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CN-122012478-A - Efficient butanediamine biosynthesis method

CN122012478ACN 122012478 ACN122012478 ACN 122012478ACN-122012478-A

Abstract

The invention discloses a high-efficiency butanediamine biosynthesis method, and belongs to the field of bioengineering. The specific enzyme activities of the arginine decarboxylase mutant provided by the invention under alkaline conditions can reach 15.6U/g, 17.8U/g and 23.4U/g respectively, and compared with wild type enzyme, the specific enzyme activities are improved by about 4.2 times, 4.9 times and 6.8 times respectively, so that the catalytic efficiency of the enzyme is obviously enhanced, and an efficient enzyme catalyst is provided for biosynthesis of butanediamine. In addition, CO 2 is introduced in the fermentation process, so that the yield of the butanediamine is further improved. The CO 2 ventilation rate is controlled within the range of 0.5-2 vvm, so that the catalytic efficiency can be ensured, the waste of resources can be avoided, and the optimization of the production process is realized.

Inventors

  • DENG YU
  • LI GUOHUI
  • Li Chouqiang
  • Shang Mingchuang
  • MAO YIN
  • ZHOU SHENGHU
  • ZHAO YUNYING

Assignees

  • 江南大学

Dates

Publication Date
20260512
Application Date
20260121

Claims (10)

  1. 1. An arginine decarboxylase mutant is characterized in that the mutant is obtained by simultaneously mutating the 95 th glutamic acid into histidine, mutating the 467 th glutamic acid into lysine and mutating the 736 th histidine into glutamic acid on the basis of the amino acid sequence shown as SEQ ID NO. 1; Or on the basis of the amino acid sequence shown as SEQ ID NO.1, simultaneously mutating the 95 th glutamic acid into histidine and mutating the 467 th glutamic acid into lysine; or on the basis of the amino acid sequence shown as SEQ ID NO.1, simultaneously mutating the 95 th glutamic acid into histidine and mutating the 736 th histidine into glutamic acid.
  2. 2. A gene encoding the arginine decarboxylase mutant of claim 1, or a vector of the gene of claim 2.
  3. 3. A recombinant cell expressing the arginine decarboxylase mutant of claim 1, or the vector of claim 2; preferably, the recombinant cell is a bacterial or fungal host cell.
  4. 4. A recombinant strain expressing the arginine decarboxylase mutant, arginine decarboxylase SpeA, and agmatinase SpeB of claim 1; Preferably, the amino acid sequence of agmatinase SpeB is shown in SEQ ID NO.3 and the amino acid sequence of arginine decarboxylase SpeA is shown in SEQ ID NO. 4.
  5. 5. A whole-cell catalyst is characterized in that, the whole cell catalyst contains the recombinant strain of claim 4.
  6. 6. A method for producing butanediamine, which is characterized in that arginine is used as a substrate, the mutant of claim 1, the recombinant strain of claim 4 or the whole cell catalyst of claim 5 is used for catalyzing and reacting to prepare butanediamine, and the whole reaction process maintains CO 2 atmosphere or inert atmosphere; preferably, the inert atmosphere comprises at least one of argon and nitrogen.
  7. 7. The method of claim 6, wherein the CO 2 atmosphere is continuously vented at a ventilation of 0.5 to 2 vvm.
  8. 8. A method for improving the enzyme activity of arginine decarboxylase is characterized in that the method is that glutamic acid at the 95 th position of the arginine decarboxylase with an amino acid sequence shown as SEQ ID NO.1 is mutated into histidine, glutamic acid at the 467 th position is mutated into lysine, and histidine at the 736 th position is mutated into glutamic acid; Or on the basis of the amino acid sequence shown as SEQ ID NO.1, simultaneously mutating the 95 th glutamic acid into histidine and mutating the 467 th glutamic acid into lysine; or on the basis of the amino acid sequence shown as SEQ ID NO.1, simultaneously mutating the 95 th glutamic acid into histidine and mutating the 736 th histidine into glutamic acid.
  9. 9. A method for increasing the yield of butanediamine, which is characterized in that CO 2 is introduced in the process of preparing butanediamine by using the recombinant strain of claim 4 or the whole-cell catalyst of claim 5; preferably, the CO 2 is continuously introduced with ventilation of 0.5-2 vvm.
  10. 10. Use of an arginine decarboxylase mutant according to claim 1, or a gene or vector according to claim 2, or a recombinant cell according to claim 3, or a recombinant strain according to claim 4, or a whole cell catalyst according to claim 5, for the preparation of butanediamine or a product comprising butanediamine, or for the preparation of a nylon product; preferably, the nylon product includes, but is not limited to PA46, PA410, PA4T.

