CN111951898A - Method for screening 2-ketolase capable of converting L-amino acid into - Google Patents
Method for screening 2-ketolase capable of converting L-amino acid into Download PDFInfo
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- 101710092506 Aspartate aminotransferase Proteins 0.000 claims description 15
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Abstract
The invention belongs to the technical field of L-amino acid fermentation. The invention provides a method for screening 2-ketolase capable of converting L-amino acid into the 2-ketolase, which comprises the following steps: scoring the sequences in the final database with each subfamily HMM profile to obtain sequences with E values below 0.001; 15 ATs belonging to classes I and II were obtained. The invention has the beneficial effects that: AT has wide source and is easy to carry out heterologous expression, and expensive cofactors do not need to be additionally added; high activity and stereoselectivity.
Description
Technical Field
The invention relates to the technical field of biocatalytic synthesis of 2-keto acid and D-amino acid.
Background
In recent years, the development of metabolic engineering and synthetic biology has led to great success in the fermentation of L-amino acids. The global amino acid production in 2017 was about 850 million tons, and it is expected that 1100 million tons will be reached by 2022. With the further development of modern biotechnology, the production cost of L-amino acids is continuously reduced. As a large country for producing and consuming amino acids, a large number of amino acid products are in a state of passing supply and demand for a long time in China. Therefore, the production of high value-added products using L-amino acids as starting materials is an urgent problem to be solved.
2-keto acids play an important role in the metabolism of humans and animals as direct precursors for the synthesis of L-amino acids, and are widely used in the fields of medicine, food, agriculture, and the like. However, the synthesis of 2-keto acids is mainly dependent on chemical methods, and these methods require expensive catalysts or special starting compounds, so that there are disadvantages of high production cost and environmental unfriendliness.
The method for catalyzing the conversion of the natural L-amino acid into the 2-keto acid by adopting the biological enzyme method has outstanding advantages in the aspect of sustainability: renewable resources are used as starting materials, mild reaction conditions and high atom economy. Based on this, four types of enzymes have been found to catalyze the production of 2-keto acids from L-amino acids, i.e., L-amino acid dehydrogenase (LADH), L-amino acid oxidase (LAAO), L-amino acid deaminase (LAAD) and L-amino acid Aminotransferase (AT).
Although the high efficiency and enantioselectivity and regioselectivity confer the ability of AT to participate in enzymatic cascades and 2-keto acid synthesis, AT generally accepts only a group of similar amino acids as substrates, and this substrate tolerance prevents widespread use of this technology. In addition, some amino acids (e.g., threonine, lysine and arginine) do not have the corresponding aminotransferases or some aminotransferases have lower activity. Therefore, how to find a new AT compatible with the above reaction system by bioinformatics means to expand the substrate acceptance range has become a difficult point in the field of research.
As a result of the recent increased interest in chiral unnatural amino acids in the pharmaceutical and agrochemical industries, there is a great need for an economically efficient method for synthesizing D-amino acids and N-methylated amino acids from the produced 2-keto acids.
Disclosure of Invention
The invention aims to provide a method for screening L-amino acid capable of being converted into 2-ketolase, which comprises the following steps: scoring the sequences in the final database with each subfamily HMM profile to obtain sequences with E values below 0.001; 15 ATs belonging to classes I and II were obtained.
Further, the method for obtaining the sequence in the final database comprises: the sequences of the unaddressed AspAT/ARAT and AlaAT from bacteria and archaea in the UniprotKB database were downloaded as data sets, and then redundant sequences were removed from the data sets to obtain the final database sequences.
Further, the method for establishing the HMM subfamily map comprises the following steps: retrieving the sequences of AspAT/ARAT and AlaAT that have been confirmed by the study from Swiss-Port; redundancy was removed from these sequences and HMM subfamily maps were created using the HMMER software package.
Furthermore, the method for removing redundant sequences is to remove sequences with the amino acid number of more than 200 and the similarity of less than 90 percent with the sequence of AspAT/ARAT or AlaAT.
The invention has the beneficial effects that:
AT has wide source, is easy to carry out heterologous expression, and does not need to additionally add expensive cofactors; high activity and stereoselectivity.
2. Cheap and reproducible substrate and cofactor are used, other cofactor regeneration systems are not required to be additionally added, the unfavorable chemical equilibrium is shifted to the synthesis direction of the product, and the inhibition effect of 2-ketoglutarate on transaminase is weakened.
3. The reaction conditions are mild (pH 8.0-8.5, 30 ℃), the substrate conversion rate is high, the HPLC yield of the target compound is high (> 92.7%), and the stereoselectivity is high (the ee value of the product is more than 99%).
Drawings
FIG. 1 is a diagram showing a reaction cascade of a plurality of enzymes for converting an L-form of an amino acid into a 2-keto acid.
FIG. 2 is a graph of the overall analysis scheme and results of screening transaminase sequences by subfamily HMM profiles.
Wherein, "in"and" jn"score for subfamily mapping analysis of AspAT/ATAR and AlaAT, respectively, with unknown sequence nThe value is obtained.
FIG. 3 is a schematic of the enzymatic cascade reverse synthesis of chiral unnatural amino acids.
Detailed Description
1. In AT families I and II, AspAT and ARAT are classified into the same subfamily (AspAT/ARAT) according to protein structure and sequence characteristics; due to the large sequence differences between AlaAT and AspAT/ARAT, AlaAT is classified as another subfamily in classes I and II.
