Method for removing pulullan polysaccharide and extracting and obtaining beta-polymalic acid by enzyme process
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A technology of pullulan polysaccharide and polymalic acid is applied in the field of biological synthesis of biodegradable polymer materials, which can solve the problems of low PMLA yield, high reaction temperature, high PMLA production cost, etc., so as to reduce production costs and improve Extraction yield, the effect of avoiding environmental problems
Inactive Publication Date: 2009-10-21
天津北洋百川生物技术有限公司
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Problems solved by technology
Although this method is direct and simple, the product is easy to separate and purify, and the yield is high, but the molecular weight of polymalic acid is low, the reaction temperature is high, mostly γ-PMLA, the practical application value is low, and the catalyst is poisonous; ring-opening polymerization synthesizes polymalic acid The molecular weight of malic acid is higher than that of direct polymerization, and the product is mainly β-polymalic acid. Acid is mainly synthesized by microbial fermentation. Current studies have found that physarum polycephalum and Aureobasidium Pullulan have a high ability to synthesize polymalic acid.
Biosynthesis of PMLA has outstanding advantages. Through microbial ferme
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Embodiment 1
[0024] Prepare β-polymalic acid by removing the by-product pullulan in the fermentation broth as follows:
[0025] Preparation of the first step medium
[0026] (1) Strain Activation Medium: Potato Dextrose Agar Medium (PDA): Potato 200g / L, Glucose 20g / L, Agar 20g / L, pH 4.0-4.5, sterilized by autoclaving at 121°C for 15min;
[0027] (2) Seed medium: glucose 8%, ammonium succinate 0.3%, succinic acid 0.2%, K 2 CO 3 0.04%, KH 2 PO 4 0.01%, MgSO 4 ·7H 2 O 0.01%, ZnSO 4 ·7H 2 O 5×10 -4 %, corn extract 0.05%, CaCO 3 2% (sterilized alone), pH 4.5, sterilized by high-pressure steam at 121°C for 15 minutes;
[0028] (3) Fermentation medium: glucose 12%, ammonium succinate 0.3%, succinic acid 0.2%, K 2 CO 3 0.04%, KH 2 PO 40.01%, MgSO 4 ·7H 2 O 0.01%, ZnSO 4 ·7H 2 O 5×10 -4 %, corn extract 0.05%, CaCO 3 3% (sterilized alone), pH 4.5, sterilized by high-pressure steam at 121°C for 15 minutes;
[0029] The second step of bacterial activation
[0030] Transfer t...
Embodiment 2
[0038] In this example, according to the method of Example 1, the pH of the fermentation filtrate (supernatant) in the fifth step was adjusted to pH 6, 8 plu / mL of pullulanase was added, stirred slowly at 40°C, and reacted for 60 minutes. To degrade the pullulan; the liquid was slowly stirred, and anhydrous methanol was added dropwise until the final concentration reached 50%; the others were the same as in Example 1.
Embodiment 3
[0040] In this example, according to the method of Example 1, the pH of the fermentation filtrate (supernatant) in the fifth step was adjusted to pH 7, 11 plu / mL of pullulanase was added, stirred slowly at 30°C, and reacted for 90 minutes. Degrading the pullulan; stirring the liquid slowly, adding anhydrous methanol drop by drop until the final concentration reaches 60%; others are the same as in Example 1.
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Abstract
The invention discloses a method for effectively degrading and removing pulullan polysaccharide in Aureobasidium pullulans fermentation broth by utilizing pullulanase so as to be favorable for extracting and obtaining beta-polymalic acid. The method can effectively degrade and remove the pulullan polysaccharide in the Aureobasidium pullulans fermentation broth, improve the yield and the purity of products, avoids a series of trivial operations caused by the interference of the pulullan polysaccharide, improve the working efficiency and reduce the production cost of the products. The method is realized by the following proposal: Aureobasidium pullulans is subjected to activation and seed culture and is inoculated into a fermentation medium; the Aureobasidium pullulans is cultured between 7 and 12 d at the temperature of between 24 and 30 DEG C and under the condition of between 120 and 200 r/m, fermentation broth is subjected to 5,000 r/m centrifugation or filtration after the fermentation is over, and thalli are removed; the pH value of a fermentation filtered fluid (a supernatant) is adjusted to be between 5 and 7, 6-11 plu/mL supernatant of the Pullulanase is added, and the reaction is performed for 30 to 90 minutes at the temperature of between 30 and 50 DEG C; subsequently dried methyl alcohol is added until the final concentration of methanol is between 40 and 60 percent, the mixture is slowly stirred and kept stand over night, and the obtained deposit is a coarse product of the beta-polymalic acid; and the coarse product of the beta-polymalic acid is dissolved in distilled water according to the ratio of 1:10(m:v), the obtained product is subjected to 5,000 r/m centrifugation, micromolecular undissolved substances are removed, and the beta-polymalic acid is obtained after vacuum concentration and freeze drying.
Description
technical field [0001] The technical scheme of the invention belongs to the research field of biodegradable polymer materials synthesized by biological methods. Background technique [0002] Polymalic acid (polyhydroxysuccinic acid ester) [Poly (malic-acid) or Poly malate, referred to as PMLA] is a homopolymer polymer with malic acid as the only monomer. Although polymalate was discovered as early as the late 1960s, it was not appreciated until 20 years later when it was isolated from several organisms. In 1969, Shimada et al. discovered that a polymer compound composed of malic acid could inhibit the acid protease of Penicillium cyclopium. At that time, the position of the carboxyl group in the ester bond had not been determined. bacteria (physarum polycephalum) was rediscovered. In the next few years, the development of polymalic acid was almost at a standstill. In 1989, Fischer et al. found that polymalic acid was an inhibitor of DNA polymerase α in slime mold (physarum...
Claims
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