Method for removing pulullan polysaccharide and extracting and obtaining beta-polymalic acid by enzyme process

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...

AI Technical Summary

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 fermentation, the products are all β-type, with high molecular weight, mild production conditions, and high purity of the product. The molecular weight of PMLA obtained by microbial fermentation can reach 200-760kDa, while the molecular weight of chemically synthesized The maximum is 174kDa, but the yield of PMLA produced by microbial fermentation is not high, and separation and purification are difficult, especially in the process of producing polymalic acid by fermentation of Aureobasidium Pullulan. The removal of the fermentation by-product pullulan has always been the extraction of PMLA. It is an important problem that cannot be ignored, and it is also an important reason for the huge production cost of PMLA