Description

Efficient butanediamine biosynthesis method Technical Field The invention relates to a high-efficiency butanediamine biosynthesis method, and belongs to the field of bioengineering. Background Butanediamine (Putrescine), also known as putrescine, belongs to amino acid derivatives. 1, 4-butanediamine is not only an important molecule in life process, but also regulates the cellular metabolism of organisms. Has important value in the production fields of engineering plastics, medicines, agrochemicals and surfactants. The preparation method is mainly used for preparing polyamide materials, comprises various products such as PA46, PA410, PA4T and the like, and has wide application in the fields of textile, mechanical engineering, electronic and electric appliances, automobile manufacturing and the like. At present, the traditional chemical synthesis is still a main production mode, the synthesis raw materials have toxicity, and the production process has the problems of severe reaction conditions, high requirements on equipment, expensive catalysts, high hydrogen pressure, high energy consumption and the like, so that the butanediamine synthesis technology barrier is high. Therefore, the synthesis biological technology is utilized, and the biomass raw material is directly converted by the microbial cells or the enzyme catalytic system to prepare the butanediamine, so that the technical bottleneck of the traditional process is effectively avoided, the obvious advantages of environmental friendliness and economy are further shown, and an important solution is provided for sustainable production of the butanediamine. Disclosure of Invention Technical scheme The first object of the invention is to provide an arginine decarboxylase mutant, which is obtained by mutating the glutamic acid at the 95 th position into histidine, mutating the glutamic acid at the 467 th position into lysine and mutating the histidine at the 736 th position into glutamic acid on the basis that the amino acid sequence is shown as SEQ ID NO. 1; Or on the basis of the amino acid sequence shown as SEQ ID NO.1, simultaneously mutating the 95 th glutamic acid into histidine and mutating the 467 th glutamic acid into lysine; or on the basis of the amino acid sequence shown as SEQ ID NO.1, simultaneously mutating the 95 th glutamic acid into histidine and mutating the 736 th histidine into glutamic acid. It is a second object of the present invention to provide a gene encoding the arginine decarboxylase mutant or a vector carrying the gene. It is a third object of the present invention to provide recombinant cells expressing the arginine decarboxylase mutant, or the vector. Preferably, the recombinant cell is a bacterial or fungal host cell. It is a fourth object of the present invention to provide a recombinant strain expressing the arginine decarboxylase mutant, arginine decarboxylase SpeA, and agmatinase SpeB. In one embodiment of the invention, the amino acid sequence of agmatinase SpeB is shown in SEQ ID NO.3 and the amino acid sequence of arginine decarboxylase SpeA is shown in SEQ ID NO. 4. It is a fifth object of the present invention to provide a whole cell catalyst comprising the recombinant strain. The sixth object of the present invention is to provide a method for producing butanediamine, wherein arginine is used as a substrate, the butanediamine is prepared by using the recombinant strain, the whole cell catalyst or the mutant to catalyze the reaction, and the CO 2 atmosphere or the inert atmosphere is maintained in the whole reaction process. In one embodiment of the present invention, the CO 2 atmosphere is continuously introduced with a ventilation of 0.5 to 2 vvm. In one embodiment of the present invention, the inert atmosphere comprises at least one of argon and nitrogen. The seventh object of the present invention is to provide a method for improving the enzymatic activity of arginine decarboxylase, wherein the method comprises the steps of mutating the glutamic acid at the 95 th position of the arginine decarboxylase with the amino acid sequence shown as SEQ ID NO.1 into histidine, mutating the glutamic acid at the 467 th position into lysine, and mutating the histidine at the 736 th position into glutamic acid; Or on the basis of the amino acid sequence shown as SEQ ID NO.1, simultaneously mutating the 95 th glutamic acid into histidine and mutating the 467 th glutamic acid into lysine; or on the basis of the amino acid sequence shown as SEQ ID NO.1, simultaneously mutating the 95 th glutamic acid into histidine and mutating the 736 th histidine into glutamic acid. An eighth object of the present invention is to provide a method for increasing the yield of butanediamine by introducing CO 2 during the preparation of butanediamine using the mutant or the whole cell catalyst. In one embodiment of the present invention, the CO 2 is continuously introduced with a ventilation of 0.5 to 2 vvm. It is a ninth object of the present inv