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Animal waste metagenome-derived alanine racemase as well as preparation and application thereof

A technology of alanine racemase and metagenomics, which is applied in the field of alanine racemase and its preparation and application, can solve the problems of non-culturable microorganisms, limit the extensiveness, effectiveness and safety of screening, and achieve expansion The effect of using space

Active Publication Date: 2021-07-13
YUNNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the research on alanine racemase is mainly to directly screen the strains producing alanine racemase from environmental samples, then extract the genomic DNA of a single strain, and design merger primers to clone the alanine racemase gene. The vast majority of microorganisms are non-culturable microorganisms, so the screening of new ALRs by traditional isolation and culture methods greatly limits the breadth, effectiveness and safety of screening.

Method used

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  • Animal waste metagenome-derived alanine racemase as well as preparation and application thereof
  • Animal waste metagenome-derived alanine racemase as well as preparation and application thereof
  • Animal waste metagenome-derived alanine racemase as well as preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0037] Experimental example 1 Acquisition of alanine racemase gene NC Alr1

[0038] (1) Screening of alanine racemase genes in gibbon fecal microbial metagenomics

[0039] According to the results of gene prediction, functional annotation and secreted protein prediction analysis from the constructed gibbon fecal microbial library, the genes annotated as alanine racemase were screened to obtain the alanine racemase gene NC Alr1. The gene sequence is as follows: Shown in SEQ ID NO.1.

[0040] (2) Cloning of alanine racemase gene NC Alr1

[0041] Take NC Alr 1-F 1 : 5'-TAAGAAGGAGATATACATATGGAATTGATGGATTCAACATTAAAGCGG-3' (SEQ ID NO.3) and NC Alr 1-R 1: 5'-GTGGTGGTGGTG GTGCTCGAGCCCCCTTAAAAGAAGCTC-3' (SEQ ID NO.4) is a primer pair, and PCR amplification is performed using the fecal microbial metagenomic DNA of the western black crested gibbon as a template.

[0042] PCR reaction system (20.0 μL): metagenomic library DNA 0.5 μL, PrimeSTAR Max 10 μL, NCAlr1-F 1 0.5 μL, NCAlr 1-R ...

experiment example 2

[0045] Experimental example 2 Preparation of alanine racemase NC ALR1

[0046] The alanine racemase gene NCAlr1 prepared in Example 1 was connected to the plasmid pEASY-E2 to obtain the recombinant expression vector pEASY-E2-NCAlr1, and then transformed into E. coli BL21 (DE3) to obtain the recombinant E. coli strain BL21 (DE3) / NCAlr1. Take the Escherichia coli strain BL21(DE3) / NC Alr1 containing the recombinant expression vector pEasy-E2-NCAlr1, and inoculate it in LB (containing 100 μg / mL Amp) culture medium with a 0.1% inoculum size, and cultivate overnight at 37° C. at 180 rpm. Then inoculate the activated bacterial solution into fresh LB (containing 100 μg / mL Amp) culture solution with 1% inoculum amount, and cultivate it at 37° C. at 180 rpm for about 5 to 6 hours (OD 600 After reaching 0.8-1.0), add IPTG with a final concentration of 0.7mmol / L to induce, and culture at 16°C and 180rpm for about 16h. Centrifuge at 5000rpm for 10min to collect the bacteria. After suspen...

experiment example 3

[0048] Experimental example 3 Determination of properties of alanine racemase NC ALR 1

[0049] Enzyme activity determination method refers to Soda K (Microdetermination of D-amino acids and D-amino acid oxidase activity with 3,methyl-2-benzothiazolone hydrazonehydrochloride, 1968) and Lida F et al. (Electrochemical Study of Iodide in the Presence of Phenol and o- Cresol: Application to the Catalytic Determination of Phenol and o-Cresol, 2004): The activity of ALR was determined by a two-step method of racemization reaction and oxidation reaction.

[0050] Racemization reaction: 200 μL reaction system contains 20 mmol / L Beritan-Robinson buffer, 50 mmol / L L-alanine, 0-100 μmol / LPLP (pyridoxal phosphate). Preheat at the optimum temperature for 5 minutes, add an appropriate amount of crude enzyme solution or purified enzyme protein (use boiled inactivated enzyme solution as blank control) to react for 10 minutes, immediately add 25 μL 2mol / L HCl to stop the reaction, and place on...

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Abstract

The invention discloses animal waste metagenome-derived alanine racemase as well as preparation and application thereof. The amino acid sequence is shown as SEQ ID NO.2, and after the alanine racemase is treated by a buffer solution with the pH value of 9.0-12.0 for 1 hour, the residual enzyme activity is 90% or above; when the alanine racemase is tolerant for 1 hour under the conditions of 30 DEG C, 37 DEG C, 40 DEG C and 45 DEG C, 90% of enzyme activity is still kept; the optimal cofactor concentration is 10 mumol / L, and 77% of relative activity is still maintained after the reaction is carried out for 10 min under the condition that no exogenous cofactor is added. Km and Vmax of the enzyme are 14.81 mmol / L and 89.06 mumol / L / min respectively under the conditions that the pH is 12.0 and the temperature is 40 DEG C NaCl, and the enzyme has thermal stability and alkali resistance, still has higher activity without adding exogenous cofactors, and has potential application value in the aspects of synthesizing D-amino acid by an enzyme method and screening novel antibacterial drugs.

Description

technical field [0001] The invention relates to an alanine racemase, in particular to an alanine racemase derived from animal feces metagenome and its preparation and application. Background technique [0002] Alanine racemase (ALR, EC5.1.1.1) is an enzyme that uses pyridoxal phosphate (PLP) as a cofactor to catalyze the interconversion of L- and D-alanine. In prokaryotes and some eukaryotes, it is closely related to diseases caused by bacteria. Studies have shown that ALR exists in many pathogenic bacteria, such as Mycobacterium tuberculosis, Streptococcus mutans, Bacillus pseudofirmus and so on. The activity of ALR determines the amount of D-alanine in bacteria, that is, controls the synthesis of bacterial cell walls, and then affects the survival of pathogenic bacteria. Therefore, ALR has become an important target for screening new antibacterial drugs. [0003] In addition, ALR is also one of the key enzymes for enzymatically synthesizing D-amino acids, and its racemiz...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12N15/61C12N15/70C12N1/21C12P13/06C12R1/19
CPCC12N9/90C12N15/70C12P13/06C12Y501/01001Y02A50/30
Inventor 许波杨金茹黄遵锡韩楠玉
Owner YUNNAN NORMAL UNIV
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