N-carbamyl-L-cysteine (L-NCC) amidohydrolase, encoding gene and application of recombinant expressed protein of L-NCC amidohydrolase

A technology of amidohydrolase and protein, applied in the fields of hydrolase, application, genetic engineering, etc., can solve the problems of few reports on L-cysteine-related enzymes, achieve good industrialization value, high catalytic efficiency, separation The effect of easy purification

Active Publication Date: 2012-04-11
天津世纪伟康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are few reports about enzymatic synthesis of L-cysteine-related enzymes

Method used

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  • N-carbamyl-L-cysteine (L-NCC) amidohydrolase, encoding gene and application of recombinant expressed protein of L-NCC amidohydrolase
  • N-carbamyl-L-cysteine (L-NCC) amidohydrolase, encoding gene and application of recombinant expressed protein of L-NCC amidohydrolase
  • N-carbamyl-L-cysteine (L-NCC) amidohydrolase, encoding gene and application of recombinant expressed protein of L-NCC amidohydrolase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Enzyme activity determination method of L-NCC amidohydrolase

[0026] (1) Principle: The acidic ninhydrin method is adopted. L-NCC amidohydrolase decomposes L-NCC to generate L-cysteine, and L-cysteine ​​reacts with acidic ninhydrin reagent under boiling conditions to generate a red substance, which has a maximum absorption at 560nm, and its color depth is the same as The amount of L-cysteine ​​is directly proportional.

[0027] (2) Method: accurately add 6% K to the solution of 1% L-NCC 2 HPO 4 Prepare a solution with a final substrate concentration of 0.75% until the pH value is 7.5, then take 1 mL of the substrate solution, add 0.5 mL of enzyme solution, and react in a 37°C water bath for 10 min.

[0028] The determination of L-cysteine ​​content in the reaction solution adopts the acidic ninhydrin method. Take 0.2mL of the reaction solution, add 0.2mL glacial acetic acid, and then add 0.2mL acidic ninhydrin reagent (weigh 250mg ninhydrin, dissolve in 6mL A mixtu...

Embodiment 2

[0032] Cloning and Primary Structure Characterization of L-NCC Amidohydrolase

[0033] The present inventor extracted genomic DNA from Pseudomonas sp.QR-101, then digested genomic DNA with HindIII, and recovered 2-9kb HindIII digests from 1.2% agarose gel with a gel recovery kit. Fragments, the recovered enzyme-digested fragments were connected to the Pucl8 vector that had been treated by the same enzyme digestion, and then transformed into E.coli JM109, and blue-white primary screening was performed on a plate coated with X-gal and IPTG.

[0034] The above-mentioned recombinant white single colony containing the insert fragment was picked into each well containing 50 μL LB medium (peptone: 10g / L, yeast powder: 5g / L, sodium chloride: 10g / L), and after overnight culture at 37°C, Add 100 μL of 0.75% L-NCC as a substrate, shake at 35° C. for 30 min, and add acidic ninhydrin reagent for color reaction.

[0035] The recombinants with high activity were re-screened through the micr...

Embodiment 3

[0037] Construction of Escherichia coli Genetic Engineering Bacteria and Expression of Recombinant L-NCC Amidehydrolase

[0038] According to the sequencing results of Example 2, primers P1 and P2 were designed according to the sequences at both ends of the protein-coding gene, wherein upstream P1: 5'-CCG GAA TTC ATG AGT GGA GTC AAC AGC ATG AA-3' contains an EcoRI restriction site; Downstream primer P2: 5'-CCC AAG CTT TCA GCC GGT GCG GCT GTC CGC CA-3' contains a HindIII restriction site.

[0039] Using the genome of strain Pseudomonas sp.QR-101 as a template, DNA amplification was performed according to the following PCR procedure:

[0040] Denaturation at 94°C for 1 min, renaturation at 66°C for 1 min, extension at 72°C for 1 min, and 30 cycles of amplification reaction.

[0041] The PCR amplified product was double digested with EcoR I and HindIII, and connected to the vector pET21a(+) after the same digestion to construct the recombinant pET-21a(+) / atcC.

[0042] The obta...

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Abstract

The invention discloses a gene sequence of N-carbamyl-L-cysteine (L-NCC) amidohydrolase, and a preparation and an application of a recombinant expressed protein of the L-NCC amidohydrolase. The L-NCC amidohydrolase derived from pseudomonad with preservation number of CGMCC (China General Microbiological Culture Collection) No.5315 is an amino acid residue sequence in SEQ ID No.1 in a sequence table, or is a protein which is derived from SEQ ID No.1 by substituting, deleting or adding one or more amino acid residues to the amino acid residue sequence in SEQ ID No.1 and has the same activity asSEQ ID No.1. According to the invention, the pseudomonad-derived L-NCC amidohydrolase is subjected to gene cloning and expression to prepare a recombinant enzyme, and the obtained recombinant enzyme can be used for biological hydrolysis of L-NCC to obtain L-cysteine, thus the L-NCC amidohydrolase can be used for enzymatic production of L-cysteine.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a coding gene of L-NCC amidohydrolase derived from pseudomonas, and expression and application of recombinant protein. Background technique [0002] N-carbamyl-L-cysteine ​​amidohydrolase (N-carbomyl-L-cysteine ​​amidohydrolase), that is, L-NCC amidohydrolase, is a catalytic N-carbamyl-L-cysteine (N-carbamyl-L-cysteine, L-NCC) is an enzyme that hydrolyzes to generate L-cysteine. Because ATC racemase catalyzes the resolution of DL-ATC to generate L-ATC; L-ATC hydrolase hydrolyzes the C-S single bond in the thiazole ring of ATC to generate the intermediate product L-NCC; -cysteine, so L-NCC amidohydrolase is one of the important members involved in the enzymatic conversion of DL-ATC to produce L-cysteine. For the specific process, see the appendix figure 1 . [0003] L-cysteine ​​(L-cysteine) is one of the 20 amino acids that make up proteins. It is an α-amino acid. Its existence can ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/80C12N15/55C12N15/63C12N15/70C12N1/21C12P13/12C12R1/38
Inventor 高智慧刘磊姚瑞娟王文芳
Owner 天津世纪伟康生物科技有限公司
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