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Method for whole-cell catalysis production of pentanediamine through self-control of pH by virtue of byproduct carbon dioxide

A whole-cell, pentamethylenediamine technology, applied in the biological field, can solve problems such as increasing the dosage of pH regulators, increasing equipment investment, and reducing product purity.

Inactive Publication Date: 2017-11-10
HEILONGJIANG EPPEN NEW MATERIALS LTD
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

[0007] Although the pH regulation and aeration process are theoretically beneficial for the enzymatic reaction to produce 1,5-pentanediamine, in actual production applications, these two process control steps will limit the production of 1,5-pentanediamine. Application of amine whole-cell catalysis in industrial production: the pH regulation process consumes a large amount of acid, which will increase the cost of auxiliary materials; inorganic acids such as hydrochloric acid and sulfuric acid are highly corrosive, and require high equipment materials, which will increase equipment investment; adipic acid Organic acids such as sebacic acid and sebacic acid are difficult to dissolve in water, and they need to be fed in the form of dry powder or prepared into a paste, which will increase the difficulty of the design of the feeding pipeline; the aeration process will lead to a rapid increase in pH, which will increase The amount of pH regulator; the aeration process will lead to partial oxidation of 1,5-pentamethylenediamine, which will reduce the product purity; the gas stripping effect of the aeration process will make the exhaust gas contain carbon dioxide and pentamethylenediamine, which will lead to air Pollution, increase the cost of environmental treatment of enterprises

Method used

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  • Method for whole-cell catalysis production of pentanediamine through self-control of pH by virtue of byproduct carbon dioxide
  • Method for whole-cell catalysis production of pentanediamine through self-control of pH by virtue of byproduct carbon dioxide
  • Method for whole-cell catalysis production of pentanediamine through self-control of pH by virtue of byproduct carbon dioxide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1 HPLC method detects lysine and 1,5-pentanediamine

[0067] Take 1mL of the whole cell catalytic solution, centrifuge at 8000g for 5min, collect the supernatant to detect the lysine content; take 10μL of the supernatant in a 2mL centrifuge tube, add 200μL of 0.5M NaHCO 3 Aqueous solution and 100 μL of 1% (volume ratio) 2,4-dinitrofluorobenzene acetonitrile solution were heated in the dark at 60°C in a water bath for 60 minutes at a constant temperature, then cooled to room temperature, and 700 μL of 0.04mol / L KH was added 2 PO 4 Dilute the aqueous solution (pH=7.2±0.05, adjust the pH with 40g / L KOH aqueous solution) and shake well, let it stand for 15min and filter before injecting, the injection volume is 15μL;

[0068] The chromatographic column used is a C18 column (ZORBAX Eclipse XDB-C18, 4.6*150mm, Agilent, USA); column temperature: 40°C; UV detection wavelength: 360nm; mobile phase A is 0.04mol / L KH 2 PO 4 Aqueous solution (pH=7.2±0.05, adjust pH wi...

Embodiment 2

[0074] Example 2 Construction of Whole Cell Catalytic Production of Pentadiamine Engineering Bacteria

[0075] (1) Construction of lysine decarboxylase gene cadA overexpression plasmid

[0076] The ORF of the optimized lysine decarboxylase gene cadA was inserted behind the T7 promoter and RBS of the pET28a(+) expression vector. Primers were designed with optimally designed sequences, using the genomic DNA of wild-type Escherichia coli K12 W3110 strain as a template, using the high-fidelity polymerase KAPAHiFi TM HotStar uses P1 and P2 as primers to amplify the cadA gene by PCR. The nucleotide sequence of the gene is shown in SEQ ID NO.1. The PCR program is: denaturation at 98°C for 30 seconds, annealing at 65°C for 15 seconds, extension at 72°C for 150 seconds, and 26 cycles. The mutation site is introduced by primer P1, thereby obtaining a fragment of the modified lysine decarboxylase gene cadA*.

[0077] P1: 5'-CATG CCATGG CAGTTATTGCAATATTGAATCATATGGGAGT-3' (SEQ ID NO. 5...

Embodiment 3

[0096] Example 3 Bacterial Culture and Production Technology Catalyzed by 1,5-Pentanediamine

[0097] Scraping Engineering Bacteria E.coli BL21(DE3)P cadB ::P T7 / pET28a-cadA * The bacterial lawn was inserted into a 500mL Erlenmeyer flask containing 50mL LB (50mg / L kanamycin (5-200mg / L is acceptable) medium), and cultured in a shaker at 37°C and 220rpm for 4h to obtain a seed solution, OD 600 4-5; the seed solution obtained by cultivation is inserted into a 10L fermenter containing 2L inorganic salt medium according to the inoculation amount of 2%, the cultivation temperature is 37°C, the DO is controlled above 30%, and the tank pressure is controlled at 0.02-0.10 MPa. The concentration of glucose in the culture medium was maintained below 5 g / L by feeding the feed solution. When the cell OD in the culture medium 600 When it reaches 30-40, add 0.1mM inducer IPTG, and after 2h, the OD of the bacterial culture solution 600 When the temperature reaches about 80, the wet cel...

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Abstract

The invention provides a method for whole-cell catalysis production of 1,5-pentanediamine through self-control of pH by virtue of a byproduct carbon dioxide. Whole-cell catalysis production of the pentanediamine is controlled by the method in which pH of whole-cell catalysis liquid is subjected to self-regulation by virtue of the catalysis byproduct CO2 and non-aeration is adopted. With the application of the pH self-control and non-aeration method provided by the invention, the catalysis production of the pentanediamine can be implemented efficiently, and in addition, adjuvant cost and waste gas processing cost in the production of the pentanediamine can be reduced, a process controlling procedure can be simplified, equipment investment can be reduced and the operability of a production process can be improved. The technique (the method) provided by the invention is practically applicable to large-scale industrial production of the 1,5-pentanediamine; and the technique is quite high in practicability and applicability.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for whole-cell catalytic production of 1,5-pentanediamine by using by-product carbon dioxide to self-control pH. Background technique [0002] 1,5-Pentanediamine (1,5-Pentanediamine), also known as Cadaverine, 1,5-Diaminopentane (1,5-Diaminopentane), and dibasic acid can be polymerized into high molecular polyamide material (i.e. nylon). The global production of about 7 million tons of polyamide materials every year consumes a lot of petrochemical resources. Therefore, the biosynthesis of 1,5-pentanediamine, an important constituent monomer of polyamide, has important economic and ecological significance. [0003] The whole-cell catalysis method uses lysine as a substrate to catalyze the production of 1,5-pentanediamine by lysine decarboxylase in bacterial cells. At present, lysine, as one of the bulk amino acid varieties, has serious excess production capacity and extreme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/00C12R1/19
CPCC12P13/001
Inventor 温廷益刘树文马吉银李戴欢梁勇商秀玲张芸邓爱华郭小炜赵春光
Owner HEILONGJIANG EPPEN NEW MATERIALS LTD
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