Method for preparing and regenerating rhizopus oryzae protoplast for producing L-lactic acid at high yield

A technology of protoplast and Rhizoplast oryzae is applied in the field of Rhizoplast oryzae protoplast preparation and regeneration, which can solve the problem of large workload of screening forward mutation, and achieve high protoplast preparation number, high regeneration rate and uniform size. Effect

Inactive Publication Date: 2011-05-18
HEFEI UNIV OF TECH
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  • Summary
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  • Claims
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AI Technical Summary

Problems solved by technology

The traditional strain improvement technology uses physical, chemical or biological mutagenesis methods to treat the starting strains, and randomly selects and breeds mutant strains with excellent performance. The disadvantage is that the changes in genetic traits induced by mutagens are random. The workload of forward mutation is larger

Method used

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Experimental program
Comparison scheme
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Embodiment

[0028] The operation steps of the preparation and the regeneration method of the Rhizopus oryzae protoplast for high production of L-lactic acid are as follows:

[0029] A, preparation Rhizopus oryzae spore suspension:

[0030] The preserved Rhizopus oryzae AS3.819 ( Rhizopus oryzae AS3.819) a loop of mycelia, inoculated onto a solid slope made of 50mL PDA medium in a 100mL Erlenmeyer flask, at 32 o C culture in a constant temperature incubator for 72 hours to obtain a bottle of mycelium containing mature Rhizopus oryzae spores, add 30 mL of sterile distilled water to wash the spores, and obtain a spore liquid containing spore clusters, pour the spore liquid into a bottle containing glass beads In a sterile Erlenmeyer flask, oscillate to break up the spore clusters, filter with 4 layers of sterile high-grade lens-cleaning paper, take the filtrate for multiple dilution, and count under a microscope to make the final concentration of spores 1×10 6 spores / mL, the concentration...

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Abstract

The invention relates to a method for preparing and regenerating a rhizopus oryzae protoplast for producing L-lactic acid at high yield, comprising the following steps of: A. preparing a rhizopus oryzae spore suspension; B. preparing a seed culture liquid; C. preparing a hypha suspension; D. enzymolyzing a cell wall; E. separating a protoplast; and F. regenerating the protoplast. The rhizopus oryzae protoplast prepared with the method disclosed by the invention is in an approximately-circular shape and has higher activity; the preparation quantity and the regeneration rate of the rhizopus oryzae protoplast can respectively reach 2.74*10<6>/mL and 6.8 percent to the maximum extent. The method is easy to operate, has low requirements for the equipment, and provides a technical reference for preparing and regenerating the rhizopus oryzae protoplast. By using the method disclosed by the invention, the protoplast can be quickly prepared with short regeneration period and high regeneration speed; and the preparation efficiency of the protoplast can be effectively improved. The protoplast prepared with the method provided by the invention completely meets the requirement for fusing the protoplast. The invention provides an operation method with practical significance for preparing and regenerating the protoplast.

Description

technical field [0001] The invention belongs to the technical field of preparation and regeneration of fungal protoplasts in cell engineering, and specifically relates to the preparation and regeneration technology of rhizopus oryzae protoplasts. Background technique [0002] Lactic acid is an important multi-purpose organic acid, one of the three major organic acids, especially L-lactic acid, which shows a wide range of application prospects due to its unique configuration advantages. L-lactic acid widely exists in plants and organisms, and has important applications in food, medicine, feed, chemical and other fields. In recent years, with the development and application of polylactic acid, the demand for L-lactic acid is increasing. Rhizopus oryzae has low nutritional requirements for fermentation, and can directly use starch without saccharification treatment; it is resistant to low pH value, and the product is L-lactic acid with high optical purity. The bacteria are lar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12R1/845
Inventor 姜绍通罗水忠潘丽军邱静郑志吴学凤
Owner HEFEI UNIV OF TECH
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