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Sequence, preparation method and application of catalytic synthesis of linear alpha-olefin biocatalyst olet-bm3r

A biocatalyst, olet-bm3r technology, applied in the field of protein engineering, can solve the problems of severe reaction system conditions, low yield of enzyme reaction system, complex reaction system, etc., and achieve the effects of simplified reaction system, low energy consumption, and reduced production cost.

Active Publication Date: 2021-10-22
YANGZHOU UNIV
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Problems solved by technology

[0006] In order to reduce the dependence of the synthesis of linear α-olefins on the petroleum industry, to make up for the shortcomings of existing industrial methods that cannot produce linear α-olefins with odd carbon chain lengths, and to overcome the high energy consumption, severe reaction system conditions, and high energy consumption of linear α-olefins based on metal catalysts To produce wastes that are not conducive to the environment, and to solve the problems of low yield, narrow substrate range, and complex reaction system of the existing enzyme reaction system, the first purpose of the present invention is to provide a catalyst with high catalytic efficiency and wide substrate range. , the biocatalyst fatty acid decarboxylase OleT-BM3R that catalyzes the synthesis of linear α-olefins with a simpler reaction system

Method used

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  • Sequence, preparation method and application of catalytic synthesis of linear alpha-olefin biocatalyst olet-bm3r

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Experimental program
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Effect test

Embodiment 1

[0030] The construction of the gene expression vector of embodiment 1 OleT-BM3R

[0031] The OleT-BM3R fragment (sequence shown in SEQ ID NO: 2, synthesized by Suzhou Jinweizhi Biological Co., Ltd.) synthesized by the whole gene was subjected to double enzymes by restriction enzymes NdeI and XhoI (New England Biolabs) according to the instructions. After cutting, T4 ligase (New England Biolabs) was used to connect to the expression vector pET28a that had been digested by NdeI and XhoI. The ligation product was used to transform Escherichia coli DH5α competent cells (Tiangen Biochemical Technology Co., Ltd.). Pick the successfully transformed monoclonal colony from the solid LB medium plate containing 50 μg / ml kanamycin, in the LB liquid medium containing the same concentration of kanamycin, at a temperature of 37°C, and the shaker speed is 220rpm cultured overnight under conditions. The recombinant plasmid was extracted from the cultured bacterial liquid with a small plasmid...

Embodiment 2

[0032] Example 2 Preparation of OleT-BM3R

[0033] (1) Transform the recombinant plasmid successfully sequenced into Escherichia coli Rosetta (DE3) (Tiangen Biochemical Technology Co., Ltd.), pick the positive clone into 5ml LB liquid containing 50μg / ml kanamycin and 34μg / ml chloramphenicol After culturing overnight in the medium, add the bacterial solution to 500ml TB liquid medium containing 50μg / ml kanamycin and 34μg / ml chloramphenicol at a ratio of 1:100, and carry out expansion cultivation at 37°C with a shaker speed of 220rpm . Waiting for OD 600 At about 0.6, the heme precursor δ-aminolevulinic acid and 0.1 mM IPTG were added at a final concentration of 0.5 mM for induction. After expressing for 20 hours at 22°C with a shaker speed of 220 rpm, the bacteria were harvested by centrifugation at a speed of 5000 rpm for 10 minutes. After discarding the supernatant medium, resuspend the bacteria with 50ml of buffer A (0.1M KPi potassium phosphate, 0.3MKCl, pH7.0), lyse the...

Embodiment 3

[0035] Example 3 Using OleT-BM3R freeze-dried powder combined with PTDH-based NADPH regeneration system to catalyze the synthesis of corresponding alpha-olefins from natural saturated fatty acids

[0036] Reaction system: buffer A, OleT-BM3R (3 μ M), fatty acid (1 mM) (fatty acid C4-C11 co-solvent: 5% EtOH (v / v), fatty acid C12-C20 co-solvent: 5% EtOH (v / v) and 1.5% Triton X-100 (v / v)), catalase (100U mL -1 ), sodium phosphite (10mM), PTDH phosphite dehydrogenase (2μM), NADPH (200μM), room temperature, shaker speed 125rpm, react for 12 hours. Yield of olefins synthesized by decarboxylation of natural fatty acids: C4: 14%, C5: 11%, C6: 68%, C7: 70%, C8: 40%, C9: 47%, C10: 70%, C11: 58%, C12: 46%, C14: 52%, C16: 60%, C18: 73%, C20: 70%.

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Abstract

The invention belongs to the field of protein engineering, and relates to the sequence, preparation method and application of a biocatalyst OleT-BM3R, which uses fatty acids to catalyze the synthesis of linear α-alkenes. The biocatalyst OleT‑BM3R is a salty sea coccus ( Jeotgalicoccus sp.ATCC 8456) fatty acid decarboxylase OleT JE with Bacillus megaterium ( Bacillus megaterium ) The reductase domain fusion of fatty acid hydroxylase CYP102A1 (P450BM3). The linear α-olefin synthase OleT-BM3R provided in the present invention uses fatty acids as substrates to achieve decarboxylation in one step. The advantages are as follows: 1) The reaction raw materials are renewable and easily available fatty acids, which can reduce the synthesis of linear α-olefins Dependence on the petroleum industry; 2) The reaction is carried out in an environmentally friendly aqueous solution at room temperature, with mild reaction conditions and low energy consumption; 3) The reaction has high catalytic efficiency, good stability, a wide range of substrates, and a simple reaction system. Therefore, it has a good application prospect in the preparation of linear α-olefins.

Description

technical field [0001] The invention belongs to the field of protein engineering. Through protein engineering transformation, an enzyme can effectively utilize various carboxylic acids to synthesize corresponding linear α-alkenes. Specifically, it relates to a biocatalyst OleT-BM3R that catalyzes the synthesis of linear α-alkenes. Sequence, preparation method and its application. Background technique [0002] Linear ɑ-olefins (LAOs) are ideal next-generation energy substances and are important industrial raw materials for the synthesis of surfactants, lubricating oils, detergents, additives, and polymers. Therefore, the application of LAOs has broad market prospects. At present, LAOs are mainly polymerized from ethylene, a traditional petroleum industry product. The olefins obtained by this method are all even-numbered carbons long, which makes the price of olefins with odd-numbered carbons extremely expensive. Furthermore, with the dwindling fossil energy reserves, it is ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N9/02C12N15/62C12N15/70C12N15/66C12P5/02
CPCC07K2319/00C12N9/0071C12N9/88C12N15/66C12N15/70C12P5/026
Inventor 王喜庆沈枫林陆晨王帅博陈浩
Owner YANGZHOU UNIV
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