture medium (glucose 3wt%, yeast extract 0.3wt%,, peptone 0.3wt%, K 2HPO 43H 2O 0.1wt%, MgSO 47H 2O 0.05wt%, KCl 0.05wt%, natural pH), 30 ℃, the aerobic cultivation of 230r/min 20h obtain seed liquor, insert 25ml fermention medium (fructose 0.2wt%, peptone 0.7wt%, (NH with 8% inoculum size then 4) 2SO 40.1wt%, KH 2PO 40.4wt%, calcium chloride 0.001wt%, D, L-phenylglycine 2wt% is adjusted to 9.0 with NaOH with the pH value), the aerobic cultivation of the same terms 6d, it is 42.4% that HPLC analyzes transformation efficiency.
Embodiment 8:
With Candida lipolytica SIPI0201 bacterial classification inoculation in the 25ml seed culture medium (glucose 3wt%, yeast extract 0.3wt%,, peptone 0.3wt%, K 2HPO 43H 2O 0.1wt%, MgSO 47H 2O 0.05wt%, KCl 0.05wt%, natural pH), 30 ℃, the aerobic cultivation of 230r/min 20h obtain seed liquor, insert 25ml fermention medium (fructose 0.2wt%, peptone 0.7wt%, (NH with 8% inoculum size then 4) 2SO 40.1wt%, KH 2PO 40.4wt%, calcium chloride 0.0075wt%, D, L-phenylglycine 2wt% is adjusted to 10.0 with NaOH with the pH value), the aerobic cultivation of the same terms 6d, it is 38.2% that HPLC analyzes transformation efficiency.
Embodiment 9:
With Candida lipolytica SIPI0201 bacterial classification inoculation in the 25ml seed culture medium (glucose 3wt%, yeast extract 0.3wt%,, peptone 0.3wt%, K 2HPO 43H 2O 0.1wt%, MgSO 47H 2O 0.05wt%, KCl 0.05wt%, natural pH), 30 ℃, the aerobic cultivation of 230r/min 20h obtain seed liquor, insert 25ml fermention medium (fructose 0.2wt%, peptone 0.7wt%, (NH with 8% inoculum size then 4) 2SO 40.1wt%, KH 2PO 40.4wt%, yeast extract 1.2wt%, calcium chloride 0.0075wt%, D, L-phenylglycine 2wt% is adjusted to 9.0 with NaOH with the pH value), the aerobic cultivation of the same terms 6d, it is 47.4% that HPLC analyzes transformation efficiency.
Embodiment 10:
With Candida lipolytica SIPI0201 bacterial classification inoculation in the 25ml seed culture medium (glucose 3wt%, yeast extract 0.3wt%,, peptone 0.3wt%, K 2HPO 43H 2O0.1wt%, MgSO 47H 2O 0.05wt%, KCl 0.05wt%, natural pH), 30 ℃, the aerobic cultivation of 230r/min 20h obtain seed liquor, insert 75ml fermention medium (fructose 0.2wt%, peptone 0.7wt%, (NH with 8% inoculum size then 4) 2SO 40.1wt%, KH 2PO 40.4wt%, yeast extract 1.2wt%, calcium chloride 0.0075wt%, D, L-phenylglycine 2.5wt% is adjusted to 9.0 with NaOH with the pH value), the aerobic cultivation of the same terms 6d, it is 46.3% that HPLC analyzes transformation efficiency.
Embodiment 11:
With Candida lipolytica SIPI0201 bacterial classification inoculation in the 25ml seed culture medium (glucose 3wt%, yeast extract 0.3wt%,, peptone 0.3wt%, K 2HPO 43H 2O 0.1wt%, MgSO 47H 2O 0.05wt%, KCl 0.05wt%, natural pH), 30 ℃, the aerobic cultivation of 230r/min 20h obtain seed liquor, insert 25ml fermention medium (fructose 0.2wt%, peptone 0.7wt%, (NH with 8% inoculum size then 4) 2SO 40.1wt%, KH 2PO 40.4wt%, yeast extract 2.1wt%, calcium chloride 0.0075wt%, D, L-phenylglycine 5wt% is adjusted to 9.0 with NaOH with the pH value), the aerobic cultivation of the same terms 6d, it is 36.8% that HPLC analyzes transformation efficiency.
Comparative Examples
With Candida lipolytica SIPI0201 bacterial classification inoculation in the 25ml seed culture medium (glucose 3wt%, yeast extract 0.3wt%,, peptone 0.3wt%, K 2HPO 43H 2O 0.1wt%, MgSO 47H 2O 0.05wt%, KCl 0.05wt%, natural pH), 30 ℃, the aerobic cultivation of 230r/min 20h obtain seed liquor, insert 25ml fermention medium (fructose 0.2wt%, peptone 0.7wt%, (NH with 8% inoculum size then 4) 2SO 40.1wt%, KH 2PO 40.4wt%, calcium chloride 0.0002wt%, D, L-phenylglycine 2wt% is adjusted to 7.0 with NaOH with the pH value), the aerobic cultivation of the same terms 6d, it is 31.2% that HPLC analyzes the benzoylformic acid yield.

Claims (10)

1. the substratum of a producing benzoylformic acid by fermentation method, described substratum comprises calcium ion and D, the L-phenylglycine is characterized in that, the initial pH value of described substratum is pH〉7 to pH=10.
2. substratum as claimed in claim 1 is characterized in that, the initial pH value of described substratum is 9.
3. substratum as claimed in claim 1 is characterized in that, described D, and the concentration of L-phenylglycine is 2～5wt%.
4. substratum as claimed in claim 3 is characterized in that, described D, and the concentration of L-phenylglycine is 2wt%.
5. substratum as claimed in claim 1 is characterized in that described substratum also comprises yeast extract.
6. substratum as claimed in claim 5 is characterized in that, the concentration of described yeast extract is 0.3～2.4wt%.
7. substratum as claimed in claim 6 is characterized in that, the concentration of described yeast extract is 1.2wt%.
8. substratum as claimed in claim 1 is characterized in that, the concentration of described calcium ion is Ca 2+0.0002wt% to Ca 2+=0.015wt%.
9. substratum as claimed in claim 8 is characterized in that, the concentration of described calcium ion is 0.0075wt%.
10. substratum as claimed in claim 1 is characterized in that, described substratum is made up of following ingredients:
Fructose 0.2wt%
Peptone 0.7wt%
(NH 4) 2SO 4 0.1wt％
KH 2PO 4 0.4wt％
Yeast extract 1.2wt%
Calcium chloride 0.0075wt%
D, L-phenylglycine 2wt%
And the initial pH value of this substratum is 9.0.
CNA2007101726400A 2007-12-20 2007-12-20 Culture medium for producing benzoylformic acid by fermentation method Pending CN101463369A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102344361A (en) * 2011-11-04 2012-02-08 合肥工业大学 High selectivity synthesis method of benzoyl formic acid
CN108552177A (en) * 2018-05-21 2018-09-21 长乐智高生物科技有限公司 A kind of cockroach attractant

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102344361A (en) * 2011-11-04 2012-02-08 合肥工业大学 High selectivity synthesis method of benzoyl formic acid
CN108552177A (en) * 2018-05-21 2018-09-21 长乐智高生物科技有限公司 A kind of cockroach attractant

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Application publication date: 20090624