Low-temperature alkaline phosphatidase A1 and coding gene thereof

A phospholipase and alkaline technology, applied in genetic engineering, plant genetic improvement, hydrolytic enzymes, etc., to achieve the effect of simplifying the production process

Inactive Publication Date: 2007-11-14
THE INST OF MICROBIOLOGY XINJIANG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the research on Serratia that produces both low-temperature phospholipase and lipase has not been reported at home and abroad.

Method used

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  • Low-temperature alkaline phosphatidase A1 and coding gene thereof
  • Low-temperature alkaline phosphatidase A1 and coding gene thereof
  • Low-temperature alkaline phosphatidase A1 and coding gene thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1: low temperature alkaline phospholipase A 1 Screening of producing bacteria

[0048] 1. Isolation and screening of strains

[0049] Collect Xinjiang Tianshan No. 1 glacier and permafrost, and apply it with 0.1% (NH 4 ) 2 SO 4 , 0.1%K 2 HPO 4 , 1% KCl, 0.05% MgSO 4 ·7H 2 O, 0.001% FeSO 4 ·7H 2 O, olive oil and polyvinyl alcohol emulsion mixed in a ratio of 1: 3, on the fat assimilation medium of 2% agar powder, cultivated at 20°C for 24 hours and screened 3 lipase-producing bacterial strains. Bacterial colonies were selected and spread on egg yolk+TYSPN solid agar medium (4% glucose, 1% peptone, 5% yeast extract, sterile egg yolk liquid, 2% agar powder, pH7.0). The strain xjF1 formed a yellow discoloration circle on the oil assimilation plate, and formed an opaque and cloudy halo on the egg yolk + TYSPN solid agar plate. Insert strain xjF1 into phospholipase production medium (5.4% peptone, 2.6% yeast extract, 1.512% NaH 2 PO 4 12H 2 O, 0.3%K ...

Embodiment 2

[0089] Example 2: Activity assay of enzymes produced by S. fonticola xjF1

[0090] low temperature alkaline phospholipase A 1 The activity was determined by KOH potentiometric titration (ie, modified Kawauchi method). The preparation of the substrate lecithin also refers to the Kawauchi method (Biochimica et Biophysica acta, 142-159, 1971). Lecithin 500mg, dissolved in 10mL diethyl ether, add 20mL double distilled water and mix well, bathe in 65℃ water for 25min to remove diethyl ether, then add 20mL double distilled water, emulsify in an ultrasonic generator for 40min in ice bath, add 1mol / L NaCl 10mL, 0.01mol / L CaCl 2 10mL, 0.01mol / L sodium deoxycholate 10mL, stir to dissolve, then dilute to 100mL with distilled water, finally adjust the pH value to 8.2-8.5 with 1.0mol / L NaOH, store at 4°C for no more than 48h. Take 10mL of substrate solution for each measurement, 200uL of enzyme solution to be tested, react at 37°C for 30min, add 6mL of 95% ethanol to terminate the reac...

Embodiment 3

[0101] Example 3: Phospholipase A of S. fonticola xjF1 1 gene cloning

[0102] 1. Extraction of Genomic DNA from Serratia genus xjF1

[0103] Genomic DNA of Serratia citiensis xjF1 was prepared by conventional methods known to those skilled in the art. Serratia genus xjF1 was cultured with shaking in LB liquid medium for 16 hours, and the supernatant was discarded by centrifugation. Take 50mg of bacteria sludge and add 500uL sterile water to wash. The pellet after centrifugation was resuspended in lysozyme mixture, incubated at 37°C for 30min, and then 10% SDS was added to a final concentration of 2%. The supernatant after centrifugation at 12000 rpm for 5 min was extracted with equal volumes of phenol, phenol:chloroform and chloroform in sequence. Take the upper layer solution and add isopropanol, precipitate at room temperature for 20 minutes, centrifuge at 12,000 rpm for 20 minutes, discard the supernatant, dissolve the DNA precipitate with sterile water, and store it a...

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Abstract

This invention open a cold tolerance of Serratia sand Habitat-chuen (Serratia fonticola) xjF1 CGMCC No.1971, through the strain characteristics, enzyme production conditions and the nature of enzyme research, proved a new phospholipase A1 gene consists of two open plA and plS reading frame, was certified plA gene coding phospholipase A1, plS gene coding phospholipase A1 auxiliary protein. This invention relates to the gene containing the recombinant plasmid and reorganization cell, the gene expression products phospholipase A1 in the show activity under low-temperature conditions, can be used in industries such as oil refining process can be simplified production process, lowering energy consumption, improve product quality , protecting the environment, reduce production costs, have important practical significance.

Description

technical field [0001] The invention belongs to the fields of enzyme genetic engineering and enzyme engineering. Specifically, the present invention relates to a kind of low-temperature alkaline phospholipase A of coding resistance Serratia cold springs 1 The gene sequence of the enzyme, the recombinant plasmid containing the enzyme gene, and the recombinant strains and derivatives expressing the corresponding enzyme. Background technique [0002] Phospholipases are enzymes that hydrolyze the ester bonds of phospholipids. Widely present in eukaryotes and prokaryotes, it affects the catabolism of phospholipids, the formation and reorganization of biofilms, and phospholipases are also involved in the cascade of signals. Several types of phospholipases are known with different specificities depending on the position of the bond that the phospholipase molecule attacks, of which phospholipase A 1 (Phospholipase A 1 , 3.1.1.32, referred to as PLA 1 ) catalyzes the hydrolysis ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/16C12N15/55C12N1/21C12N1/19C12R1/425C12R1/19C12R1/885
Inventor 付建红姚斌石玉瑚孟昆欧阳平凯袁铁铮
Owner THE INST OF MICROBIOLOGY XINJIANG ACADEMY OF AGRI SCI
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