CN104726374A - Preparation method of bacterial cell catalyst for catalyzing naringin ester synthesis reaction - Google Patents

Preparation method of bacterial cell catalyst for catalyzing naringin ester synthesis reaction Download PDF

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Publication number
CN104726374A
CN104726374A CN201510118619.7A CN201510118619A CN104726374A CN 104726374 A CN104726374 A CN 104726374A CN 201510118619 A CN201510118619 A CN 201510118619A CN 104726374 A CN104726374 A CN 104726374A
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preparation
naringin
cell catalyst
bacterial cell
catalyzing
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李晓凤
赖学能
赵光磊
吴晖
余以刚
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South China University of Technology SCUT
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South China University of Technology SCUT
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

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Abstract

The invention discloses a preparation method of a bacterial cell catalyst for catalyzing naringin ester synthesis reaction. The method comprises the following steps: inoculating a bacterium seed solution into an enzyme-inducer-containing liquid culture medium, and culturing at 30-50 DEG C for 12-144 hours, wherein the inoculum size is 0.5-20% (v/v); and centrifuging, collecting the thallus, washing with distilled water twice, carrying out vacuum freeze drying for 12-36 hours, and collecting the thallus dry powder to obtain the bacterial cell catalyst. The catalyst is used for catalyzing ester conversion reaction between esculine and an acyl reagent at high selectivity. Compared with the enzyme preparation, the obtained bacterial full-cell catalyst avoids the expensive and complex enzyme separation purification process, is easy to prepare and recover, is more beneficial to large-scale production, and has higher stability in organic solvents.

