CN105063111A - Method for preparing L-rhamnose acid through microbial conversion - Google Patents
Method for preparing L-rhamnose acid through microbial conversion Download PDFInfo
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- CN105063111A CN105063111A CN201510531257.4A CN201510531257A CN105063111A CN 105063111 A CN105063111 A CN 105063111A CN 201510531257 A CN201510531257 A CN 201510531257A CN 105063111 A CN105063111 A CN 105063111A
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- saccharic acid
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- sandlwood
- rhamnose
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Abstract
The invention relates to a method for preparing L-rhamnose acid through microbial conversion. The method comprises the steps of 1, adding L-rhamnose to a resting cell solution system, wherein the concentration of resting cells in the system is OD600=25-50, the mass percent concentration of L-rhamnose is 10-40%, and Na2CO3 or CaCO3 is added during conversion to enable pH to be maintained between 5.0 and 7.0; 2, extracting L-rhamnose acid, wherein centrifugation is conducted to obtain supernatant after conversion, L-rhamnose acid calcium salt is obtained through evaporation and concentration, ethanol precipitation and drying, and finally L-rhamnose acid is obtained through sulfuric acid replacement reaction. Pseudomonades are provided and can be used for converting L-rhamnose into L-rhamnose acid, and conversion rate is increased by preparing L-rhamnose acid through conversion by means of the resting cells obtained after thallus fermentation.
Description
Technical field
The invention belongs to technical field of microbial fermentation, relate to a kind of method that L-sandlwood saccharic acid is prepared in microbial transformation.
Background technology
Vegetable active high-intensity wood glue is a kind of forest-friendly building timbers glue of formaldehydeless pollution, has application prospect widely.And L-sandlwood saccharic acid is the initiator that solidification occurs vegetable active high-intensity wood glue, there is vital effect.
The preparation of current L-sandlwood saccharic acid has mainly through chemical catalysis synthesis method, and due to the production of chemical synthesis often with multiple by product in oxidising process, make separation, purification step becomes complicated, therefore production cost is relatively high.And it is less to utilize fermentable conversion method to produce the research of L-sandlwood saccharic acid, only find that less yeast can utilize rhamnosyl to generate sandlwood saccharic acid, but its fermentation time is long, productive rate is low and have the generation of by product 1,2-PD.The present invention utilizes pseudomonas microbe conversion method to produce L-sandlwood saccharic acid, and the high and no coupling product of transformation efficiency, effectively can overcome the problems referred to above, have good application prospect.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art part, provide a kind of and there is with short production cycle, that product purity is high, L-sandlwood saccharic acid is prepared in the remarkable advantage microbial transformation such as safe and reliable method.
The technical scheme that the present invention realizes object is:
A method for L-sandlwood saccharic acid is prepared in microbial transformation, and step is as follows
(1) add L-rhamnosyl in resting cell solution system, in system, resting cell concentration is OD
600=25 ~ 50, L-rhamnosyl mass percent concentration is 10% ~ 40%, and invert point is 28 DEG C ~ 32 DEG C, and shaking speed is 150rpm ~ 220rpm, and the reaction times is 24h ~ 72h, in conversion process, add Na
2cO
3or CaCO
3pH is made to be held constant at 5.0-7.0;
(2) the extraction of L-sandlwood saccharic acid: transform after terminating, centrifuging and taking supernatant, obtains L-rhamnosyl acid calcium salt through steps such as evaporation concentration, alcohol settling, oven dry, finally obtains L-sandlwood saccharic acid with sulfate substitution reaction.
And described resting cell is the one in beet pseudomonas AS1.6397, Pseudomonas fluorescens ATCC13525, chrysanthemum huge pseudomonas AS1.3385 or pseudomonas syringae NBRC14078.
And the fermentation culture method of described resting cell is: be inoculated into by pseudomonas in liquid fermentation medium, in shaking table, cultivate 16h ~ 30h for 28 DEG C ~ 32 DEG C.
And the fermention medium of described resting cell is, can be: carbon source 0 ~ 40, nitrogenous source 0 ~ 35 in g/L, be O when carbon source or nitrogenous source difference.
