Protoplast Preparation and Regeneration of Actinobacillus succinogenes

doi:Q93-33

China Biotechnology

2010, Vol. 30

Issue (06): 103-108 DOI: Q93-33

Protoplast Preparation and Regeneration of Actinobacillus succinogenes

MAO Yu²,WANG Dan^1,3,LI Qiang^1,3,XING Jian-min¹,HUANG Zhan-bin²

1.National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China
2.School of Chemical and Evironmental Engineering, China University of Mining & Technology, Beijing 100083, China
3.Graduate University of Chinese Academy of Sciences, Beijing 100049, China

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Abstract

Genome shuffling was an important technology of strain improvement. The effects of glycin concentration, cell age, lysozyme concentration, the operational time and temperature were conducted to optimize the conditions of protoplast formation and regeneration of Actinobacillus succinogenes CGMCC 2650. The effects of the different osmotic stabilizing agents on regeneration were also examed. The protoplast yield was the highest under these conditions: The bacteria was cultivated in Tryptic Soy Broth (TSB) medium containing 0.6 mg/ml glycin for 5 h, then collected and diluted with SMM until the optical density (660nm) reached 1.0. The diluent was treated by 0.025 mg/mL lysozyme at 37℃ for 45 min and plated on the TSB medium with 0.3 mol/L sucrose as osmotic stabilizer. The regeneration rate was up to 40.9%. This study provides the optimized conditions of protoplast preparation and regeneration for genome shuffling of A.succinogenes.

Key words： Actinobacillus succinogenes Succinic acid Protoplast Genome shuffling

Received: 28 January 2010 Published: 12 June 2010

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Cite this article:

MAO Yu, WANG Dan, LI Jiang, GENG Jian-Min, HUANG Tie-Bin. Protoplast Preparation and Regeneration of Actinobacillus succinogenes. China Biotechnology, 2010, 30(06): 103-108.

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https://manu60.magtech.com.cn/biotech/Q93-33 OR https://manu60.magtech.com.cn/biotech/Y2010/V30/I06/103

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