Phospholipase B from pseudomonas fluorescens and production method thereof

A technology of Pseudomonas fluorescens and a production method, which are applied in the fields of enzyme genetic engineering and enzyme engineering to achieve the effects of good activity and stability, low production cost and high product quality

Inactive Publication Date: 2011-04-06
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, the research on Pseudomonas fluorescens producing low-tem

Method used

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  • Phospholipase B from pseudomonas fluorescens and production method thereof
  • Phospholipase B from pseudomonas fluorescens and production method thereof
  • Phospholipase B from pseudomonas fluorescens and production method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] 1. Screening of phospholipase-producing strains

[0022] 1. Separation and screening of strains

[0023] Collected from Shihezi City in Xinjiang Uygur Autonomous Region, Datong City in Shanxi Province, Yuncheng City in Henan Province, Zhengzhou City in Henan Province, including soil samples around soybean oil, rapeseed oil, cottonseed oil processing workshops and soybean and rapeseed planting bases, all below the surface Collect 18 copies at 10cm. Take 10 g of soil sample, put it into a triangular flask containing 90 mL of sterile water and glass beads, shake at room temperature for 12 hours and then stand still. Aspirate 1mL of the upper liquid and inoculate it in 49mL enrichment medium (soy phospholipid 4.0g / L, KN0 3 1.0g / L, K 2 HPO 4 1.0g / L, NaCl 0.5g / L, MgSO 4 ·7H 2 O 0.5g / L), shake flask culture at 30°C for 24h, and then apply a small amount of enriched culture solution to the primary screening medium (soy phospholipid 4.0g / L, peptone 10.0g / L, K 2 HPO 4 1.0g / L, NaCl 0....

Embodiment 2

[0079] 1. Cloning of the phospholipase B gene of Pseudomonas fluorescens BIT-18

[0080] 1. PCR amplification of conservative fragments of phospholipase B gene

[0081] A pair of degenerate primers P1-F (5'-GVSAACAACGGCGGCTACGC-3') and P2-R (5'-GCCARYTCCAYTGCGGRTGC-3') were designed according to the conservative sequence of bacterial phospholipase B published on the NCBI, and the fluorescent pseudo single The genomic DNA of the bacterium BIT-18 was used as a template for PCR amplification. The PCR reaction conditions were: pre-denaturation at 94°C for 4 minutes, denaturation at 94°C for 40 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 2 minutes, and extension at 72°C for 10 minutes after 35 cycles. The PCR reaction solution is separated by 1% agarose gel electrophoresis, such as Image 6 Shown. The results showed that a DNA fragment with a size of about 450 bp was amplified.

[0082] The DNA fragment amplified by PCR with a size of about 450 bp was ligated to pM...

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Abstract

The invention relates to a phospholipase B from pseudomonas fluorescens and a production method thereof, which belong to the field of enzyme gene engineering and enzyme engineering. The phospholipase B is produced from the pseudomonas fluorescens, has the total gene length of 1272bp and codes of 423 amino acids, and the zymoprotein theoretical molecular weight is 45.8kDa, wherein 1 to 69bp code phospholipase B signal peptide, and 70 to 1272bp code phospholipase B mature peptide are comprised. An expression vector and a recombinant host of the phospholipase B can be obtained by the traditional molecular biological method, i.e. colibacillus recombinant plasmid and recombinant colibacillus containing genes of the phospholipase B are obtained. The phospholipase B has good activity and stability at low temperature, does not have the lipase activity, can be hydrolyzed for catalyzing two fatty acyl group radicals in complete phospholipids, has wide application in the industrial process of grease refining, phospholipids emulsifying agent modification and the like, and also has simple preparation method, high product quality and low production cost.

Description

Technical field [0001] The invention relates to a phospholipase B derived from Pseudomonas fluorescens and a production method thereof, belonging to the fields of enzyme genetic engineering and enzyme engineering. Background technique [0002] Phospholipids are widely distributed in nature, and all cells contain phospholipids, which are the basic components of biological membranes. Phospholipids play a role in metabolism and structure formation in the process of life, and are important life substances. Phospholipase is an enzyme that hydrolyzes the ester bond of phospholipids. It is widely present in eukaryotes and prokaryotes. It affects the catabolism of phospholipids, the formation and reorganization of biological membranes, and phospholipases are also involved in the signal cascade. According to different sites of action, phospholipases are classified into phospholipase A 1 , A 2 , B, C, D, etc. Of which phospholipase A 1 And A 2 Respectively hydrolyze Sn-1 and Sn-2 acyl gr...

Claims

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Application Information

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IPC IPC(8): C12N9/16C12N15/55C12N15/63C12R1/39
Inventor 李春姜芳燕王金梅戴大章
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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