Levulose valine oxidizing enzyme of high activity

A technology of valine oxidase and high activity, which is applied in the field of high activity fructose valine oxidase to achieve the effects of easy implementation, reduced enzyme amount and high activity

Active Publication Date: 2011-04-27
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
  • Levulose valine oxidizing enzyme of high activity
  • Levulose valine oxidizing enzyme of high activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Using the FVO gene coding sequence of Corynebacterium sp.2-4-1 as a template, error-prone PCR amplification was performed.

[0023] 1. Primer sequence:

[0024] Forward primer: 5'-TTGTTCGGATCCATGTCCTCCACCGCTAC-3'

[0025] Reverse primer: 5'-TTGTTCAAGCTTCTAGGAGAACCGGCCCG-3'

[0026] 2. Error-prone PCR reaction system and reaction conditions:

[0027] PCR reaction system:

[0028] 100μL system contains:

[0029] 10mM Tris-HCl pH8.30, 50mM KCl, 6.5mM MgCl 2 ,

[0030] 0.15mM MnCl 2 , 0.2mM dGTP / dATP, 0.8mM dTTP / dCTP,

[0031] 2.2μg SSB Protein, 0.5μM forward / reverse primer, 10ng template DNA

[0032] 2.5 units of Taq DNA polymerase.

[0033] PCR reaction conditions:

[0034] 94°C for 5min; 94°C for 30sec, 55°C for 30sec, 72°C for 2min, 35 cycles; 72°C for 10min; 4°C forever.

[0035] 3. Take 3 μL of the error-prone PCR product and electrophoresis on 1% agarose gel. It can be seen that there is a product band of about 1 kb. The error-prone PCR product was purifi...

Embodiment 2

[0037] Initial screening of mutant strains:

[0038] Extract the plasmid DNA in the mutant library, digest it with BamH I and Hind III, and recover the target fragment of about 1kb. The vector pGEX-4T-1m is also digested with BamH I and Hind III, recover, and connect the two at 4°C Overnight, transformed into BL-21 strain, spread on LB containing IPTG, fructose valine and N-(carboxymethylaminocarbonyl)-4,4-bis(methylamino)-benzidine [DA-64] Plates, after 12-24 hours of incubation at 37 ° C, obvious discoloration can be seen. The clones with fast discoloration and large discoloration circle were picked for liquid medium culture, and a total of 48 clones were picked.

Embodiment 3

[0040] Quinone method detection of mutant enzyme activity:

[0041] 1. After inducing expression for 4 hours, collect the bacterial liquid, measure and record the OD600 value of the cell culture.

[0042] 2. Prepare bacterial lysate for detecting enzyme activity. In 374 μL B-PER TM Cells were resuspended in bacterial protein extraction reagent (Pierce product78248), and then 50uL of protease and phosphorylase inhibitors and bacterial cell extract (Sigma product P8465), and 1uL of 34mg / mL chloramphenicol (prepared with methanol) were added. Vortex quickly for one minute. Place on ice for 5 min. Centrifuge for 1 min and place on ice.

[0043] 3. Determination of protein content by Bradford method.

[0044] 4. Take 50 μL 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 composition of the reaction mixture is: 100mM potassium phosphate buffer (pH8.0), 1purpurogallin unit / ml p...

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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 invention relates to a high activity fructose valine oxidase. Background technique [0002] Glycosylated hemoglobin in blood (hereinafter referred to as: glycosylated Hb) reflects the blood sugar level in a living body for a period of time, and has been used as an important indicator for the diagnosis and treatment of diabetes in recent years. At present, glycosylated Hb can be determined by methods such as high performance liquid chromatography, microcolumn method, immunoassay, and pigment method, but these methods either require special instruments, are expensive, or take a long time to detect and have poor measurement accuracy. It is detected by redox reaction and does not require special measuring instruments. It is easy to operate, high in precision and short in time, so it is widely used in biochemical analysis and clinical examination. [0003] During the determination of glycosylated Hb using the above redox reaction, a special type of en...

Claims

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Application Information

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