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Technology of screening Prulan high yield strains from protoplast of bacillus brevis induced by laser

A protoplast, high-yield strain technology, applied in horticulture, horticultural methods, botanical equipment and methods, etc., can solve the problems of low positive mutation rate, poor sensitivity of Aureobasidium pullulans, etc., and achieves high positive mutation rate, Sensitive effect

Inactive Publication Date: 2005-07-27
JILIN UNIV
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AI Technical Summary

Problems solved by technology

However, the sensitivity of Aureobasidium pullulans to ultraviolet light is not good, and the positive mutation rate is low

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1 best technological process

[0019] 1. Preparation of Aureobasidium pullulans Protoplasts

[0020] (1) Bacterial culture: Aureobasidium pullulans was inoculated in liquid basic medium, and cultured on a shaker (28°C, 120r / min) until the logarithmic growth phase.

[0021] (2) Pretreatment: Add an equal volume of glycine solution (glycine dissolved in 0.1mol / L pH6.0 citric acid-sodium citrate buffer solution containing 0.7mol / L NaCl) to the liquid basic medium with a concentration of 1%. hypertonic stable solution) for hypertonic pretreatment for 40 min.

[0022] (3) Enzyme hydrolysis: centrifuge the pre-cultured bacterial solution at 4000r / min for 4.5min, wash the precipitate twice with hypertonic stabilizing solution, then centrifuge at 4000r / min for 4.5min, and make the precipitate into a bacterial suspension. Take 1mL of the bacterial suspension and add 2 times the volume of the mixed enzyme solution with the enzyme concentration ratio of 0.2% helicase,...

Embodiment 2

[0026] The optimal test of embodiment 2 protoplast preparation conditions

[0027] (1) The selection of the optimal mixed enzyme concentration ratio.

[0028] Adopt orthogonal design experimental method (three factors three levels), its factor is helicase, cellulase and lysozyme, carry out protoplast preparation experiment on three levels of 0.1%, 0.2%, 0.5% at concentration, helicase, cellulase and lysozyme, When the enzyme concentration ratios of cellulase and lysozyme are both 0.1-0.5%, the enzymatic hydrolysis effect is better; and the combined protoplast formation rate and regeneration rate product of 0.2% helicase, 0.1% cellulase, and 0.2% lysozyme The highest is determined to be the best enzyme concentration ratio.

[0029] (2) The choice of hypertonic stable solution.

[0030] These 6 kinds of hypertonic stable solutions were used respectively, namely ①0.1mol / L pH6.0 phosphate buffer containing 0.7mol / L NaCl; ②0.1mol / L pH6.0 phosphate buffer containing 0.5mol / L sucro...

Embodiment 3

[0035] Example 3 Laser mutagenesis selection test for high-yielding pullulan strains

[0036] The prepared protoplasts were diluted 10 4times, take 0.3ml respectively and place them in 16 small test tubes, use four power densities (5mW / cm 2 、10mW / cm 2 、20mW / cm 2 、40mW / cm 2 ) He-Ne laser (wavelength 632.8nm) was irradiated for 5min, 10min, 20min and 30min respectively. The irradiated protoplast suspension in the small test tube was coated on the regenerated double-layer plate for regeneration for 48 hours.

[0037] Single colonies of surviving strains on the regeneration plate were picked out and inoculated in liquid medium for fermentation and culture for 5 consecutive days. Samples were taken every day to measure the pullulan content in the fermentation broth, and the conversion rate of sucrose was calculated.

[0038] As a result, the intensity is 10~30mW / cm 2 The mutated strains were obtained by irradiating the laser for 10-30 minutes, and the conversion rate of sucr...

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Abstract

A process for screening the high-output pullulan strain by laser mutagenesis to the protoplasm of Aureobasidium pullulans arnaud includes such steps as inoculating Aureobasidium pullulans arnaud to the liquid culture medium, shake culturing, adding glycine solution to the fermented liquid, hyperosmotic preculturing, adding mixed enzyme liquid to remove cell walls and obtaining protoplasm, and He-Ne laser mutagenesis. Its advantages are high mutagenesis sensitivity, high positive mutation rate and high conversion rate of cane sugar.

Description

technical field [0001] The invention belongs to the technical field of microbial mutagenesis, and in particular relates to a technique for screening pullulan high-yield strains by laser mutagenesis of Aureobasidium pullulans protoplasts. Background technique [0002] Aureobasidium Pullulans is a polymorphic fungus whose yeast-like cells can secrete exopolysaccharide-pullulan, also known as pullulan, whose molecules are composed of maltotriose and α- 1,6-glycosidic linkages. [0003] Pullulan is a colorless, tasteless and odorless polymer substance, which has the characteristics of non-toxic, safe, heat-resistant, salt-resistant, acid-alkali-resistant, low viscosity, strong plasticity, and good film-forming properties. It has broad application prospects in product preservation, food processing industry, environmental protection field, packaging industry, medicine, petroleum, etc. [0004] The market prospect of pullulan polysaccharide is good, and the economic benefit is re...

Claims

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Application Information

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IPC IPC(8): A01G7/00A01H3/00
Inventor 刘兰英王雪松王莹陈佳于晓迪初宇卓范豪
Owner JILIN UNIV
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