Description

The preparation method of the bacterial cell catalyzer of catalysis naringin ester synthesis reaction
Technical field
The invention belongs to microorganism culturing, biocatalysis and biological medicine preparation field, be specifically related to the preparation method of the bacterial cell catalyzer of catalysis naringin ester synthesis reaction.
Background technology
Flavonoid compound is that two phenyl ring are interconnected by three carbon atoms and a series of important polyphenolic compound formed, and is extensively present in nearly all plant, especially vegetables and fruit; Medicine, foods and cosmetics there is very large using value.Flavones has multiple biological activity to comprise anti-oxidant activity, anti-inflammatory, anticancer, antibacterial, antianaphylaxis, and antiviral etc., but due to its polyhydroxy structure, most of flavones biological activity is limited to that it is low fat-soluble or water-soluble.Be positioned at the biological and chemical activity of the hydroxyl on phenyl ring or glycoside skeleton on flavones and have important impact.The esterification of flavones is considered to a kind of very promising method for improving the solubleness of flavones, thus makes flavones show more physiologically active.There are some researches show, modifying flavonoid compound structure not only can improve physico-chemical properties selectively, such as, and thermostability and photoresistance coefficient; And strengthen its biological activity, as anti-oxidant activity; Anti-microbial activity; Premeabilisation of cells ability; Fat-reducing and hypolipemic function etc.Naringin has another name called naringin, isohesperidin, naringenin-7-O-neohesperidoside, it is a kind of flavanone compounds, mainly being present in rutaceae natsudaidai, tangerine, orange pericarp and pulp, is also one of principle active component of the Chinese medicines such as Rhizome of Fortune's Drynaria, the dried immature fruit of citron orange, Fructus Aurantii, Pummelo Peel.Naringin has and promotes the effect such as bone growth, anti-oxidant, decreasing cholesterol and reducing blood-fat, antitumor, the bioavailability that improves other drug.
The synthesis of current flavones ester derivative all adopts chemical catalyst or unorganized ferment catalyzer.Because on the phenyl ring of flavones or sugared ring, hydroxyl is numerous, and reactive behavior is similar, adopts the subject matter existed during chemical catalyst to be that conversion zone selectivity is very low, easily produces a large amount of by products and alkali waste, causes severe contamination to environment.Enzyme catalyst is more green than chemical catalyst, and regioselectivity is high, reaction conditions is gentle, but the separation and purification process due to enzyme complicated and in the process enzymic activity have partial loss, unstable in steric environment, the shortcomings such as easy in inactivation, and enzyme is expensive, cause enzyme process reaction cost higher, these aspects all seriously constrain it in industrialized application.Therefore, research and development novel green catalyzer is of great immediate significance for the preparation of flavones ester.
Summary of the invention
The present invention is directed to the deficiency of the existing catalyzer for naringin Lipase absobed, a kind of preparation method of bacterial cell catalyzer of catalysis naringin ester synthesis reaction is provided.Its preparation method is simple to operation, and the inexpensive easy purchase of starting material, does not relate to the separation and purification of enzyme.With this law prepare can the cell catalyst of catalysis naringin ester synthesis reaction, alternative catalyzes and synthesizes naringin 6 ' '-monoesters, its regioselectivity reaches more than 97%, the low substrate utilization ratio that causes of the selectivity overcoming traditional chemical routes is low, product purity is low, easily generates the shortcomings such as by product.Be all green, the biological catalyst of reaction conditions gentleness, cell catalyst is than the advantage of unorganized ferment catalyzer high, inexpensive, the easy acquisition that has stability.
The object of the invention is achieved through the following technical solutions:
A preparation method for the bacterial cell catalyzer of catalysis naringin ester synthesis reaction, comprises the following steps:
1) bacterium seed liquor be seeded to containing producing in the liquid nutrient medium of enzyme inducer, inoculum size is 0.5% ~ 20%(v/v), culture condition is 30 DEG C ~ 50 DEG C, and incubation time is 12 ~ 144h;
2) collected after centrifugation thalline, distilled water wash twice, through vacuum lyophilization 12h-36h, results thalline dry powder, is bacterial cell catalyzer.
In aforesaid method, described bacterium comprises Pseudomonas stutzeri pseudomonas stutzerior Pseudomonas aeruginosa pseudomonas aeruginosa.
In aforesaid method, described liquid culture based component is: 0. 5 ~ 100 g/L produce enzyme inducer, 0.5 ~ 50 g/L organic nitrogen source, 0.5 ~ 10 g/L MgSO 47H 2o, 5 g/L (NH 4) 2sO 4, 1 g/L K 2hPO 4.
In aforesaid method, described product enzyme inducer comprise saccharide compound, saturated vegetable oil fat, polyunsaturated vegetable oil fat, lipid acid, Tween series or Span serial in one or both.
In aforesaid method, described organic nitrogen source comprises extractum carnis, yeast extract paste, peptone, Tryptones, casein food grade, acid hydrolyzed casein peptone or without one or both in amino yeast extract paste.
The present invention bacterium public used can buy from Culture Collection or oneself screening obtains.
The present invention has following advantage compared with prior art:
1. bacterial cell catalyzer is biological catalyst,, environmental friendliness gentle than chemical catalyst reaction conditions, regioselectivity is high, 6 ' of selectivity synthesis naringin '-monoesters, its regioselectivity is more than 90%, thus it is low to overcome traditional chemical routes product purity, easily generate the shortcomings such as by product.
2. bacterial cell catalyzer nutritional requirement is very low, easily cultivates, and culture medium prescription is simple, and prepare easy to operate, preparation cost is low.
Embodiment
