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Preparation method for obtaining phospholipase D through fermentation of genetically engineered streptomyces

A technology of genetic engineering and Streptomyces, which is applied in the field of fermentation engineering, can solve the problems affecting the application of PLD enzymes, low catalytic activity, and low PLD secretion, and achieve the effects of good preparation effect, good preparation quality and high cultivation efficiency

Pending Publication Date: 2021-05-25
HENAN BUSINESS SCI RES INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] In recent years, researchers have begun to pay attention to the preparation of PS by enzymatic conversion, that is, the synthesis of phosphatidylcholine and L-serine by catalyzing the substrates phosphatidylcholine and L-serine through phospholipase D; microorganisms, especially Streptomyces, synthesize phospholipase enzyme D from PS (hereinafter referred to as PLD ) has the activity of transphosphatidyl, can catalyze the synthesis of PS in an aqueous environment, the reaction conditions are mild, the by-products are few, the product yield is high, and the quality is good, it will become the main production method of industrialization in the future; The catalytic activity of wild bacteria is low, and the PLD secretion is far lower than the industrial production standard, which seriously affects the application of PLD enzyme in the synthesis of PS industrialization

Method used

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  • Preparation method for obtaining phospholipase D through fermentation of genetically engineered streptomyces
  • Preparation method for obtaining phospholipase D through fermentation of genetically engineered streptomyces
  • Preparation method for obtaining phospholipase D through fermentation of genetically engineered streptomyces

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] A preparation method for obtaining phospholipase D by fermentation of genetically engineered streptomyces, the specific steps are as follows:

[0044] Step 1, fermentation and acquisition, specifically includes the following steps:

[0045] (1) Fermentation culture: Streptomyces was inoculated into MM medium, placed in an incubator and cultivated until the bacterial liquid was turbid.

[0046] Wherein the MM medium is the basic fermentation medium, and the specific components include soluble starch 10g / L, (NH 4 ) 2 SO 4 3g / L, K 2 HPO 4 ·3H 2 O 3.4g / L, NaH 2 PO 4 2H 2 O 2.34g / L, MgSO 4 ·7H 2 O0.6g / L, casamino acid 5g / L; four kinds of trace metal ions ZnSO 4 ·7H 2 O, FeSO 4 ·7H 2 O, MnCl 2 4H 2 O, CaCl 2 Both are 0.001g / L, and they are packed in a fluted shaker bottle.

[0047] (2) Cooling and centrifugation: Pour the bacterial solution into a centrifuge tube and cool down to 0-4°C, then use a centrifuge at 6000rpm to centrifuge at 4°C for 30min, and th...

Embodiment 2

[0061] With the preparation method in embodiment 1, the fermenting temperature of test streptomyces is to the influence of enzyme activity and thalline quantity, and specific mode is to set the cultivating temperature in (1) fermenting culturing in step 1, cultivating time is set as Fixed value, uniform Streptomyces inoculum, and under this condition, the enzyme activity and the number of bacteria were measured.

[0062] Set the temperature of the incubator at 26°C, 28°C, 30°C, 32°C, 34°C, and 36°C respectively, repeat the preparation method in Example 1 to inoculate 1% Streptomyces for 6 days, and measure the enzyme live and the number of bacteria, and the results are shown in the table below, and the line graph is made as figure 2 shown.

[0063]

[0064] From the table above and figure 2 It can be seen that the enzyme activity of phospholipase D and the concentration of Streptomyces reached the peak value when Streptomyces was cultured at 32°C, so 32°C was the optimu...

Embodiment 3

[0066] With the preparation method among the embodiment 1, the fermenting time of test Streptomyces is to the influence of enzyme activity and thalline quantity, and specific mode is that the culture temperature in the (1) fermentation culture in step 1 is fixedly set, and under this condition On the 3rd day, 4th day, 5th day, 6th day, 7th day, and 8th day of fermentation, enzyme activity monitoring and cell count monitoring were carried out.

[0067] That is, according to the preparation method in Example 1, inoculate 1% Streptomyces, cultivate at 32°C, track and detect the number of bacteria and enzyme activity data in the culture medium, and obtain the results as shown in the table below, and draw a line graph as image 3 shown.

[0068]

[0069] From the table above and image 3 It can be seen that the enzyme activity of phospholipase D and the thalline concentration of Streptomyces reached a peak value when fermented to the 6th day, so 6 days of fermentation culture w...

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Abstract

The invention discloses a preparation method for obtaining phospholipase D through fermentation of genetically engineered streptomyces, which comprises the following steps: step 1, obtaining of fermentation through fermentation culture, cooling centrifugation, suction filtration and precipitation; step 2, separation and purification: centrifugal liquid removal, protein redissolution, centrifugal impurity removal and ultrafiltration concentration; and step 3, content detection, including protein content detection and gel leakage enzyme activity detection. According to the fermentation method for preparing phospholipase D by utilizing the gene engineering streptomyces, a basic method is provided for industrial production of phospholipase D, and a good scientific research achievement transformation basis is provided for ensuring the enzyme activity of phospholipase D preparation and ensuring the yield of phospholipase Dpreparation at the same time.

Description

technical field [0001] The invention belongs to the field of fermentation engineering, and relates to a preparation method for obtaining phospholipase D by fermenting genetic engineering streptomyces. Background technique [0002] In recent years, researchers have begun to pay attention to the preparation of PS by enzymatic conversion, that is, the synthesis of phosphatidylcholine and L-serine by catalyzing the substrates phosphatidylcholine and L-serine through phospholipase D; microorganisms, especially Streptomyces, source PS to synthesize phospholipase enzyme D (hereinafter referred to as PLD ) has the activity of transphosphatidyl, can catalyze the synthesis of PS in an aqueous environment, the reaction conditions are mild, the by-products are few, the product yield is high, and the quality is good, it will become the main production method of industrialization in the future; The catalytic activity of the wild bacteria is low, and the PLD secretion is far lower than the...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/16C12R1/465
CPCC12N1/20C12N9/16C12Y301/04004
Inventor 朱海华王鋆坦刘红伟周莉王慧王永李井泉
Owner HENAN BUSINESS SCI RES INST
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