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Method for producing pentanediamine through fermentation and method for extracting pentanediamine

A technology of pentamethylenediamine and extractant, which is applied in the field of bio-fermentation engineering, can solve the problems such as the decrease of catalytic efficiency of lysine sulfate, and achieve the effect of high added value and high catalytic efficiency

Active Publication Date: 2019-03-01
HEILONGJIANG EPPEN NEW MATERIALS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although various salts of lysine (such as hydrochloride and sulfate) in theory do not have a substantial impact on this fermentation, the inventors have found that using lysine hydrochloride as a substrate with There are many differences in the fermentative production of lysine sulfate as the substrate, including the catalytic efficiency of the enzymes used in the above technologies has a certain degree of decline for lysine sulfate, and the production of solid waste will cause environmental pressure

Method used

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  • Method for producing pentanediamine through fermentation and method for extracting pentanediamine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Material testing methods and instruments

[0065] The following methods and instruments are used to detect the content and purity of 1,5-pentanediamine and the performance indicators of nylon 56.

[0066] (1) Analysis of 1,5-pentanediamine content (unit: g / L)

[0067] Chromatographic column: American Eclipse XDB-C18column (4.6×150nm; Agilent Technologies, USA) column.

[0068] Detector: secondary array detector DAD (detection wavelength 360nm, reference wavelength 400nm).

[0069] Mobile phase: A: pH 7.2 potassium dihydrogen phosphate aqueous solution; B: 66% acetonitrile aqueous solution

[0070] Gradient ratio A: B=5%: 95%

[0071] Flow rate: 1.0ml / min

[0072] Column temperature: 40℃

[0073] Injection volume: 15.0μL

[0074] Detection method: using 2,4-dinitrofluorobenzene (DNFB) HPLC pre-column derivatization method.

[0075] (2) Purity analysis of 1,5-pentanediamine (unit: wt%)

[0076] Instrument: Shimadzu GC-2010 gas chromatograph

[0077] Chromatographic column: Quartz...

Embodiment 2

[0094] Example 2 Construction of engineering bacteria

[0095] With reference to the detailed description of Chinese Patent Application No. 201610322421.5, an engineered bacteria producing 1,5-pentanediamine was constructed and named E. coli BL21 (DE3) P cadB :: P T7 / pet28a- cadA * , Wherein the amino acid sequence of lysine decarboxylase is shown in SEQ ID NO: 2, and the nucleotide sequence of the coding gene is shown in SEQ ID NO: 1.

[0096] In addition, based on our preliminary screening of alanine, we found a lysine decarboxylase that is more suitable for catalyzing lysine sulfate. Its amino acid sequence is shown in SEQ ID NO: 4, with an A at position 315. For amino acid mutation, the nucleotide sequence of the coding gene is shown in SEQ ID NO:3. Basically referring to the above method, the only difference is that the mutant gene was substituted to construct an engineered bacteria producing 1,5-pentanediamine, named E. coli BL21 (DE3) P cadB :: P T7 / pet28a- cadA *3...

Embodiment 3

[0097] Example 3 Catalytic production based on lysine sulfate

[0098] Take the two engineering bacteria constructed in Example 2 respectively, refer to the detailed description of Chinese Patent Application No. 201610322421.5, and obtain OD by seed solution culture, fed-feed culture and IPTG induction culture. 600 The cell culture solution that reached about 80 was centrifuged to obtain the respective wet cells.

[0099] Prepare a catalytic liquid system containing 208 g / L lysine sulfate and 0.2 mmol / L pyridoxal phosphate (PLP), adjust the temperature to 37°C, set the fermenter stirring speed to 500 rpm, and add 20 g / L respectively The above-mentioned two kinds of wet bacteria (equivalent to the dry cell weight 4 g / L) start whole-cell catalysis. No acidic substances are added to adjust pH, and no air is passed through, using by-product CO 2 Adjust the pH of the self-regulating catalytic system. Take out the catalytic liquid from the fermenter every 1h, 12000 × g Centrifuge for 5 ...

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Abstract

The invention belongs to the field of biological fermentation engineering, and provides a method for producing 1,5-pentanediamine through fermentation with lysine sulfate and a method for extracting the 1,5-pentanediamine without solid waste. The method for preparing the 1,5-pentanediamine through fermentation comprises the steps that the pH value of a conversion liquid is adjusted by using cell catalyzing lysine sulfate expressing lysine decarboxylase, extraction is performed to obtain an oil phase and an aqueous phase, the oil phase is rectified, and the 1,5-pentanediamine capable of being used for generating polyamide is obtained. Waste is comprehensively utilized, and therefore no solid waste is generated, and the aqueous phase is concentrated to obtain sulfate capable of being used for preparing an amino acid fermentation culture medium. Additionally, the invention also provides the improved lysine decarboxylase.

Description

Technical field [0001] The present invention belongs to the field of biological fermentation engineering. Specifically, the present invention relates to a method for producing 1,5-pentanediamine by fermentation of lysine sulfate and an extraction method without solid waste, that is, using lysine sulfate as The technology of producing 1,5-pentanediamine by fermentation of raw materials and comprehensive utilization of waste. Background technique [0002] 1, 5-Pentanediamine (1,5-Pentanediamine), also known as Cadaverine, 1, 5-Diaminopentane (1, 5-Diaminopentane), can be polymerized with dibasic acid to form a polymer Amide material (ie nylon). The world produces about 7 million tons of polyamide materials every year, which consumes a lot of petrochemical resources. Therefore, 1,5-pentanediamine, an important monomer for the synthesis of polyamide by biological methods, has important economic and ecological significance. [0003] The whole-cell catalysis method uses lysine as a sub...

Claims

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Application Information

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IPC IPC(8): C12P13/00C12P13/08C12P13/14C12P13/22C12P13/06C12P13/10C07C209/86C07C211/09C08G69/28
CPCC07C209/86C08G69/28C12P13/001C12P13/06C12P13/08C12P13/10C12P13/14C12P13/227C07C211/09
Inventor 赵春光孟刚郭小炜马文友田斌张坤
Owner HEILONGJIANG EPPEN NEW MATERIALS LTD
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