For better understanding the present invention, to do the present invention below in conjunction with embodiment and describe in detail further, it should be noted that, these embodiments do not form limiting the scope of the invention.
embodiment 1
Pseudomonas aeruginosa will be comprised pseudomonas aeruginosa GIM1.46seed liquor is forwarded to cultivates containing in liquid nutrient medium, and inoculum size is 0.5%(v/v), culture condition is 30 DEG C, and liquid culture based component is: 0.5 g/L soybean oil, 0.5 g/L yeast extract paste, 0.5 g/L MgSO 47H 2o, 5 g/L (NH 4) 2sO 4, 1 g/L K 2hPO 4, pH is 7.0.Collected by centrifugation wet thallus after cultivation 60h, distilled water wash twice, obtains cell catalyst after vacuum lyophilization.This cell catalyst is used in the acylation reaction of naringin and propionate, 6 ' '-naringin propyl ester regioselectivity is 98.7%.
The regioselectivity of the catalyzer that the inventive method obtains is reacted with naringin and propionate regioselective acylation and is measured; reaction conditions is add the thalline dry powder of 80mg as catalyzer in the naringin esterification system of 2mL; using 65.8 μ l propionate as acry radical donor; naringin consumption is 17.4mg; reaction system is 90% (v/v) acetonitrile-tertiary amyl alcohol; temperature of reaction is 40 DEG C, and hunting speed 180rpm reacts under normal pressure.Conversion zone selectivity is characterized by: the ultimate production of the output/flavones ester of 6 '-naringin propyl ester.
embodiment 2
Pseudomonas aeruginosa will be comprised pseudomonas aeruginosa GIM1.46seed liquor is forwarded to cultivates containing in liquid nutrient medium, and inoculum size is 0.5%(v/v), culture condition is 50 DEG C, and liquid culture based component is: 0.5 g/L Tween 80,0.5 g/L yeast extract paste, 0.5 g/L MgSO 47H 2o, 5 g/L (NH 4) 2sO 4, 1 g/L K 2hPO 4, pH is 7.0.Collected by centrifugation wet thallus after cultivation 60h, distilled water wash twice, obtains cell catalyst after vacuum lyophilization.This cell catalyst is used in the acylation reaction of naringin and propionate, 6 ' '-naringin propyl ester regioselectivity is 97.7%.
embodiment 3
Pseudomonas aeruginosa will be comprised pseudomonas aeruginosa GIM1.46seed liquor is forwarded to cultivates containing in liquid nutrient medium, and inoculum size is 20%(v/v), culture condition is 30 DEG C, and liquid culture based component is: 50 g/L Tween 80,10 g/L yeast extract pastes, 0.2 g/L MgSO 47H 2o, 5 g/L (NH 4) 2sO 4, 1 g/L K 2hPO 4, pH is 6.8.Collected by centrifugation wet thallus after cultivation 48h, distilled water wash twice, obtains cell catalyst after vacuum lyophilization.This cell catalyst is used in the acylation reaction of naringin and propionate, 6 ' '-naringin propyl ester regioselectivity is 98.2%.
embodiment 4
Pseudomonas aeruginosa will be comprised pseudomonas aeruginosa GIM1.46seed liquor is forwarded to cultivates containing in liquid nutrient medium, and inoculum size is 0.5%(v/v), culture condition is 30 DEG C, and liquid culture based component is: 0.5 g/L Tween 80,0.5 g/L yeast extract paste, 0.5 g/L MgSO 47H 2o, 5 g/L (NH 4) 2sO 4, 1 g/L K 2hPO 4, pH is 6.8.Collected by centrifugation wet thallus after cultivation 12h, distilled water wash twice, obtains cell catalyst after vacuum lyophilization.This cell catalyst is used in the acylation reaction of naringin and propionate, 6 ' '-naringin propyl ester regioselectivity is 98.4%.
embodiment 5
Pseudomonas aeruginosa will be comprised pseudomonas aeruginosa GIM1.46seed liquor is forwarded to cultivates containing in liquid nutrient medium, and inoculum size is 0.5%(v/v), culture condition is 30 DEG C, and liquid culture based component is: 0.5 g/L Tween 80,0.5 g/L yeast extract paste, 0.5 g/L MgSO 47H 2o, 5 g/L (NH 4) 2sO 4, 1 g/L K 2hPO 4, pH is 6.8.Collected by centrifugation wet thallus after cultivation 144h, distilled water wash twice, obtains cell catalyst after vacuum lyophilization.This cell catalyst is used in the acylation reaction of naringin and propionate, 6 ' '-naringin propyl ester regioselectivity is 99.1%.
embodiment 6
Pseudomonas aeruginosa will be comprised pseudomonas aeruginosa GIM1.46seed liquor is forwarded to cultivates containing in liquid nutrient medium, and inoculum size is 0.5%(v/v), culture condition is 30 DEG C, and liquid culture based component is: 0.5 g/L glucose, 0.5 g/L yeast extract paste, 0.5 g/L MgSO 47H 2o, 5 g/L (NH 4) 2sO 4, 1 g/L K 2hPO 4, pH is 6.8.Collected by centrifugation wet thallus after cultivation 48h, distilled water wash twice, obtains cell catalyst after vacuum lyophilization.This cell catalyst is used in the acylation reaction of naringin and propionate, 6 ' '-naringin propyl ester regioselectivity is 99.2%.
embodiment 7
Pseudomonas aeruginosa will be comprised pseudomonas aeruginosa GIM1.46seed liquor is forwarded to cultivates containing in liquid nutrient medium, and inoculum size is 0.5%(v/v), culture condition is 30 DEG C, and liquid culture based component is: 0.5 g/L soybean oil, 0.5 g/L yeast extract paste, 0.5 g/L MgSO 47H 2o, 5 g/L (NH 4) 2sO 4, 1 g/L K 2hPO 4, pH is 6.8.Collected by centrifugation wet thallus after cultivation 48h, distilled water wash twice, obtains cell catalyst after vacuum lyophilization.This cell catalyst is used in the acylation reaction of naringin and propionate, 6 ' '-naringin propyl ester regioselectivity is 98.9%.
embodiment 8
Pseudomonas aeruginosa will be comprised pseudomonas aeruginosa GIM1.46seed liquor is forwarded to cultivates containing in liquid nutrient medium, and inoculum size is 0.5%(v/v), culture condition is 30 DEG C, and liquid culture based component is: 0.5 g/L span60,0.5 g/L yeast extract paste, 0.5 g/L MgSO 47H 2o, 5 g/L (NH 4) 2sO 4, 1 g/L K 2hPO 4, pH is 6.8.Collected by centrifugation wet thallus after cultivation 48h, distilled water wash twice, obtains cell catalyst after vacuum lyophilization.This cell catalyst is used in the acylation reaction of naringin and propionate, 6 ' '-naringin propyl ester regioselectivity is 98.3%.
The above embodiment of the present invention is only for example of the present invention is clearly described, and is not the restriction to embodiments of the present invention.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without the need to also giving all embodiments.All any amendments done within the spirit and principles in the present invention, equivalent to replace and improvement etc., within the protection domain that all should be included in the claims in the present invention.

