CN105063111A - Method for preparing L-rhamnose acid through microbial conversion - Google Patents

Abstract

The present invention relates to a method for preparing L-rhamnonic acid by microbial conversion. The steps are as follows (1) Add L-rhamnose to the resting cell solution system, the resting cell concentration in the system is OD ₆₀₀ =25-50, L- The rhamnose mass percentage concentration is 10% to 40%, and Na ₂ CO ₃ or CaCO ₃ is added during the conversion process to keep the pH constant at 5.0-7.0; (2) Extraction of L-rhamnonic acid: after the conversion, centrifuge and take Clear, through the steps of evaporation concentration, ethanol precipitation, drying and other steps to obtain L-rhamnonic acid calcium salt, and finally use sulfuric acid displacement reaction to obtain L-rhamnonic acid. The pseudomonas provided by the invention is a bacterium capable of converting L-rhamnose into L-rhamnonic acid, and the resting cells obtained after bacterial body fermentation are transformed into L-rhamnonic acid to improve the conversion rate.

Description

A method for preparing L-rhamnonic acid by microbial conversion

technical field

The invention belongs to the technical field of microbial fermentation, and relates to a method for preparing L-rhamnonic acid through microbial transformation.

Background technique

Plant-active high-strength wood glue is an environmentally friendly wood glue without formaldehyde pollution and has a very wide application prospect. And L-rhamnonic acid is an initiator for the curing of plant-active high-strength wood glue, which plays a vital role.

At present, the preparation of L-rhamnolic acid is mainly through the chemical catalytic synthesis method. Since the chemical synthesis method is often accompanied by the production of various by-products during the oxidation process, the separation and purification steps become complicated, so the production cost is relatively high. However, there are few studies on the production of L-rhamnonic acid by microbial fermentation transformation, and only a few yeasts have been found to be able to use rhamnose to produce rhamnonic acid, but the fermentation time is long, the yield is low and there are by-products 1, 2-Propanediol formation. The invention utilizes the pseudomonas bacterium fermentation conversion method to produce L-rhamnonic acid, has a high conversion rate and no by-products, can effectively overcome the above problems, and has good application prospects.

Contents of the invention

The purpose of the present invention is to overcome the disadvantages of the prior art, and provide a method for preparing L-rhamnonic acid by microbial conversion with significant advantages such as short production cycle, high product purity, safety and reliability.

The technical scheme that the present invention realizes purpose is:

A method for preparing L-rhamnonic acid by microbial conversion, the steps are as follows

(1) Add L-rhamnose to the resting cell solution system, the resting cell concentration in the system is OD ₆₀₀ =25-50, the mass percentage concentration of L-rhamnose is 10%-40%, and the conversion temperature is 28°C- 32°C, the shaker speed is 150rpm-220rpm, the reaction time is 24h-72h, add Na ₂ CO ₃ or CaCO ₃ during the conversion process to keep the pH at 5.0-7.0;

(2) Extraction of L-rhamnolic acid: after the transformation, centrifuge to take the supernatant, and then obtain L-rhamnolic acid calcium salt through evaporation and concentration, ethanol precipitation, drying and other steps, and finally use sulfuric acid displacement reaction to obtain L-rhamnose acid.

Moreover, the resting cell is one of Pseudomonas beta AS1.6397, Pseudomonas fluorescens ATCC13525, Pseudomonas chrysanthemis AS1.3385 or Pseudomonas syringae NBRC14078.

Moreover, the method for fermenting and cultivating the resting cells is as follows: inoculating Pseudomonas into a liquid fermentation medium and culturing in a shaker at 28° C. to 32° C. for 16 hours to 30 hours.

Moreover, the fermentation medium of the quiescent cells may be, in g/L, 0-40 carbon source, 0-35 nitrogen source, and the carbon source or nitrogen source is not O at the same time.

Moreover, the carbon source is any one of fructose syrup, sucrose, glucose, maltose, and lactose, and the nitrogen source is at least one of yeast extract, corn steep liquor, beef extract, and peptone.

Moreover, the preferred fermentation culture conditions of the quiescent cells are as follows: the temperature is 28°C to 32°C, the liquid volume is 20 to 30mL/250mL, the inoculum size is 4% to 8%, the shaking table speed is 150rpm to 220rpm, and the culture time It is 16h ~ 30h.

Advantage and positive effect of the present invention are:

1. Pseudomonas provided by the present invention is a bacterium that can convert L-rhamnose into L-rhamnonic acid, and the conversion of resting cells obtained after bacterial fermentation to prepare L-rhamnonic acid improves the transformation Rate.

2. The L-rhamnonic acid obtained by the method provided by the present invention has significant advantages such as short production cycle, high product purity, safety and reliability.

3. The L-rhamnonic acid product prepared by the present invention can be used in food additives and other fields, and the product can be purified or post-processed to different degrees according to needs to meet the purity requirements of different application fields.

Detailed ways

The method of the present invention is described below through specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be considered as illustrative rather than limiting the scope of the invention, the spirit and scope of which is defined only by the claims. For those skilled in the art, on the premise of not departing from the spirit and scope of the present invention, various changes or modifications to the material components and dosage in these embodiments also belong to the protection scope of the present invention.

A method for preparing L-rhamnonic acid by microbial conversion may comprise the following steps:

(1) Fermentation culture of Pseudomonas: inoculate Pseudomonas into liquid fermentation medium, and culture in a shaker at 28°C to 32°C for 16h to 30h;

Fermentation medium component can be in g/L meter: carbon source 0～40, nitrogen source 0～35, described carbon source can be any in fructose syrup (42% fructose), sucrose, glucose, maltose, lactose One, the nitrogen source can be at least one of yeast extract, corn steep liquor, beef extract, peptone or other organic or inorganic nitrogen sources.

