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Novel proline 3-hydroxylase and application thereof

A proline, hydroxylase technology, applied in the biological field, can solve the problems of low industrial production, low selectivity and the like

Active Publication Date: 2020-02-18
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the low enzymatic activity of GloF and HtyE and the low selectivity of trans-3-hydroxy-L-proline, the current research on this enzyme is limited to the analytical scale to catalyze very low concentrations of substrates, and has not been used for the reaction. Formula-3-Hydroxy-L-proline preparation, so the possibility of industrial production of trans-3-hydroxy-L-proline using this enzyme is very low

Method used

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  • Novel proline 3-hydroxylase and application thereof
  • Novel proline 3-hydroxylase and application thereof
  • Novel proline 3-hydroxylase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1. Synthesis and clone expression of proline 3-hydroxylase

[0064] First, the inventor synthesized the genes shown in the sequences SEQ ID NO: 1 and SEQ ID NO: 3 through the whole gene (the encoded amino acid sequences are SEQ ID NO: 2 and 4, respectively), and respectively adopted the Nde I inner Dicer site, the 3' end was connected to the Gaokaobei plasmid pET21a using the Hind III endonuclease site, and the obtained recombinant plasmids were named pET21a-Ubp4h and pET21a-Amp3h, and then the recombinant plasmids pET21a-Ubp4h, pET21a-Ubp4h, pET21a-Amp3h was introduced into Escherichia coli BL21a(DE3) competent cells to obtain recombinant Escherichia coli BL21a(DE3)(pET21a-Ubp4h) and BL21a(DE3)(pET21a-Amp3h). Proline 3-hydroxylase was induced to express as follows.

[0065] Take 5 μl of glycerol bacteria solution and inoculate 5ml of LB medium (10g / l peptone, 10g / l NaCl, 5g / l yeast extract) containing antibiotics (100μg / ml ampicillin), and culture overnight...

Embodiment 2

[0066] Embodiment 2. Enzyme activity assay of proline 3-hydroxylase

[0067] The cultured BL21a(DE3)(pET21a-Ubp4h) and BL21a(DE3)(pET21a-Amp3h) cells were centrifuged at 4°C and 7000rpm for 6min to collect the cells, and then washed with 100mM Tris-HCl, 100mM NaCl, 10% glycerol , pH 7.4 buffer solution to wash the cells three times, break up immediately or store in a -80°C refrigerator for later use. The collected cells were resuspended with an appropriate amount of buffer solution of 50 mM MES, 100 mM NaCl, 10% glycerol, pH 6.5 and then sonicated. The cell lysate was centrifuged at 4°C and 8000rpm for 35min, then the supernatant was transferred to a pre-cooled centrifuge tube, and then His SpinTrap was used to TM columns (manufactured by GE Healthcare) nickel column to purify the protein and elute the impurity protein with concentration gradient imidazole, after removing the imidazole to obtain the pure enzyme of proline-3-hydroxylase, aliquot into pre-cooled small centrifug...

Embodiment 3

[0074] Embodiment 3. Catalyzed production of trans-3-hydroxyl-L-proline with L-proline as substrate

[0075] First, as described in Example 1, the strain BL21a(DE3)(pET21a-Ubp4h) was cultured and induced to express in shake flasks, and the above-mentioned cultured cells were centrifuged at 4°C and 7000rpm for 6min to collect the cells, and then 100mM Tris- Wash the cells three times with HCl, 100mM NaCl, 10% glycerol, pH 7.4 buffer and collect the cells. Suspend the collected cells with 10ml reaction buffer / 100ml shaker flask and culture them in a shaker at 30°C for 24h. The reaction buffer contains 50mM ES (pH 6.5), 0.5mM FeSO 4 , 1.5mM ascorbic acid, 20mM L-Pro, 14mM α-ketoglutarate. After the reaction, centrifuge at 4°C and 7000rpm for 7 minutes to separate the supernatant, and transfer the supernatant to a clean centrifuge tube for detection of the production of trans-3-hydroxyl-L-proline. The blank used in this example The control strain was BL21a(DE3)(pET21a). Detect ...

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Abstract

The invention discloses a novel proline 3-hydroxylase. The proline 3-hydroxylase has an amino acid sequence as shown in SEQ ID NO:2 or SEQ ID NO:4. The invention also discloses an expression vector and a host cell containing the enzyme or a coding gene thereof as well as applications of the expression vector and the host cell in production of trans-3-hydroxy-L-proline. The proline 3-hydroxylase disclosed by the invention has high catalytic performance, so that the trans-3-hydroxy-L-proline can be produced by taking L-proline or glucose as a substrate, the production cost is greatly reduced, and the industrial production of the trans-3-hydroxy-L-proline is further realized.

Description

technical field [0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to a novel proline 3-hydroxylase, host cells containing the enzyme or its coding gene and their application in the production of trans-3-hydroxyl-L-proline. Background technique [0002] Trans-3-hydroxy-L-proline is a naturally occurring non-protein amino acid and an important intermediate in organic synthesis. Its structure is as follows: [0003] [0004] Trans-3-hydroxy-L-proline was first isolated from the hydrolyzate of Mediterranean Sponge in 1968 (Sheehan et al.1968), which is some biologically active chemicals, such as cyclothialidine, mucrorin-D, telomycin , an important component of polyhydroxylated alkaloids and a chiral building block (Sinha et al.2005). Houwaart et al. also found trans-3-hydroxy-L-proline in the antifungal substance pneumocandins, which has been used clinically in the treatment of antifungal infections (L.Houwaart...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12P13/24
CPCC12N9/0071C12P13/24C12Y114/11028
Inventor 孙际宾赵晶刘超周文娟郑平郭轩刘娇马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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