Levulose valine oxidizing enzyme of high activity and method of producing the same

A valine oxidase and high-activity technology, which is applied in the field of preparation of high-activity fructose valine oxidase, can solve the problems of long time and high demand for enzyme amount, and achieve the goals of reduced enzyme amount, high activity, and shortened reaction time Effect

Active Publication Date: 2009-02-18
NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004]However, using the existing fructosyl amino acid oxidase to catalyze this reaction, the time is still a bit long, and the demand for the amount of enzyme is relatively high

Method used

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  • Levulose valine oxidizing enzyme of high activity and method of producing the same
  • Levulose valine oxidizing enzyme of high activity and method of producing the same
  • Levulose valine oxidizing enzyme of high activity and method of producing the same

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The FVO gene coding sequence of Corynebacterium sp.2-4-1 was used as a template for error-prone PCR amplification.

[0022] 1. Primer sequence:

[0023] Forward primer: 5’-TTGTTCGGATCCATGTCCTCCACCGCTAC-3’

[0024] Reverse primer: 5’-TTGTTCAAGCTTCTAGGAGAACCGGCCCG-3’

[0025] 2. Error-prone PCR reaction system and reaction conditions:

[0026] PCR reaction system:

[0027] The 100μL system contains:

[0028] 10mM Tris-HCl pH 8.30, 50mM KCl, 6.5mM MgCl 2 ,

[0029] 0.15mM MnCl 2 , 0.2mM dGTP / dATP, 0.8mM dTTP / dCTP,

[0030] 2.2μg SSB Protein, 0.5μM forward / reverse primer, 10ng template DNA

[0031] 2.5 units of TaqDNA polymerase.

[0032] PCR reaction conditions:

[0033] 94℃ 5min; 94℃ 30sec, 55℃ 30sec, 72℃ 2min, 35 cycles; 72℃ 10min; 4℃forever.

[0034] 3. Take 3μL of the error-prone PCR product on a 1% agarose gel electrophoresis, and a product band of about 1kb can be seen. The error-prone PCR product was purified and recovered with a purification and recovery kit, connected...

Embodiment 2

[0036] Initial screening of mutant strains:

[0037] The plasmid DNA in the mutant library was extracted and digested with BamH I and Hind III to recover a target fragment of about 1 kb. The vector pGEX-4T-1m was also digested with BamH I and Hind III and recovered, and the two were connected at 4°C. Overnight, transform into BL-21 strain and spread on LB containing IPTG, fructose valine and N-(carboxymethylaminocarbonyl)-4,4-bis(methylamino)-benzidine [DA-64] Plate: After culturing at 37°C for 12-24 hours, obvious discoloration can be seen. The clones with fast discoloration and large discoloration circles were picked and cultured in liquid medium. A total of 48 clones were picked.

Embodiment 3

[0039] Quinone method detection of mutant enzyme activity:

[0040] 1. After 4 hours of inducing expression, collect the bacterial liquid, measure and record the OD600 value of the cell culture.

[0041] 2. Prepare bacterial lysate for detecting enzyme activity. In 374 μL B-PER TM Resuspend the cells in bacterial protein extraction reagent (Pierce product78248), then add 50uL protease and phosphorylase inhibitors and bacterial cell extract (Sigma product P8465), 1uL 34mg / mL chloramphenicol (prepared with methanol). Vortex quickly for one minute. Place on ice for 5 minutes. Centrifuge for 1 min and place on ice.

[0042] 3. Use the Bradford method to determine the protein content.

[0043] 4. Take 50μL of the above bacterial lysate and add it to the quinone method reaction mixture, incubate at 37°C for 1-3min, and measure the absorbance at 555nm. The components of the reaction mixture are: 100mM potassium phosphate buffer (pH8.0), 1purpurogallin unit / ml peroxidase, 0.45mM 4-aminoan...

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Abstract

The invention relates to high-activity fructose valine oxidase and a preparation method thereof, the amino acid sequence of the fructose valine oxidase is the sequence of SEQ ID. No.2, and the nucleotide sequence is shown in SEQ ID.No.1. The preparation steps are: (1) error-prone PCR amplification is carried out with the FVO gene coded sequence of Corynebacterium sp.2-4-1 as the template to establish the mutation bank of the fructose valine oxidase; (2) the mutation bank is transferred into escherichia coli and the clone, reconstruction, conversion and expression are carried out to the gene of the transferred escherichia coli; and (3) the activity of enzyme is determined by utilizing the quinine method and the high-activity fructose valine oxidase is screened out. The activity of the obtained fructose valine oxidase is about 6 times of the activity of the ordinary fructose valine oxidase, the fructose valine oxidase can be used for testing saccharification Hb kit, the used amount of enzyme is reduced, and the reaction time is shortened.

Description

Technical field [0001] The present invention relates to a high-activity fructose valine oxidase. The invention also relates to a preparation method of high-activity fructose valine oxidase. Background technique [0002] The glycosylated hemoglobin (hereinafter referred to as glycosylated Hb) in the blood reflects the blood glucose level in the organism over a period of time, and has been used as an important indicator for the diagnosis and treatment of diabetes in recent years. At present, glycated Hb can be determined by high-performance liquid chromatography, microcolumn method, immunoassay, pigment method and other methods, but these methods either require special instruments, are expensive, or have long detection times and poor measurement accuracy. Recently, the popular enzyme Method detection, using oxidation-reduction reaction, does not require special measuring equipment, easy to operate, high precision, and short time, so it is widely used in biochemical analysis and cli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/70
Inventor 邹炳德姜云飞
Owner NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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