Method for biocatalytic synthesis of alkylpyrazine containing monomethyl semi ring

A technology for the synthesis of alkylpyrazines catalyzed by monomethylsemicycloalkylpyrazine and threonine dehydrogenase, which is applied in fermentation and other fields, and can solve problems such as poor selectivity, difficult separation, and lack of biochemical pathways

Inactive Publication Date: 2019-09-06
JIANGNAN UNIV
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AI Technical Summary

Problems solved by technology

This method is a commonly used method for synthesizing alkylpyrazines, but the reaction steps are many, and it is not suitable for large-scale industrial production from an economic point of view.
[0006] (2) In the presence of ammonium salt, it is prepared by the reaction of acrolein in glycerin heating: the yield of this method is low
The disadvantage of this two-step process for producing pyrazine is that if the second step of dehydrogenation is not complete, the product will have an unpleasant smell because the condensation product and pyrazine are difficult to separate by physical means, so the dehydrogenation catalyst and reaction conditions are limited. The requirements are high, and the application in industry is gradually reduced
[0008] (4) α-diamine and α-ethanolamine synthesis method: 2,5-DMP is synthesized from α-diamine and α-ethanolamine. The reaction route is short and the process is simple, but its selectivity is poor, and a single product cannot be obtained, and the products are separated. Difficult to purify
The raw material propylene oxide used in this method is cheap and easy to get, but the reaction product is a mixture of 2,5-DMP and 2,6-DMP, and the product separation is difficult
[0011] (7) Synthesis of α-diamine and α-diol: This method has cheap raw materials, easy access, easy preparation of catalyst, short reaction route and simple process, but the reaction products are 2,5-DMP and 2,6-DMP mixture, the product separation is more difficult
Although there are microbial strains capable of synthesizing potentially valuable flavor compounds, their yields are often low; and the development of biotechnological production of flavor compounds is hampered by a lack of knowledge about biochemical pathways, enzymes, and metabolic regulation

Method used

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  • Method for biocatalytic synthesis of alkylpyrazine containing monomethyl semi ring
  • Method for biocatalytic synthesis of alkylpyrazine containing monomethyl semi ring
  • Method for biocatalytic synthesis of alkylpyrazine containing monomethyl semi ring

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1: L-threonine can be used as the only substrate source for 2,5-DMP microbial synthesis

[0072] 1. Isotope tracer experiment

[0073] Add 1g·L respectively -1 L-threonine or [U- 13 C, 15 Add N]-L-threonine to LB medium, cultivate B. subtilis 168 to 2d at 37°C and 200rpm, use GC-MS to qualitatively analyze the 2,5-DMP in the fermentation broth, and obtain the total ion pattern ( TIC) and mass spectrometry (MS) such as figure 1 shown. After adding L-threonine and [U- 13 C, 15 In the fermentation sample of N]-L-threonine, it is 13.5min ( figure 1 There are chromatographic peaks appearing at b,c), which is consistent with the retention time of the standard 2,5-DMP ( figure 1 a). Mass spectra of compounds detected in L-threonine spiked samples ( figure 1 b) with 2,5-DMP standard mass spectrum ( figure 1 a) It is completely consistent, indicating that the compound at this peak time in the fermentation sample is 2,5-DMP.

[0074] [U- 13 C, 15 The mass sp...

Embodiment 2

[0079] Example 2: Effect of TDH on the synthesis of 2,5-DMP by microorganisms using L-threonine

[0080] TDH (NP_389581) derived from B. subtilis 168 can convert L-threonine to 2,5-DMP in vitro.

[0081] 1. Exogenous expression and purification of TDH

[0082] Construct the recombinant plasmid pET-28a-tdh as follows:

[0083] Using B. subtilis 168 genomic DNA as a template, the primer tdh-F / R was used to amplify the tdh gene by PCR to obtain the gene fragment tdh. The purified gene fragment tdh and plasmid pET-28a were subjected to double digestion (BamHI / HindIII) respectively, and the digested products were column purified. The digested gene fragment tdh was ligated with plasmid pET-28a, and transformed into E.coli BL21(DE3) competent cells. The recombinant plasmids were extracted from the transformants, and the extracted plasmids were verified by double enzyme digestion (BamHI / HindIII), and the positive clones were selected and sent to Tianlin Biotechnology (Wuxi) Co., Lt...

Embodiment 3

[0110] Example 3: TDH is involved in the microbial synthesis of trimethylpyrazine (TMP)

[0111] 1. Isotope tracer verification of the substrate source of TMP microbial synthesis

[0112] Whether TMP can be derived from L-threonine and D-glucose was verified by using the method of isotope tracing. 4 + Add [U- 13 C 4 , 15 N]-L-threonine and [U- 13 C 6 ]-D-glucose, L-threonine and [U- 13 C 6 ]-D-glucose, [U- 13 C 4 , 15 N]-L-threonine and D-glucose, L-threonine and D-glucose, cultured B.subtilis 168 (cultivation conditions: B.subtilis 168 was activated to obtain seed liquid, and transferred to 1% inoculum Connect to 2mL and add [U- 13 C 4 , 15 N]-L-threonine (1g L -1 ) and [U- 13 C 6 ]-D-glucose (2g·L -1 ), [U- 13 C 4 , 15 N]-L-threonine (1g L -1 ) and D-glucose (2g·L -1 ), L-threonine (1g L -1 ) and [U- 13 C 6 ]-D-glucose (2g·L -1), L-threonine (1g L -1 ) and D-glucose (2g·L -1 ) containing 3g·L -1 Diammonium hydrogen phosphate in LB test tubes, cu...

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Abstract

The invention discloses a method for biocatalytic synthesis of an alkylpyrazine containing a monomethyl semi ring, which belongs to the technical field of biosynthesis. The method adopts whole cell catalysis, isotope tracer, pure enzyme catalysis, gene knockout and replenishment to determine that biosynthesis of alkylpyrazine containing monomethyl semi ring by threonine dehydrogenase (including 2,5-DMP, TMP) has important functions. The method also found that the step of catalyzing the threonine by threonine dehydrogenase TDH to generate aminoacetone is not only applicable to 2,5-DMP, but alsoto other alkylpyrazine containing the monomethyl semi ring, and the method proves that the threonine dehydrogenase TDH is involved in the biosynthesis process of 2,3,5-trimethyl containing the monomethyl semi ring.

Description

technical field [0001] The invention relates to a biocatalytic method for synthesizing an alkylpyrazine containing a monomethyl semicycle, in particular to a method for biocatalyzing the synthesis of an alkylpyrazine containing a monomethyl semicycle by using threonine dehydrogenase (TDH). The field of biosynthesis technology. Background technique [0002] Alkylpyrazines are a kind of branched nitrogen-containing heterocyclic compounds with alkyl groups. As important flavor substances, they mainly contribute to the nutty, barbecue and toasty flavors in food. Alkylpyrazine has an extremely low threshold, the threshold of tetramethylpyrazine (2,3,5,6-Tetramethylpyrazine, TTMP) in fermented soybean is 10ppm, 2,5-dimethylpyrazine (2,5 -Dimethylpyrazine, 2,5-DMP) only adds 1-2ppm in food, and it can play a significant role in flavor enhancement. Due to the low threshold value, alkylpyrazine can show strong odor characteristics, and it is a flavor substance allowed to be used in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/12
CPCC12P17/12
Inventor 徐岩张丽杰
Owner JIANGNAN UNIV
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