And, described carbon source be in high fructose syrup, sucrose, glucose, maltose, lactose any one, described nitrogenous source is at least one in yeast extract paste, corn steep liquor, extractum carnis, peptone.
And the preferred fermentation culture conditions of described resting cell is: temperature is 28 DEG C ~ 32 DEG C, liquid amount 20 ~ 30mL/250mL, inoculum size 4% ~ 8%, and shaking speed is 150rpm ~ 220rpm, and incubation time is 16h ~ 30h.
Advantage of the present invention and positively effect are:
1, pseudomonas provided by the invention a kind ofly can transform the bacterium that L-rhamnosyl is L-sandlwood saccharic acid, and the conversion of resting cells obtained after thalline fermentation is prepared L-sandlwood saccharic acid and improve transformation efficiency.
2, the L-sandlwood saccharic acid that method provided by the invention obtains have with short production cycle, product purity is high, the remarkable advantage such as safe and reliable.
3, the L-rhamnosyl acid product that prepared by the present invention can be applicable to the fields such as foodstuff additive, can carry out purifying in various degree or post-treatment again to meet the purity requirement in different application field as required to product.
Embodiment
The inventive method is described below by specific embodiment.Unless stated otherwise, technique means used in the present invention is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, but not limits the scope of the invention, and the spirit and scope of the invention only limited by claims.To those skilled in the art, under the prerequisite not deviating from essence of the present invention and scope, the various change carry out the material component in these embodiments and consumption or change also belong to protection scope of the present invention.
A method for L-sandlwood saccharic acid is prepared in microbial transformation, can comprise the following steps:
(1) fermentation culture of pseudomonas: be inoculated into by pseudomonas in liquid fermentation medium, cultivates 16h ~ 30h for 28 DEG C ~ 32 DEG C in shaking table;
Fermention medium component in g/L can be: carbon source 0 ~ 40, nitrogenous source 0 ~ 35, described carbon source can be in high fructose syrup (fructose of 42%), sucrose, glucose, maltose, lactose any one, described nitrogenous source can be in yeast extract paste, corn steep liquor, extractum carnis, peptone at least one or other source organic or inorganic nitrogenous source.
Preferred fermentation culture conditions: temperature is 28 DEG C ~ 32 DEG C, liquid amount 20 ~ 30mL/250mL, inoculum size 4% ~ 8%, and shaking speed is 150rpm ~ 220rpm, and incubation time is 16h ~ 30h.
(2) resting cell preparation: in fermention medium yeast culture good after, 5000 ~ 8000rpm centrifugal 10min collection thalline, it is resuspended as resting cell solution system to add stroke-physiological saline solution.Described resting cell has the ability that conversion L-rhamnosyl is L-sandlwood saccharic acid.
(3) conversion of resting cells: add L-rhamnosyl in resting cell solution system, in system, resting cell concentration is OD
600=25 ~ 50, L-rhamnosyl mass percent concentration is 10% ~ 40%, and invert point is 28 DEG C ~ 32 DEG C, and shaking speed is 150rpm ~ 220rpm, and the reaction times is 24h ~ 72h.Na can be added in conversion process
2cO
3or CaCO
3pH is made to be held constant at about 6.0.
(4) extraction of L-sandlwood saccharic acid: transform after terminating, centrifuging and taking supernatant, obtains L-rhamnosyl acid calcium salt through steps such as evaporation concentration, alcohol settling, oven dry, finally obtains L-rhamnosyl acid product with sulfate substitution reaction.
Mentioned reagent and culture medium prescription, if no special instructions, be this area conventional reagent and substratum.
Specific description is below adopted to carry out proved invention.
Pseudomonas used herein preferably can make the one in garden beet pseudomonas AS1.6397, Pseudomonas fluorescens ATCC13525, the huge pseudomonas AS1.3385 of chrysanthemum, pseudomonas syringae NBRC14078, is commercially available business bacterial classification.
A method for L-sandlwood saccharic acid is prepared in microbial transformation, and step is as follows:
The microorganism that the present embodiment uses is pseudomonas syringae NBRC14078.
