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Method for producing 1,5-pentanediamine through immobilized lysine decarboxylase

A technology of lysine decarboxylase and immobilized enzyme, which is applied in the field of immobilized lysine decarboxylase to produce pentamethylenediamine, can solve the problems of chemical pollutant discharge, unfavorable industrialization process, large reaction discharge pollution, etc., and achieve The effect of high productivity, simplified separation process of downstream products, and small loss of enzyme activity

Inactive Publication Date: 2019-06-14
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the preparation of the polymer carrier and the immobilization of lysine decarboxylase in this method, the designed epoxy group modification and covalent bonding are cumbersome and require the participation of various organic reagents, which will cause large chemical pollution in the whole process. The emission of pollutants is not conducive to the process of large-scale industrialization
[0009] Based on comprehensive literature and patent reports, it can be concluded that the currently prepared immobilized lysine decarboxylase mostly uses polymer gel carriers, the steps in the actual operation process are cumbersome, the reaction discharges pollution, and there are also problems with low repetition rate. Happening

Method used

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  • Method for producing 1,5-pentanediamine through immobilized lysine decarboxylase
  • Method for producing 1,5-pentanediamine through immobilized lysine decarboxylase
  • Method for producing 1,5-pentanediamine through immobilized lysine decarboxylase

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Experimental program
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Effect test

Embodiment 1

[0046] Construction of embodiment 1 fusion protein particles

[0047] Bacterial Genome Extraction Kit (TianGen) was used to extract Escherichia coli MG1655 (purchased from Wuhan Miaoling Biotechnology Co., Ltd.) and from Jiangnan University Jiangnan University Hao Zhikui donated Chitinolytic bacteria meiyuanensis SYBC-H1 (ATCC BAA-2140 T ), the lysine decarboxylase gene CadA and the chitin-binding domain gene ChBD were amplified by PCR respectively.

[0048] Use the following primers

[0049] 1: 5'-CGGGATCCTTCTTTCAATACCTTAACGG-3'

[0050] 2: 5'-CATGCCATGGATGAACGTTATTGCAATATTG-3'

[0051] by E. coli The total DNA of MG1655 was used as a template for PCR amplification. After the PCR reaction, the target fragment with a size of 2.1 kb was obtained by digestion at the Ncol I and Bam HI restriction sites CadA After nucleic acid gel verification, the target gene was recovered by gel recovery kit (TianGen).

[0052] Use the following primers

[0053] 3: 5'-CCCAAGCTTCGGCG...

Embodiment 2

[0059] Example 2 Preparation of lysine decarboxylase crude enzyme solution

[0060] Prepare the seed solution of the constructed lysine decarboxylase-producing bacteria, the medium is LB medium, culture at 37°C and 200rpm for 6-7h, inoculate the seed solution with a volume fraction of 2% in the fermentation medium containing 500ml LB , cultivated at 37°C and 200rpm until the OD was 0.6, added the inducer IPTG at an amount of 1‰, induced at 30°C and 200rpm for 12h, and then finished the fermentation to collect the fermentation broth.

[0061] After centrifuging the fermentation broth at 8000g, collect the bacteria, wash twice with distilled water, resuspend in 25ml of citric acid-disodium hydrogen phosphate (100mM pH6.8), ultrasonically crush and centrifuge at 8000g, and collect the supernatant as Lai Amino acid decarboxylase crude enzyme solution, stored at 4°C for later use.

Embodiment 3

[0062] Example 3 Affinity adsorption of lysine decarboxylase and chitin

[0063] Take 10 ml of the crude lysine decarboxylase enzyme solution obtained in Example 2, add 0.1 g of high-pressure homogenized chitin powder to perform affinity adsorption of lysine decarboxylase. After the adsorption is completed, centrifuge at 6000g to remove the supernatant and collect the precipitate, which is the chitin powder (immobilized enzyme) with lysine decarboxylase adsorbed as we need. After washing twice with distilled water, the washed immobilized lysine decarboxylase was collected and stored at 4°C for future use. The adsorption environment parameters were temperature 20°C, stirring speed 200rpm, and pH 6.8.

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Abstract

The invention discloses a method for producing 1,5-pentanediamine through immobilized lysine decarboxylase. The method comprises the following steps of connecting a chitin binding domain (ChBD) gene to the N end of a lysine decarboxylase gene (CadA) to construct a fusion protein; fermenting the fusion protein to obtain a crude lysine decarboxylase enzyme solution; adding chitin into the crude enzyme solution to obtain immobilized lysine decarboxylase; finally, enabling the obtained immobilized lysine decarboxylase to participate in a decarboxylation reaction of L-lysine to prepare 1,5-1,5-pentanediamine. According to the method, the specific affinity effect of the substrate chitin and the fusion protein is skillfully utilized to adsorb the lysine decarboxylase, the cost of the immobilizedcarrier chitin is low, the immobilization efficiency is high, the activity stability of enzymes is high, and meanwhile, the procedure of protein purification can be omitted through specific affinity adsorption. The provided method achieves repeated production of 1,5-1,5-pentanediamine and can reduce the production cost, simplify the separation procedure of downstream products and enzymes and achieve remarkable economic benefits.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for producing pentamethylenediamine by immobilizing lysine decarboxylase. Background technique [0002] Polyamide (PA), also known as nylon, is an important polymer that can be widely used in textiles, automobiles, electronic appliances, medical materials and other fields. At present, the annual consumption of polyamide (nylon, PA) products in the world is about 30 million tons, and the transaction volume is expected to reach 43.7 billion US dollars by 2020. Among them, the most widely used polyamide materials are mainly polycaprolactam (PA6) and polyhexamethylene adipamide (PA66), the consumption of which accounts for about 90% of the total consumption of polyamide. Hexamethylenediamine, one of the monomers required for the polymerization of PA66, is an important compound derived from fossil fuels. Excessive reliance on the synthesis of petroleum resources will inevitably le...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/00C12N15/70C12N9/88C12N11/10
Inventor 陈可泉周宁张阿磊杨赛魏衍鹏莫晓芳王莹莹欧阳平凯
Owner NANJING UNIV OF TECH
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