Preferred fermentation culture conditions: temperature is 28°C-32°C, liquid volume is 20-30mL/250mL, inoculum size is 4%-8%, shaker speed is 150rpm-220rpm, culture time is 16h-30h.

(2) Preparation of resting cells: After the bacteria were cultured in the fermentation medium, the bacteria were collected by centrifugation at 5000-8000 rpm for 10 minutes, and sterile physiological saline was added to resuspend as a resting cell solution system. The quiescent cells have the ability to convert L-rhamnose to L-rhamnonic acid.

(3) Transformation of resting cells: adding L-rhamnose to the resting cell solution system, the concentration of resting cells in the system is OD ₆₀₀ =25-50, and the mass percentage concentration of L-rhamnose is 10%-40% , the conversion temperature is 28°C-32°C, the rotation speed of the shaker is 150rpm-220rpm, and the reaction time is 24h-72h. Na ₂ CO ₃ or CaCO ₃ can be added during the conversion to keep the pH constant at around 6.0.

(4) Extraction of L-rhamnolic acid: After the conversion, centrifuge to take the supernatant, and obtain L-rhamnolic acid calcium salt through evaporation and concentration, ethanol precipitation, drying and other steps, and finally use sulfuric acid displacement reaction to obtain L- Rhamnolic Acid Products.

The above-mentioned reagents and medium formulations are conventional reagents and medium in the art unless otherwise specified.

The following specific descriptions are used to demonstrate the present invention.

The Pseudomonas used in the present invention can preferably use one of Pseudomonas beta AS1.6397, Pseudomonas fluorescens ATCC13525, Pseudomonas chrysanthemum AS1.3385, Pseudomonas syringae NBRC14078, both Commercially available strains.

A method for microbial transformation and preparation of L-rhamnonic acid, the steps are as follows:

The microorganism used in this example is Pseudomonas syringae NBRC14078.

1. The fermentation of L-rhamnose, the steps are as follows:

Streak inoculation of Pseudomonas on solid medium and culture in a constant temperature incubator at 30°C. Pick and grow a single colony from the plate and inoculate them into 30mL liquid medium (the components of the fermentation medium are calculated in g/L: 10 sucrose, 10 peptone, 30 yeast extract), and shake at 30°C and 180r/min. Cultivate in the bed, after the thalli are cultured in the fermentation medium, 8000rpm10min centrifugal collection thalli, add sterile normal saline and resuspend as transformation liquid, add different concentrations of L-rhamnose (10%, 20%, 30%, 40%) into the resting cell transformation solution system, the resting cell concentration in the system is OD _{600 =} 45, adding CaCO during the transformation process to keep the pH constant, the transformation temperature is 30°C, the shaking table speed is 180rpm, the reaction time It is 24h～72h. The results are shown in Table 1.

Table 1 Effects of different concentrations of rhamnose on conversion

The results show that the conversion time prolongs with the increase of the L-rhamnose concentration, and finally the same yield is reached. Considering the conversion time and conversion yield, 20% L-rhamnose is selected as the preferred conversion substrate concentration .

2, the preparation of L-calcium rhamnolate, the steps are as follows:

(1) Collect the supernatant after centrifugation of the resting cell transformation solution, and evaporate and concentrate to 1/3 of the original volume at 100°C. At this time, the solution is viscous, and then cooled to room temperature;

(2) Add 10 times the volume of 95% ethanol and keep stirring. At this time, a large amount of colloidal calcium L-rhamnolate precipitates out, and the ethanol solution is removed by suction filtration after fully stirring;

(3) Add the same volume of 95% ethanol to the colloidal precipitate, and precipitate after fully stirring;

(4) Suction filtration to dryness obtains L-calcium rhamnolate crude product;

(5) Add water to the crude product to the same volume as that after concentration, heat to dissolve, then suction filter, add the same volume of 95% ethanol to stir and crystallize the filtrate after cooling to room temperature, and then drain it to obtain the L-calcium rhamnolate fine product.

3, the preparation of L-rhamnolic acid, the steps are as follows:

Adjust the obtained L-calcium rhamnolate into an aqueous solution, place it in a suitable reactor, slowly add equimolar concentrated sulfuric acid under stirring, heat in a water bath at 60°C to 90°C for 1h to 3h, and filter out the precipitated calcium sulfate Precipitation, the L-rhamnonic acid obtained after the filtrate is cooled is purified with barium hydroxide and oxalic acid as a precipitating agent to remove a small amount of sulfate and calcium ions, and excessive barium and oxalic acid are removed to form a barium oxalate precipitate; or the filtrate is cooled Then flow through the exchange columns of anion and cation exchange resins at an appropriate flow rate to obtain high-purity L-rhamnonic acid. L-rhamnonic acid products should be understood as L-rhamnonic acid and its salts, such as L-calcium rhamnoate and sodium L-rhamnolate.

The method for detecting L-rhamnolic acid is as follows:

HPLC conditions: Agilent1100 chromatographic column: amino column (4.6 × 250mm); mobile phase is acetonitrile: water = 60:40 (containing 0.1% phosphoric acid); detector: UV detector 210nm; column temperature: 25 ° C; flow rate: 1mL /min; injection volume: 10μL; retention time: 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.