1, the fermentation of L-rhamnosyl, step is as follows:
By pseudomonas streak inoculation on solid medium, cultivate in 30 DEG C of constant incubators.The single bacterium colony that picking has been grown from flat board inoculates in 30mL liquid nutrient medium that (fermention medium component is counted with g/L: sucrose 10 respectively, peptone 10, yeast extract paste 30), in 30 DEG C, cultivate in the shaking table of 180r/min and support, in fermention medium yeast culture good after, 8000rpm10min collected by centrifugation thalline, add stroke-physiological saline solution resuspended as conversion fluid, add different concns L-rhamnosyl (10%, 20%, 30%, 40%) in conversion of resting cells liquid system, in system, resting cell concentration is OD
600=45, in conversion process, add CaCO
3make pH keep constant, invert point is 30 DEG C, and shaking speed is 180rpm, and the reaction times is 24h ~ 72h.Result is as shown in table 1.
Table 1 different concns rhamnosyl is on the impact transformed
Result shows, and transformation time extends with the increase of L-rhamnosyl concentration, finally all reaches identical productive rate, considers transformation time and conversion yield, select the L-rhamnosyl of 20% as preferred conversion of substrate concentration.
2, the preparation of L-sandlwood Calciofon, step is as follows:
(1) conversion of resting cells liquid collected after centrifugation supernatant, under the condition of 100 DEG C evaporation concentration to original volume 1/3 now solution be thick, then cool to room temperature;
(2) add the ethanol of 10 times of volumes 95% and constantly stir, now having a large amount of gluey L-sandlwood saccharic acid calcium deposit to separate out, suction filtration removing ethanol after fully stirring;
(3) in gelatinous precipitate at the ethanol adding same volume 95%, fully stir postprecipitation;
(4) suction filtration obtains L-sandlwood Calciofon crude product to dry;
(5) added water to by crude product and above-mentioned concentrated rear identical volume, suction filtration after heating for dissolving, suction filtration liquid cool to room temperature adds after same volume 95% ethanol stirred crystallization is separated out to be drained, and obtains L-sandlwood Calciofon fine work.
3, the preparation of L-sandlwood saccharic acid, step is as follows:
The obtained L-sandlwood Calciofon furnishing aqueous solution, be placed in suitable reactor, the equimolar vitriol oil is slowly added under stirring, in 60 DEG C ~ 90 DEG C heating in water bath 1h ~ 3h, the calcium sulfate precipitation that elimination is separated out, the L-sandlwood saccharic acid hydrated barta obtained after filtrate cooling and oxalic acid are that precipitation agent carries out purifying, and remove a small amount of sulfate radical and calcium ion, too much barium and oxalic acid oxalic precipitated barium are removed; Or the exchange column of anion and cation exchange resin after filtrate cooling, is flow through with suitable flow velocity, obtain highly purified L-sandlwood saccharic acid.L-rhamnosyl acid product is interpreted as L-sandlwood saccharic acid and salt thereof, as L-sandlwood Calciofon, L-sandlwood sodium saccharate.
The method detecting L-sandlwood saccharic acid is as follows:
HPLC condition: Agilent1100 chromatographic column: nh 2 column (4.6 × 250mm); Moving phase is acetonitrile: water=60:40 (phosphoric acid containing 0.1%); Detector: UV-detector 210nm; Column temperature: 25 DEG C; Flow velocity: 1mL/min; Sample size: 10 μ L; Retention time is 4.26min.
Claims (6)
1. a method for L-sandlwood saccharic acid is prepared in microbial transformation, it is characterized in that: step is as follows
(1) add L-rhamnosyl in resting cell solution system, in system, resting cell concentration is OD
600=25 ~ 50, L-rhamnosyl mass percent concentration is 10% ~ 40%, and invert point is 28 DEG C ~ 32 DEG C, and shaking speed is 150rpm ~ 220rpm, and the reaction times is 24h ~ 72h, in conversion process, add Na
2cO
3or CaCO
3pH is made to be held constant at 5.0-7.0;
(2) the extraction of L-sandlwood saccharic acid: transform after terminating, centrifuging and taking supernatant, obtains L-rhamnosyl acid calcium salt through steps such as evaporation concentration, alcohol settling, oven dry, finally obtains L-sandlwood saccharic acid with sulfate substitution reaction.
