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Engineering bacillus subtilis capable of expressing phospholipase D

A technology of Bacillus subtilis and phospholipase, applied in the field of genetic engineering, can solve the difficulties of expressing phospholipase D, target gene identification and other problems, and achieve the effect of high reactivity and compact catalytic structure

Inactive Publication Date: 2018-11-20
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] However, Streptomyces belongs to the family of Actinomycetes, and Bacillus subtilis belongs to the genus Bacillus. The two belong to different strains, and there may be some genetic gaps, which may lead to the fact that the target gene derived from Streptomyces may not be recognized. Recognized by subtilis, there are certain difficulties in expressing phospholipase D in Bacillus subtilis

Method used

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  • Engineering bacillus subtilis capable of expressing phospholipase D
  • Engineering bacillus subtilis capable of expressing phospholipase D
  • Engineering bacillus subtilis capable of expressing phospholipase D

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Experimental program
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Effect test

Embodiment 1

[0051] Embodiment 1: Construction of recombinant plasmid

[0052] According to the codon optimization of the phospholipase D gene (PLD) in Streptomyces racemochromogenes (GenBank: AB573232) published on NCBI, the optimized gene was synthesized and ligated to the plasmid pUC by restriction enzyme ligation, and then according to Plasmid pPUC-PLD sequence information, the designed primer sequence is 2-F of SEQ ID NO.3: 5'-CG GAATTC GAATTCGCATCACCTACACCTCCA-3', with the sequence being 2-R of SEQ ID NO.4: 5'-CG GGATCC GTGGTGGTGGTGGTGGTGTACGCCTGGCAAAGGCCT-3', (where the horizontal lines are the introduced EcoRI and BamHI restriction sites respectively) use the above primers to amplify the phospholipase D coding gene (PLD) using the plasmid pPUC-PLD as a template.

[0053] The plasmid pSTOP and the above amplified gene fragments were respectively digested with SpeI and BamHI and ligated to construct a recombinant plasmid. Double restriction digestion and sequencing confirmed the s...

Embodiment 2

[0056] Embodiment 2: fermentation produces phospholipase D

[0057] The recombinant plasmids pSTOP-PLD, pMA0911-PLD, and pP43-PLD obtained in Example 1 were introduced into Bacillus subtilis WB600, and then the bacteria were inoculated in the seed medium containing kana antibiotic 50 μg / mL, at 37°C, 200rpm Cultivate the logarithmic growth phase as the seed solution; then transfer the seed solution to 20 mL of fermentation medium containing 50 μg / mL of kanamycin at an inoculation amount of 3%, and cultivate at 37 ° C and 200 rpm for 36 h. The fermentation broth was centrifuged at 4°C and 8000r / min for 10min to obtain the supernatant to obtain the crude phospholipase D enzyme solution.

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Abstract

The invention discloses engineering bacillus subtilis capable of expressing phospholipase D, and belongs to the technical field of gene engineering. The engineering bacillus subtilis capable of expressing the phospholipase D is constructed by taking bacillus subtilis (WB600) as an expression host and taking a phospholipase D encoding gene (PLD) from streptomyces racemochromogenes as a target gene.The activity of the phospholipase D produced by the engineering bacteria can be up to 5.916 U / mL and has an extremely high application value in the field of medicines, food and health care products;meanwhile, the phospholipase D adopted by the engineering bacteria is actinomyces-derived endogenous phospholipase D, which has high reaction activity, high phosphatidyl substrate selectivity and highorganic matter stability; furthermore, a catalysis structure is the most compact; the phospholipase D is more suitable for efficient synthesis of phospholipid and a phospholipid derivative in the industrial production.

Description

technical field [0001] The invention relates to a bacillus subtilis engineering bacterium capable of expressing phospholipase D, belonging to the technical field of genetic engineering. Background technique [0002] Phospholipase D belongs to the phosphatidyl diester synthetase class and has the obvious characteristics of this family, that is, it contains two intervals of H(X)K(X)4D (H stands for histidine; K stands for lysine; D stands for Aspartic acid; X represents any amino acid) conserved sequence. It can catalyze the transesterification reaction similar to ester condensation between the phospholipids in the substrate phase and the nucleophilic donor in the water phase. It is a good tool enzyme for the synthesis of phosphatidylserine, phosphatidylinositol and other rare phospholipids. [0003] Utilizing the high specificity of phospholipase D to convert and modify crude phospholipids from a wide range of sources, some rare phospholipids can be prepared, which has great...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N9/16C12P9/00C12R1/125
CPCC12N9/16C12N15/70C12P9/00C12Y301/04004
Inventor 刘龙孙怡夏洪志黄婷婷李江华堵国成陈坚
Owner JIANGNAN UNIV
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