Novel lysine decarboxylase and application thereof

An amino acid, application technology, applied in the biological field, can solve problems such as poor thermal stability

Pending Publication Date: 2017-09-19
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

On the other hand, although the optimum pH of LdcC is relatively high, about 7.6, and it has high activity between pH 6-8, its thermal stability is very poor. Elevated enzyme activity rapidly decreased

Method used

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  • Novel lysine decarboxylase and application thereof
  • Novel lysine decarboxylase and application thereof
  • Novel lysine decarboxylase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Example 1. Expression plasmid and bacterial strain construction of lysine decarboxylase

[0101] The lysine decarboxylase sequence of the present invention is shown in the following SEQ ID NO: 1-2:

[0102] SEQ ID NO:1(命名为Ldc6)–MNIFAILNHSGVFFKEEPVRELHASLEKAGYKVVYPVDAQDLYKMVEMNPRICGVLFDWDKYSLDLCTEINVLNEKLPLYAFANQHSTLDISLTDLRLNLHFFEYALGMADDIALKINQATEEYIDQIMPPFTKALFKYVEEGKYTFCTPGHMGGTAFQKSPAGSIFYDFYGPNAFKADVSISMPELGSLLDHSGPHKEAEEYIARTFNADSSYIVTNGTSTSNKIVGMFSAPAGSTVLVDRNCHKSLTHMMMMSDVTPIYFRPTRNAYGILGGIPQSEFTREVIEAKVAATPNATMPGYAVITNSTYDGLLYNTQYIKETLDTKFIHFDSAWVPYTNFNSIYEGKCGMSGEAMPGKVFYETQSTHKLLAAFSQASMIHVKGEFDKESFNEAFMMHTSTSPQYGIVASTEIAAAMMRGNTGKKLIQDSIDRAIRFRKEIKRLESESDSWFFDVWQPENIDTTECWKLDPKDTWHGFKDIDDDHMYLDPIKVTLLTPGMNENSEMSETGIPASIVAKYLDEHGIVVEKTGPYNLLFLFSIGIDKSKAMQLLRALTDFKRGYDLNLTVKNFLPSLYNEDPSFYEGMRIQELAQGIHDLTRQYRLPELMFKAFDVLPELKVTPHAAWQEELRGNVEEVKLEEMVGRVSANMILPYPPGVPLVLPGEMVTTESRPVLDFLEMLCAIGAHYPGFETDIHGVYAQKDGSYTVKVLKED;

[0103] SEQ ID NO:2(命名为Ldc14)–MKDILFLCNPSP...

Embodiment 2

[0105] Embodiment 2. Separation and purification of enzyme

[0106] The recombinant bacteria SKEcC2, SKEcC3 and SKEcC4 were respectively inserted into 5 mL LB liquid medium containing 100 μg / mL ampicillin, and cultured at 37 °C and 200 r / min for about 10 h. Take 0.5mL of seed solution and put it into a 500mL shake flask containing 100μg / mL ampicillin in 50mL LB liquid medium, and culture at 37°C and 200r / min. When grown to OD 600 When it is 0.6-0.8, add ITPG to a final concentration of 0.1 mM, induce expression at 20°C, 200r / min for 20h. The induced bacterial solution was centrifuged at 4°C to collect the bacterial cells, and the 4°C pre-cooled resuspension buffer (20mM potassium phosphate, 20mM imidazole, 0.1mM pyridoxal phosphate, 1mM DTT, 100mM NaCl, adjusted to pH=7.4) was used for induction After the cells were resuspended and washed 3 times, they were disrupted by ultrasound. The broken solution was centrifuged at 12,000rpm for 20min, and the obtained soluble supernat...

Embodiment 3

[0107] Embodiment 3. Different pH conditions enzyme activity assay

[0108] In a 100ul enzyme reaction system (100mM potassium phosphate, 0.1mM pyridoxal phosphate, 1mM DTT, 100mM NaCl) the amount of LDC pure enzyme is 0.5ug, the concentration of substrate L-lysine is 15mM, and the pH of the buffer solution is adjusted to 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, react at 37°C for 3 minutes. Take 30ul of the reaction solution and add it to 70ul of 1M sodium carbonate stop solution to stop the reaction, take 50ul of 10mM TNBS into the reaction stop solution, develop color at 42°C for 5min, and finally extract with 500ul of toluene, draw 200μL of the supernatant on the microplate reader Read the absorbance at 340nm. The enzyme activity unit U is defined as: under the above enzyme reaction conditions, the amount of enzyme required to catalyze the production of 1 μmol 1,5-pentanediamine per minute is defined as an enzyme activity unit.

[0109] The specific enzyme activities of Ldc6...

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Abstract

The invention discloses novel lysine decarboxylase. The amino acid sequence of the novel lysine decarboxylase is shown as SEQ ID NO: 1 or SEQ ID NO: 2. The lysine decarboxylase can be expressed efficiently in a host cell, has superior thermal stability, and has the characteristics of high activity under a neutral to weakly-alkaline condition, so that the novel lysine decarboxylase is superior to conventional lysine decarboxylase. The invention further provides an expression vector including an encoding sequence of the lysine decarboxylase, a host cell capable of expressing proteins, application of the expression vector and the host cell to production of 1,5-pentamethylene diamine, and a production method.

Description

technical field [0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to the application of a protein with lysine decarboxylase activity, an expression vector containing the gene encoding the protein, and a genetically engineered bacterium capable of expressing the protein in the production of 1,5-pentanediamine. Background technique [0002] 1,5-Pentanediamine, also known as cadaverine, 1,5-diaminopentane, pentamethylenediamine and cadaveric toxin, is a nitrogenous base with biological activity widely present in organisms, and it is a lysine Produced by decarboxylation under the action of lysine decarboxylase (E.C.4.1.1.18), the reaction is L-lysine+H + →CO 2 +cadaverine. [0003] 1,5-Pentanediamine has many uses. For example, in agriculture, 1,5-pentanediamine can be used to regulate the aging process of plants, promote the development of pistils, improve the development of plant fruits, and increase fruit yield...

Claims

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Application Information

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IPC IPC(8): C12P13/00C12N9/88C12N15/60C12N15/70C12N15/75C12N15/77C12N15/74C12N1/21
CPCC12N9/88C12P13/00C12Y401/01018
Inventor 孙际宾刘娇赵晶寇风雨郑平马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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