Pseudoalteromonas sp.B3 and application thereof in biological oxidation of L-amino acid

A technology of alternating pseudomonas and biological oxidation, applied in the directions of microorganism-based methods, microbial determination/inspection, biochemical equipment and methods, etc. Effect

Active Publication Date: 2013-04-24
菏泽建数智能科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Domestic research on LAAO is only more than ten years old, only LAAO derived from snake venom has been studied, and there is no report on other species. Moreover, there is no domestic use of microbial source LAAO for biooxidation of L-amino acids. see the report

Method used

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  • Pseudoalteromonas sp.B3 and application thereof in biological oxidation of L-amino acid
  • Pseudoalteromonas sp.B3 and application thereof in biological oxidation of L-amino acid
  • Pseudoalteromonas sp.B3 and application thereof in biological oxidation of L-amino acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 strain screening and identification

[0040] a) Strain screening

[0041] Collect sea mud (30.03°N, 122.11°E) from 50cm below the sea level in Dinghai sea area of ​​Ningbo, put it in a sterile sample bottle, then weigh 10g of sea mud sample, add it to 90mL of sterile seawater, mix well That is, the concentration is 10 -1 The samples were serially diluted with sterile seawater to a concentration of 10 -2 、10 -3 、10 -4 、10 -5 、10 -6 100 μL samples of different concentrations were spread on MM solid medium (yeast extract 3g / L, peptone 5g / L, sea salt 30g / L, agar 20g / L; pH 7.2, solvent is water) plate, in Cultivate at 28°C for 4 days to obtain a single colony; then place the single colony obtained by primary screening on MM solid medium (yeast extract 3g / L, peptone 5g / L, sea salt 30g / L, agar 20g / L; pH 7.2, solvent is Water) on the plate, carry out twice streak re-screening; then inoculate the single colonies obtained after re-screening into 5mL MM liquid med...

Embodiment 2

[0054] Example 2 Verification of Pseudomonas alternata B3 Biooxidation of L-Amino Acids——Detection of Ketoacids

[0055] Inoculate a single colony of strain B3 into 5 mL of MM liquid medium (yeast extract 3g / L, peptone 5g / L, sea salt 30g / L; pH 7.2, solvent is water), shake at 28°C and 200rpm After bed culture for 4 days, centrifuge at 8000 rpm for 5 minutes to collect the fermentation supernatant to obtain the enzyme source;

[0056] The reaction is divided into 4 groups: group 1 is the positive control group (directly react with α-phenylpyruvate and 2,4-dinitrophenylhydrazine), group 2 is the experimental group, and group 3 is the no-substrate group (without adding the bottom). Substance L-amino acid, other operations are the same as the experimental group), group 4 is the enzyme inactivation group (the supernatant containing the enzyme is inactivated at high temperature, other operations are the same as the experimental group);

[0057] The operation of group 1 is: 1.5 mL o...

Embodiment 3

[0059] Example 3 Verification of Pseudomonas alternata B3 Biological Oxidation of L-Amino Acid——Detection of Ammonia

[0060] The experiment was divided into 3 groups, the experimental group, the positive control group and the negative control group.

[0061] Positive control group: Add 3 mL of ammonia water with a volume concentration of 4% to 47 mL of uncultured MM liquid medium, store at room temperature for 30 min, add 0.5 mL of 1 M NaOH aqueous solution, and react for 1 min, the color of the pH test strip changes from yellow ( nature) turns green (alkaline);

[0062] Negative control group: 50mL uncultured MM liquid medium as enzyme solution, other operations are the same as the experimental group;

[0063] Experimental group: collect the supernatant according to the method in Example 2, get 50mL of Pseudomonas alternata B3 supernatant (wet cell concentration: 0.1mg wet cell / mmol substrate) as enzyme liquid, and pack into 250mL Erlenmeyer flask, after reacting with 50mL...

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Abstract

The invention discloses pseudoalteromonas sp.B3 and application thereof to biological oxidation of L-amino acid. During the application of the pseudoalteromonas sp.B3, an enzyme cultured by fermenting the pseudoalteromonas sp.B3 is taken as an enzyme source, the L-amino acid is taken as a substrate, and the enzyme source and the substrate react for 0.5-2h at a temperature of 25-50 DEG C and the pH value of 6-8 so as to oxidize the L-amino acid and release hydrogen peroxide. The invention provides a novel microbial strain for producing L-amino acid oxidase, and the strain can be use for biologically oxidizing the L-amino acid, has the characteristics of wide substrate spectrum, mild reaction, environment friendliness and the like, and has a wide application prospect in the aspects of quantitative analysis of the L-amino acid, splitting of the DL-amino acid and the like.

Description

(1) Technical field [0001] The invention relates to a new bacterial strain producing L-amino acid oxidase, in particular to Pseudomonas alternata B3 and its application in biological oxidation of L-amino acid. (2) Background technology [0002] L-amino acid oxidase (LAAO for short, enzymatic number: EC1.4.3.2) is a flavoproteinase with flavin adenine dinucleotide (FAD) as the prosthetic group. This enzyme can specifically catalyze the oxidative deamination of L-amino acids to generate α-keto acids, ammonia and hydrogen peroxide. Therefore, it can be applied to the biooxidation of L-amino acids. In recent years, it has been reported that LAAO has the functions of interacting with platelets, cytotoxicity and inducing apoptosis. It is also reported that it has hemorrhagic or hemolytic activity, causes edema, antibacterial, and anti-AIDS virus (Zhang Yun et al. Application of snake venom L-amino acid oxidase in the preparation of AIDS treatment drugs. Application number: 03117...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P41/00C12Q1/26C12R1/01
Inventor 余志良乔华裘娟萍
Owner 菏泽建数智能科技有限公司
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