2. the method for L-sandlwood saccharic acid is prepared in microbial transformation according to claim 1, it is characterized in that: described resting cell is the one in beet pseudomonas AS1.6397, Pseudomonas fluorescens ATCC13525, chrysanthemum huge pseudomonas AS1.3385 or pseudomonas syringae NBRC14078.
3. the method for L-sandlwood saccharic acid is prepared in microbial transformation according to claim 2, it is characterized in that: the fermentation culture method of described resting cell is: be inoculated into by pseudomonas in liquid fermentation medium, in shaking table, cultivate 16h ~ 30h for 28 DEG C ~ 32 DEG C.
4. the method for L-sandlwood saccharic acid is prepared in microbial transformation according to claim 3, it is characterized in that: the fermention medium of described resting cell is, can be: carbon source 0 ~ 40, nitrogenous source 0 ~ 35 in g/L, is O when carbon source or nitrogenous source difference.
5. the method for L-sandlwood saccharic acid is prepared in microbial transformation according to claim 4, it is characterized in that: described carbon source be in high fructose syrup, sucrose, glucose, maltose, lactose any one, described nitrogenous source is at least one in yeast extract paste, corn steep liquor, extractum carnis, peptone.
6. the method for L-sandlwood saccharic acid is prepared in microbial transformation according to claim 3, it is characterized in that: the preferred fermentation culture conditions of described resting cell is: temperature is 28 DEG C ~ 32 DEG C, liquid amount 20 ~ 30mL/250mL, inoculum size 4% ~ 8%, shaking speed is 150rpm ~ 220rpm, and incubation time is 16h ~ 30h.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108129290A (en) * | 2017-12-25 | 2018-06-08 | 武汉三江航天固德生物科技有限公司 | A kind of method of sulfate radical in removal lactic acid |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0282942A2 (en) * | 1987-03-17 | 1988-09-21 | University Of Iowa Research Foundation | Method for producing rhamnose |
| JP2012005401A (en) * | 2010-06-24 | 2012-01-12 | San-Ei Sucrochemical Co Ltd | Method of producing carboxylic acid and/or carbohydrate carboxylic acid and/or salt thereof belonging to genus pantoea |
| CN104531810A (en) * | 2015-01-14 | 2015-04-22 | 天津科技大学 | Method for preparing maltonic acid through efficient microbial conversion |
-
2015
- 2015-08-26 CN CN201510531257.4A patent/CN105063111A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0282942A2 (en) * | 1987-03-17 | 1988-09-21 | University Of Iowa Research Foundation | Method for producing rhamnose |
| JP2012005401A (en) * | 2010-06-24 | 2012-01-12 | San-Ei Sucrochemical Co Ltd | Method of producing carboxylic acid and/or carbohydrate carboxylic acid and/or salt thereof belonging to genus pantoea |
| CN104531810A (en) * | 2015-01-14 | 2015-04-22 | 天津科技大学 | Method for preparing maltonic acid through efficient microbial conversion |
Non-Patent Citations (3)
| Title |
|---|
| HAUSER G等: "Rhamnose and rhamnolipide biosynthesis by Pseudomonas aeruginosa", 《JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| TOSHIYUKI SUZUKI等: "Aerobic Dissimilation of l-Rhamnose and the Production of l-Rhamnonic Acid and 1,2-Propanediol by Yeasts", 《AGRICULTURAL AND BIOLOGICAL CHEMISTRY》 * |
| 杨大娇 等: "静息细胞在发酵工业中的研究进展", 《食品与发酵工业》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108129290A (en) * | 2017-12-25 | 2018-06-08 | 武汉三江航天固德生物科技有限公司 | A kind of method of sulfate radical in removal lactic acid |
| CN108129290B (en) * | 2017-12-25 | 2021-01-22 | 武汉三江航天固德生物科技有限公司 | Method for removing sulfate radical in lactic acid |
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