Mutant aureobasidium pullulans TKPM00006 for massively producing beta-poly(malic acid) and culture method thereof

A technology of Aureobasidium pullulans and polymalic acid, which is applied in the field of fermentation engineering, can solve the problems of low PMLA yield, low yield and high production cost in industrial production, so as to optimize fermentation and culture conditions, reduce production costs, and improve work efficiency. The effect of efficiency

Active Publication Date: 2011-02-23
天津慧智百川生物工程有限公司
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

Although the direct polymerization method has simple steps, easy product separation and purification, and high yield, the polymalic acid has low molecular weight, high reaction temperature, mostly γ-PMLA, low practical application value, and poisonous catalyst; ring-opening polymerization synthesis Although the main product is β-polymalic acid, there are many steps in the synthesis process and the yield is low, and the separation and purification of intermediate products is cumbersome; polymalic acid prepared by biological pathways is mainly synthesized by microbial fermentation, and biosynthesis of PMLA has outstanding advantages. They are all β-type, high molecular weight, mild production conditions, and high product purity. The molecular weight of PMLA obtained by microbial fermentation can reach 200-760kDa, while the molecular weight of chemical synthesis is up to 174kDa, but the yield of PMLA produced by microbial fermentation is not high, and it is extremely difficult. High production costs failed to achieve large-scale industrial production, which affected the application of the substance

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Aureobasidium pullulans TKPM10017, a bacterial strain producing β-polymalic acid screened from soil

[0033] 1. Bacteria morphology: The strain has two forms of yeast-like and fungal mycelium. Yeast-like cells secrete polymalic acid. Mycelium-like cells lack macromolecules. Young vegetative cells are oval to lemon-shaped, with several Individual bacteria can be connected into rods, often with arthrospores, chlamydospores, and bud conidia. Asexual reproduction can be performed in a variety of ways, mainly from the polygonal budding form of yeasts to the formation of distinct mycelial filaments.

[0034] 2. Colony morphology: Initially viscous, dirty white, quickly turning to light green, and finally black. The texture of the colony ranges from viscous to hard and leathery. The edge of the colony is obviously root-like, and the depression in the center of the colony is relatively moist. If it is cultivated for a long time, the periphery will turn from milky whi...

Embodiment 2

[0039] Example 2 Taking the high-yield strain TKPM10017 as the starting strain, the strain TKPM00006 was screened by multiple groups of compound mutagenesis techniques

[0040] 1. Obtaining of mutagenized strains

[0041] (1) Select the well-grown β-polymalic acid production strain TKPM10017 slant strain, wash the spores with sterile saline, and make 10 6 Each / mL spore suspension was irradiated under 30W ultraviolet light at a distance of 30cm for 1, 2, 3, 4, 5, 6, 7, and 8 minutes respectively, and the bacterial suspension was diluted and spread on a plate, and cultured at 25°C in the dark. Calculate the fatality rate;

[0042] (2) Choose 1min, 3min, 5min three different doses of irradiation time to carry out ultraviolet irradiation treatment to the spore suspension of bacterial strain TKBO16 respectively;

[0043] (3) Then the bacterial suspensions of three different irradiation times were mixed evenly, and serially diluted 10 times for 10 -1 ~10 -6 , take 10 -4 、10 -5...

Embodiment 3

[0057] The cultivation of embodiment 3 Aureobasidium pullulans TKPM00006

[0058] 1. Preparation of culture medium

[0059] (1) Strain Activation Medium: Potato Dextrose Agar Medium (PDA): Potato 200g / L, Glucose 20g / L, Agar 20g / L, pH4.0~4.5, 121°C autoclaved for 15min;

[0060] (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;

[0061] (3) Fermentation medium: glucose 12%, 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 3% (sterilized alone), pH 4.5, sterilized by high-pressure steam at 121°C for 15 minutes;

[0062] 2. Activation of strains

[0063] Aureobasidium pullulan (Aureobasidium, Pullulan) TKPM00006 was transferred to PD...

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Abstract

The invention provides mutant aureobasidium pullulans TKPM00006 for massively producing beta-poly(malic acid) and a method for producing poly(malic acid) by using the mutant through fermentation. The strain is the mutant for producing beta-poly(malic acid) with high yield, which is cultured by multiple groups of composite mutagenesis technology based on beta-poly(malic acid) producing TKPM10017 screened in the soil of Wang Taibao orchard, Yinchuan, Ningxia Hui Autonomous Region, China. Under the optimization condition, the yield of beta-poly(malic acid) reaches 18.4+/-1.3g/l.

Description

technical field [0001] The invention belongs to the technical field of fermentation engineering, and in particular relates to a mutagenic strain Aureobasidium pullulans TKPM00006 capable of producing a large amount of β-polymalic acid and a cultivation method thereof. Background technique [0002] Polymalic acid [Poly (malic-acid) or Poly malate, referred to as PMLA] is a homopolymer polymer with malic acid as the only monomer, and is a new type of fully biodegradable polymer material. The degradation product of this kind of material is non-toxic and harmless, and will not cause secondary pollution to the environment. In recent years, the development and research of this kind of polymer material has been developed rapidly. There are three main structures of PMLA: α-type, β-type, and γ-type PMLA. The only one that exists in the human body is β-type PMLA. As an aliphatic polyester, PMLA is highly water-soluble, biocompatible, biodegradable, bioabsorbable, non-immunogenic and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/62C12N13/00C12N1/14C12N15/01C12R1/645
Inventor 乔长晟钟堃王凤荣蒋磊徐勇虎贾士儒
Owner 天津慧智百川生物工程有限公司
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