Claims (5)

1. a preparation method for the bacterial cell catalyzer of catalysis naringin ester synthesis reaction, is characterized in that, comprise the following steps:
1) bacterium seed liquor be seeded to containing producing in the liquid nutrient medium of enzyme inducer, inoculum size is 0.5% ~ 20%(v/v), culture condition is 30 DEG C ~ 50 DEG C, and incubation time is 12 ~ 144h;
2) collected after centrifugation thalline, distilled water wash twice, through vacuum lyophilization 12h-36h, results thalline dry powder, is bacterial cell catalyzer.
2. preparation method according to claim 1, is characterized in that, described bacterium comprises Pseudomonas stutzeri pseudomonas stutzerior Pseudomonas aeruginosa pseudomonas aeruginosa.
3. preparation method according to claim 1, is characterized in that, described liquid culture based component is: 0. 5 ~ 100 g/L produce enzyme inducer, 0.5 ~ 50 g/L organic nitrogen source, 0.5 ~ 10 g/L MgSO 47H 2o, 5 g/L (NH 4) 2sO 4, 1 g/L K 2hPO 4.
4. preparation method according to claim 3, is characterized in that, described product enzyme inducer comprise saccharide compound, saturated vegetable oil fat, polyunsaturated vegetable oil fat, lipid acid, Tween series or Span serial in one or both.
5. according to the preparation method described in claim 3, it is characterized in that, described organic nitrogen source comprises extractum carnis, yeast extract paste, peptone, Tryptones, casein food grade, acid hydrolyzed casein peptone or without one or both in amino yeast extract paste.
CN201510118619.7A 2015-03-18 2015-03-18 Preparation method of bacterial cell catalyst for catalyzing naringin ester synthesis reaction Pending CN104726374A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105002238A (en) * 2015-07-28 2015-10-28 华南理工大学 Naringin hydroxyl protective reaction method based on Pseudomonas stutzeri cell catalysis
CN105063137A (en) * 2015-07-28 2015-11-18 华南理工大学 Aesculin hydroxyl protecting reaction method based on catalysis of pseudomonas stutzeri cells

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CN102250785A (en) * 2011-05-20 2011-11-23 华南理工大学 Preparation method of bacterial cell catalyst for catalyzing starasid synthesis reaction
CN102851336A (en) * 2012-08-31 2013-01-02 华南理工大学 Method for realizing protective reaction for hydroxyl groups of cytidine compound through catalysis of pseudomonas fluorescens

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105002238A (en) * 2015-07-28 2015-10-28 华南理工大学 Naringin hydroxyl protective reaction method based on Pseudomonas stutzeri cell catalysis
CN105063137A (en) * 2015-07-28 2015-11-18 华南理工大学 Aesculin hydroxyl protecting reaction method based on catalysis of pseudomonas stutzeri cells
WO2017016175A1 (en) * 2015-07-28 2017-02-02 华南理工大学 Hydroxyl protection reaction method of aesculin based on pseudomonas stutzeri cell catalysis

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Application publication date: 20150624