Method for obtaining pyruvate oxidase in recombinant escherichia coli

A technology of recombinant Escherichia coli and pyruvate oxidase, applied in the field of bioengineering, can solve the problem of low fermentation yield, achieve high yield, convenient operation, and good industrial value

Inactive Publication Date: 2018-11-13
UNIV OF SHANGHAI FOR SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the fermentation yield of pyruvate oxidase derived from coccus viridans is as low as 120U / L, so it is necessary to develop new technologies to increase its yield

Method used

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  • Method for obtaining pyruvate oxidase in recombinant escherichia coli
  • Method for obtaining pyruvate oxidase in recombinant escherichia coli
  • Method for obtaining pyruvate oxidase in recombinant escherichia coli

Examples

Experimental program
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Embodiment 1

[0029] Step 1, inoculate the recombinant Escherichia coli pET28a-pod containing the expression gene of pyruvate oxidase on a solid medium, and activate it after culturing at 37° C. for 12 hours to obtain activated recombinant Escherichia coli pET28a-pod.

[0030] Step 2, inoculate the activated recombinant Escherichia coli pET28a-pod into LB medium, then place the LB medium on a shaker and shake it at 200 rpm for 12 hours at a temperature of 37°C to obtain activated recombinant Escherichia coli pET28a- pod's seed fluid.

[0031] Step 3, inoculate 5% of the seed liquid into the TB medium, and cultivate the seed liquid at a temperature of 37°C until the concentration of the seed liquid is 7.5×10 7 -8.8×10 7 After cfu / mL, add the inducer isopropyl-β-D-thiogalactoside for induction, shake at a speed of 200rpm at a temperature of 20-30°C for 12-20h until the concentration is 0.5mmol / L, and then continue to induce, Obtain the bacterial solution I.

[0032]Step 4, take 12000g of b...

Embodiment 2

[0045] Step 1: inoculate the recombinant Escherichia coli pET28a-pod containing the expression gene of pyruvate oxidase on a solid medium, and activate it after culturing at 37° C. for 12 hours to obtain activated recombinant Escherichia coli pET28a-pod.

[0046] Step 2, inoculate the activated recombinant Escherichia coli pET28a-pod into LB medium, then place the LB medium on a shaker and shake it at 200 rpm for 12 hours at a temperature of 37°C to obtain activated recombinant Escherichia coli pET28a- pod's seed fluid.

[0047] In step 3, 5% of the total amount of seed liquid will be inoculated into TB medium, and the seed liquid will be cultivated at a temperature of 37°C until the concentration of the seed liquid is 7.5×10 7 -8.8×10 7 After cfu / mL, add the inducer isopropyl-β-D-thiogalactoside for induction, shake at 20°C at a speed of 200rpm for 16h until the concentration is 0.1mmol / L, and then continuously add hydrochloric acid solution for fermentation Until the pH va...

Embodiment 3

[0060] Step 1: inoculate the recombinant Escherichia coli pET28a-pod containing the expression gene of pyruvate oxidase on a solid medium, and activate it after culturing at 37° C. for 12 hours to obtain activated recombinant Escherichia coli pET28a-pod.

[0061] Step 2, inoculate the activated recombinant Escherichia coli pET28a-pod into LB medium, then place the LB medium on a shaker and shake it at 200 rpm for 12 hours at a temperature of 37°C to obtain activated recombinant Escherichia coli pET28a- pod's seed fluid.

[0062] Step 3, inoculate TB medium with 5% of the total amount of seed liquid, and cultivate the seed liquid at a temperature of 37°C until the concentration of the seed liquid is 7.5×10 7 -8.8×10 7 After cfu / mL, add the inducer isopropyl-β-D-thiogalactoside for induction, shake at 25°C at a speed of 200rpm for 16h until the concentration is 0.5mmol / L, and then continuously add hydrochloric acid solution for fermentation Until the pH value is 6.0, and then ...

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Abstract

The invention provides a method for obtaining pyruvate oxidase in recombinant escherichia coli. The method comprises the steps as follows: inoculating recombinant escherichia coli pET28a-pod containing an expressed gene of pyruvate oxidase onto a solid culture medium for activation to obtain activated recombinant escherichia coli pET28a-pod; inoculating the activated recombinant escherichia coli pET28a-pod onto an LB culture medium placed on a shaker and shaking to obtain a seed solution of the activated recombinant escherichia coli pET28a-pod; inoculating the seed solution onto a TB culture medium for culture, adding an inducer for induction after the concentration is cultured to be a first cell concentration, continuously adding a hydrochloric acid solution for fermentation until a pH value is 5.5-6.5 when the concentration reaches a second cell concentration, and then performing induction continually to obtain a bacteria solution I; taking the bacteria solution I for centrifugation,pouring out supernatant, then washing twice with a PBS solution, and then resuspending with an equal volume of the PBS solution to obtain a resuspension solution; performing ultrasonication on the resuspension solution to obtain a ultrasonicated bacteria solution II; and taking the bacteria solution II for centrifugation, and collecting the supernatant containing the pyruvate oxidase.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a method for obtaining pyruvate oxidase in recombinant Escherichia coli. Background technique [0002] Pyruvate oxidase can be used for the detection of pyruvate, pyruvate kinase, alanine aminotransferase, aspartate aminotransferase, inorganic phosphorus, sialic acid, sialic acid kinase and urea, and is very important in biochemical detection kits components. In recent years, some scholars have reported wild-type strains that can fermentably produce pyruvate oxidase, such as Lactobacillus plantarum, Chlorococcus viridans and Escherichia coli. Among these wild-type strains, the pyruvate oxidase derived from A. viridans was used as the main component of biochemical detection reagents because of its good performance. However, the fermentation yield of pyruvate oxidase derived from coccus viridans is as low as 120U / L, so it is necessary to develop new technologies to incre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12R1/19
CPCC12N9/0008C12Y102/03003
Inventor 张建国卢俊文
Owner UNIV OF SHANGHAI FOR SCI & TECH
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