D-lactate oxidase and application thereof in D-lactic acid detection

A lactate oxidase and lactic acid technology, applied to lactate oxidase and its application fields, achieves the effects of easy fixation, good stability and good application potential

Active Publication Date: 2015-07-01
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the search found that there is no report on D-lactate oxidase that uses molecular oxygen as a...

Method used

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  • D-lactate oxidase and application thereof in D-lactic acid detection
  • D-lactate oxidase and application thereof in D-lactic acid detection
  • D-lactate oxidase and application thereof in D-lactic acid detection

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1: Construction of Escherichia coli recombinant expression strain comprising Gluconobacter oxidans 621H D-lactate oxidase gene

[0028] 1. PCR amplification of GOX2071 gene of Gluconobacter oxidans 621H D-lactate oxidase

[0029] Genomic DNA of Gluconobacter oxydans 621H is prepared by conventional methods. This process can refer to the method of small-scale preparation of bacterial genomes in the "Guide to Molecular Biology" published by Science Press to extract the genomic DNA of Gluconobacter oxydans 621H ;

[0030] Primers were designed to introduce the NdeI and XhoI restriction enzyme sites that can be inserted into the plasmid pET25b (Novagen). The primer sequences are as follows:

[0031] Upstream primer 5'-AAG CATATGCCGGAACCAGTCATGA-3', carrying an NdeI site;

[0032] Downstream primer 5'-CAA CTCGAG GCCCGTGTAAACAGCA-3', carrying an XhoI site.

[0033] To extract the genomic DNA of Gluconobacter oxydans 621H, PCR amplification was performed using t...

Embodiment 2

[0040] Example 2: Expression of Gluconobacter oxydans 621H recombinant D-lactate oxidase in Escherichia coli recombinant strain

[0041] Inoculate the glycerol tube of the recombinant strain Escherichia coli BL21 (pET25b-GOX2071) obtained in Example 1 into 5 mL of LB liquid medium containing 100 μg / mL ampicillin with a 1% inoculum size, and culture on a shaker at 37° C. for 6 to 12 hours Afterwards, transfer to 100mL LB liquid medium containing 100μg / mL ampicillin with 1% inoculum amount, culture on a shaker at 37°C for 6-12 hours, and then transfer to 1L containing 100μg / mL ampicillin at 2.5% inoculum amount Penicillin LB liquid medium, cultured on a shaker at 37°C, when OD 600nm After reaching 0.4-0.8, IPTG with a final concentration of 1 mmol / L was added to induce expression, and the expression was induced at 37°C for 6 hours.

[0042] After the induction culture, the cells were collected by centrifugation at 6,000rpm for 10 minutes, and the cell pellet was washed twice wi...

Embodiment 3

[0043] Example 3: Purification of Gluconobacter oxydans 621H recombinant D-lactate oxidase

[0044] 1. Preliminary purification of D-lactate oxidase using Souce30Q anion exchange column

[0045] The crude enzyme liquid containing recombinant D-lactate oxidase obtained in Example 1 was filtered through a filter head with a pore size of 0.22 μm, and then placed on an anion exchange column Source30Q equilibrated with Buffer A, and an anion exchange column was used to elute with a gradient of Buffer B Elution, the elution gradient is 40%, 50%, 65%, 100%, collect the 65% gradient eluate containing D-lactate oxidase, promptly obtain the recombinant D-lactate oxidase ( figure 1 ).

[0046] The formula of the above anion exchange column elution buffer Buffer B is: 20mmol / L sodium phosphate, 500mmol / L sodium chloride, pH 7.4.

[0047] 2. Further purification of D-lactate oxidase using HisTrap Ni affinity chromatography column

[0048] Concentrate the above-mentioned eluate containin...

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Abstract

The invention discloses a D-lactate oxidase. An amino acid sequence of the D-lactate oxidase is as shown in SEQ ID NO.2, and the D-lactate oxidase is soluble protein, wherein a cofactor is FAD; the optimum temperature is 55 DEG C; the optimal pH is 8.0; the D-lactate oxidase has over 60% enzyme activity within a pH range of 7.0-9.0; the D-lactate oxidase is capable of catalyzing D-lactic acid oxidation to generate pyruvic acid and hydrogen peroxide by employing molecular oxygen as a direct electron acceptor; when the concentration of D-lactic acid is within the range of 0.1-1.3mmol/L, the concentration of hydrogen peroxide generated by enzyme catalysis has a linear correlation with the initial D-lactic acid concentration; the appearance Km and Vmax of the D-lactate oxidase to the D-lactic acid are respectively 3.62+/-0.53mmol/L and 0.52+/-0.05U/mg; and the appearance Km and Vmax of the D-lactate oxidase to another substrate O2 are respectively 0.16+/-0.01mmol/L and 0.97+/-0.03U/mg. The D-lactate oxidase disclosed by the invention can be used for preparing a biosensor for detecting D-lactic acid by determining the concentration of hydrogen peroxide; and simple and rapid quantitative determination of D-lactic acid is realized.

Description

technical field [0001] The invention relates to a lactate oxidase and its application, in particular to a novel D-lactate oxidase and its application in D-lactic acid detection. Background technique [0002] Lactic acid widely exists in nature, such as yogurt, molasses, wine, apples, and animals and plants. Lactic acid has two optical isomers, L-lactic acid and D-lactic acid. Abnormal accumulation of D-lactate in human blood can lead to D-lactatemia, which must be confirmed by rapid measurement of D-lactate level when acute symptoms occur. In addition, D-lactic acid is also an important platform compound, which is widely used in medicine, chemical industry, biosynthesis and other fields, so the detection of D-lactic acid is often involved in industrial production. However, the current lactic acid biosensors for rapid determination of lactic acid concentration can only detect L-lactic acid, so a method for specific and rapid detection of D-lactic acid, such as D-lactic acid...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12Q1/26
CPCC12N9/0006C12Q1/26
Inventor 高超盛彬彬马翠卿许平
Owner SHANDONG UNIV
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