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Caballeros strain with high pha production, screening method and method for producing pha

A technology of Caballeros strains and Caballeros bacteria, which is applied in the field of high-yield PHA strains, can solve the problems of low proportion of PHA accumulation, unsatisfactory strain yield, unstable recombinant strains, etc., and achieves a scientific and reasonable screening method and easy operation Effect

Active Publication Date: 2021-11-23
HEBEI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] One of the purposes of the present invention is to provide a high-yield PHA-producing Caballeronia sp. Y5702 strain to solve the problems of unsatisfactory yield of existing strains for fermenting and producing PHA, low proportion of PHA accumulation, instability of recombinant strains, etc.

Method used

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  • Caballeros strain with high pha production, screening method and method for producing pha
  • Caballeros strain with high pha production, screening method and method for producing pha
  • Caballeros strain with high pha production, screening method and method for producing pha

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Experimental program
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Effect test

Embodiment 1

[0034] Preliminary screening and verification of high-yielding strains

[0035] 1.1 Screening method

[0036]Based on the excellent performance of Nile blue stain, avoiding the complicated operation of using Nile blue stain to directly stain the bacteria, cooperate with the optimized YMA medium in our laboratory. YMA medium has a relatively high carbon-nitrogen ratio and low phosphorus content, which is suitable for the growth of rhizobia and can also promote the accumulation of a large amount of PHA.

[0037] YMA solid medium: mannitol 10g, yeast powder 5g, KH 2 PO 4 0.25g, K 2 HPO 4 0.25g, NaCl 0.1g, MgSO 4 ·7H 2 O 0.2g, agar 18g, the rest is deionized water, pH 7.0.

[0038] Nile blue YMA solid medium: Weigh 10mg of Nile blue dye and dissolve it in 1ml dimethyl sulfoxide solution, filter and sterilize and add to the melted YMA solid medium to prepare 5% Nile blue YMA solid medium base.

[0039] The Nile blue stain can specifically bind to the PHA particles in the...

Embodiment 2

[0066] PHA was produced using the Caballeronia sp. Y5702 strain.

[0067] YMA solid medium and beef extract peptone liquid medium are the same as in Example 1 in this embodiment.

Embodiment 2-1

[0069] Inoculate the high-yielding strain Caballeronia sp.Y5702 on a YMA solid medium plate, culture it in a 25°C incubator for 3 days, inoculate its single colony in beef extract peptone liquid medium, and place it on a shaker at 25°C and 220rpm Cultured in medium for 24h, prepared as seed solution.

[0070] Then insert 15% of the inoculum into the preparation fermentation medium, culture in a shaker at 30° C. and 220 rpm for 45 hours to obtain the fermentation liquid of the corresponding bacterial strain.

[0071] Centrifuge the fermentation broth at 12,000 rpm for 12 minutes in a high-speed centrifuge, collect the bacteria and discard the supernatant, wash with distilled water and ethanol three times each to remove the extracellular polysaccharides in the bacteria. The PHA in the bacteria was extracted by SDS-NaClO method.

[0072] Each liter of medium contains 10-50 g of carbon source substances (respectively selected from glucose, starch and sucrose), (NH 4 ) 2 SO 4 3...

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Abstract

The invention provides a Caballeros strain with high PHA production, a screening method and a method for producing PHA, and the preservation name of the strain is Caballeronia sp.Y5702, the preservation unit Guangdong Microbial Culture Collection Center (GDMCC), the preservation number is GDMCC No.61153, and the preservation date is August 14, 2020. The address of the preservation unit is Guangdong Institute of Microbiology, 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou City. The accumulation of PHA has reached 88%-95% of the dry weight of the bacteria. The higher proportion saves a lot of resources for the extraction work after fermentation. It is an excellent strain that can be used in industrial fermentation to produce PHA.

Description

technical field [0001] The present invention relates to a high-yielding PHA strain, in particular to a high-yield PHA-yielding Caballeronia sp. Y5702, a screening method and a method for producing PHA. Background technique [0002] Polyhydroxyalkanoate (PHA) is a common substance in organisms. It participates in various life activities and also accumulates in cells as a carbon source storage substance for microorganisms. As a new material, its good thermal processability, excellent biocompatibility and degradability make it have great application prospects in cutting-edge medicine and food fields. [0003] In recent years, my country's industrial fermentation of PHA has many problems such as unsatisfactory strain yield, low proportion of PHA accumulation, and unstable recombinant strains. In the prior art, the following method is usually used to screen PHA strains: 10 mg of Nile blue stain is dissolved in 0.5 ml of acetone, diluted to 20 ml with deionized water, and the mot...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P7/62C12R1/01
CPCC12N1/20C12P7/625
Inventor 刘晓云李伟康刘昱王北南高军王乐
Owner HEBEI UNIVERSITY