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A kind of leucine-5-hydroxylase mutant and its application

A leucine and mutant technology, applied in the fields of genetic engineering and enzyme engineering, can solve the problems of many by-products, complex process routes, harsh reaction conditions, etc.

Active Publication Date: 2021-05-07
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many disadvantages in the chemical process to realize the hydroxylation of the distal carbon position of amino acid, including complex process route, harsh reaction conditions, many by-products, low yield, etc.

Method used

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  • A kind of leucine-5-hydroxylase mutant and its application
  • A kind of leucine-5-hydroxylase mutant and its application
  • A kind of leucine-5-hydroxylase mutant and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1. Obtaining of the gene mLEH encoding the leucine-5-hydroxylase mutant V77A

[0051] Obtaining mutant V77A: the gene of wild-type leucine-5-hydroxylase derived from Nostoc sp. was codon-optimized (SEQ ID NO.3) and constructed in pET28a(+) expression vector (wild-type leucine- Genbank sequence number RCJ32143.1 of 5-hydroxylase) to obtain LEH-pET28a; using LEH-pET28a plasmid as template, V77A_F (SEQ ID NO.5), V77A_R (SEQ ID NO.6) as primers, reverse PCR site-directed mutagenesis; the experiment in this example uses the KOD-Plus mutant kit (purchased from Toyobo (Shanghai) Biotechnology Co., Ltd.):

[0052] (1) The PCR reaction system is as follows:

[0053] Template (53ng / μL) 1μL Primer F (10pmol / μL) 1.5μL Primer R (10pmol / μL) 1.5μL 2mM dNTPs 5μL 10×Buffer for iPCR 5μL KOD-Plus 1μL wxya 2 o

35μL

[0054] PCR reaction conditions: pre-denaturation at 94°C for 2min; denaturation at 98°C for 10sec; exte...

Embodiment 2

[0061] Embodiment 2, expression of leucine-5-hydroxylase

[0062] The recombinant vectors LEH-pET28a and mLEH-pET28a obtained by constructing the wild-type coding gene LEH and the mutant coding gene mLEH respectively on the pET28a(+) expression vector were transformed into Escherichia coli BL21(DE3), and BL21 / LEH and BL21 / V77A recombinant bacteria.

[0063] The above two recombinant bacteria were respectively inoculated in 5 mL LB medium (containing 50 μg / mL kanamycin), and cultured at 220 r / min at 37 ° C for 12 h; Kanamycin), to be bacteria concentration OD 600 =0.6-0.8, add IPTG, the final concentration is 0.75mM, induce at 16°C, 180r / min for 20h.

Embodiment 3

[0064] Example 3, Purification and Refining of Leucine-5-Hydroxylase

[0065] The bacterium solution obtained in Example 2 was centrifuged at 5000r / min and 15min to collect the thalline, and after resuspending with solution A (20mM Tris-HCl, pH 8.0, 300mM NaCl, 20mM imidazole, 1.5mM DTT), add lysozyme ( The final solubility is 200μg / mL), protease inhibitor (final solubility is 1mM) placed on ice for 30min, sonicated on ice (3s on, 5s off, 350W power), low temperature and high-speed centrifugation (4°C, 18000r / min) Cell debris was removed to obtain the supernatant.

[0066] Ni affinity chromatography: take 4 open columns (Open-Column), and add 1 mL Ni-NTA resin (QIAGEN) to each. Equilibrate the resin with 20 mL of Solution A. The high-speed centrifuged supernatant was combined with 1 mL of resin at 4°C for 40-60 min. Pass the mixture through an open column, and the resin bound to the protein will be retained. Rinse the resin with 20 mL of solution A. Finally, the protein w...

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Abstract

The invention belongs to the technical field of genetic engineering and enzyme engineering, and in particular relates to a leucine-5-hydroxylase mutant with improved activity and an application thereof. The present invention obtains the leucine-5-hydroxylase encoding gene derived from Nostoc punctiforme NIES-2108 by means of molecular biology, adopts inverse PCR to carry out site-directed mutation to obtain the gene encoding the mutant, and then uses the encoding mutant The leucine-5-hydroxylase mutant V77A was obtained by heterologous expression of the gene. When the mutant used leucine and methionine as substrates, its relative enzyme activity was increased by 69.53% and 23.26%, respectively, compared with the wild type. The variant with improved activity screened out by the present invention can more efficiently realize the hydroxylation of the 5th carbon position at the far end of leucine, and can catalyze the generation of methionine sulfoxide from methionine, which is the enzymatic catalytic production of industrial pharmaceutical intermediates Provide broad prospects.

Description

Technical field: [0001] The invention belongs to the technical fields of genetic engineering and enzyme engineering, and in particular relates to a leucine-5-hydroxylase mutant with improved activity and its application. Background technique: [0002] Hydroxylated amino acids are widely used in the synthesis of multifunctional biomacromolecules, pharmaceuticals and fine chemicals because they can be used as chiral precursors, intermediates and final products in the synthesis of various chemical materials and medical drugs. In addition to being used as intermediates in drug synthesis in the pharmaceutical field, hydroxyamino acids are also widely used in many fields such as feed additives, food fortifiers, spices, etc. For example, L-β-hydroxyalanine is not only used in amino acid diet drinks and In addition to sports drinks, it can also be used as an animal feed additive; L-hydroxyproline is often used as a nutritional enhancer and flavor enhancer in fruit juices, nutritiona...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12P13/04C12R1/19
CPCC12N9/0071C12N15/70C12P13/04C12Y114/11
Inventor 秦慧民路福平孙登岳李玉毛淑红
Owner TIANJIN UNIV OF SCI & TECH
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