TABLE 1 class of transaminases and their substrate profiles
2. AspAT/ARAT and AlaAT sequences that have been confirmed by experimental studies were retrieved from Swiss-Port for the construction of family sequence maps.
3. The redundancy of these sequences was removed, and the sequences in which the degree of similarity was greater than 90% were removed. The remaining sequences were aligned within subfamilies using MAFFT, V7.419 software package for multiple sequences, and HMMER3.2.1 software package was used to create HMM subfamily maps.
4. Downloading sequences of the non-researched AspAT/ARAT and AlaAT from bacteria and archaea in a UniprotKB database as a data set, and removing redundant sequences from the data set to obtain a sequence of a final database;
the method for removing redundant sequences is to use a CD-HIT software package to remove sequences with the amino acid number of less than 200 and the similarity of more than 90 percent with the sequence of AspAT/ARAT or AlaAT.
5. Sequences in the final database were scored with each subfamily HMM map (parameters set to default values, sequences with E values below 0.001 were retained) and plotted in a 2-dimensional plane according to the final scoring value (figure 2).
The number of sequences drawn on a two-dimensional space is 2667; if the scoring of the sequences is strongly biased towards AspAT/ARAT or AlaAT, these sequences are typically AspAT/ARAT or AlaAT.
From the distribution of sequences, 15 ATs belonging to classes I and II were selected as candidate sequences.
TABLE 2 information extracted from 15 ATs from families I and II
The related protein sequence can be obtained by searching for the access ID on the Unit.
6. The candidate sequences were cloned into pET-28a vector and expressed in E.coli BL21(DE3), followed by affinity purification using His tag. Two proteins, AlaAT7 and ATI2, which are not sufficiently expressed in E.coli, were removed. The remaining 13 ATs were expressed in soluble form and purified to obtain higher purity proteins.
7. The activities and specificities of the above 13 ATs were determined for 18L-amino acids other than L-Pro and L-Glu (Table 3).
It can be seen that AT from different sources show complementary substrate specificity, and these transaminases are able to tolerate most L-amino acid substrates (14 species).
TABLE 3 Activity of the selected AT on 18L-amino acids.
TABLE 3 continuation
Activity unit: u/mg (1U equals the amount of enzyme required to catalyze the production of 1. mu. mol product per minute)
Activity measurement conditions: 30 ℃ at 200rpm in 1mL reaction system consisting of 0.2M phosphate buffer (pH7.5), 20mM L-amino acid, 10mM 2-ketoglutarate, 10. mu.g/mL ATs and 20. mu.MPLP.
8. Catalytic ability of enzymes in 2-keto acid synthesis
AT with higher activity on a single substrate is selected as a candidate enzyme. The 9L-amino acids indicated in Table 4 were tested and all enzymatic reactions were carried out in a shaker to ensure oxygen supplementation. Almost all of the tested L-amino acids can be efficiently converted to the corresponding 2-keto acids by an enzymatic cascade with a high conversion (> 99%) in 24 hours (Table 4).
It is noteworthy that the maximum yields of the above 2-keto acids by kinetic control reach 31.9%, 40% and 78.9%, respectively, since glyoxylic acid, oxalacetic acid and B-indolypyruvic acid tend to undergo spontaneously beta-decarboxylation side reactions at neutral pH.
TABLE 4 enzymatic cascade oxidation of L-amino acids to the corresponding 2-keto acids.
Oxidizing the L-amino acid to form 2-keto acid by AT-based cascade reaction, wherein the reaction is carried out AT 30 ℃, 200rpm and 1mL of reaction system, and the reaction system mainly comprises the following components: 0.2M phosphate buffer (pH7.5), 30mM L-amino acid, 1mM 2-oxoglutarate, 0.25mg/ml ATs, 0.1mg/ml LGOX, 0.1mg/ml catalase and 20. mu.M PLP.
[a]Product yield by HPLC analysis;
[b]due to the lack of commercial standards, no quantification of product yield was performed.
9. A one-pot two-step enzymatic cascade is used to synthesize chiral unnatural amino acids.
The first step, oxidizing L-amino acid to form 2-keto acid in an AT-based cascade reaction system to obtain 2-keto acid reaction liquid;
wherein the conditions of the reaction system are as follows: 30 ℃, 200rpm, 1mL of reaction volume, and the reaction system comprises the following components: 0.2M phosphate buffer (pH7.5), 30mM L-amino acid, 1mM 2-oxoglutarate, 0.25mg/ml ATs, 0.1mg/ml LGOX, 0.1mg/ml catalase and 20. mu.M PLP.
Secondly, 0.3mg of StDAPDH W121L/H227I or PfNMAADH, 0.2mg of GDH to the reaction solution of 2-keto acid to a final concentration of 1mM NADP +; 1.3 equivalents of glucose and 5 equivalents of amine at a pH of 8.0-8.5. During the reaction, the pH of the second reaction was adjusted to 8.0-8.5 with NaOH.
The method for determining the product yield and the spatial configuration comprises the following steps: the sample is derivatized with 2, 4-dinitrofluorobenzene (derivatization method: after sample solution is diluted to make total concentration of ammonia water and amino acid lower than 50mM, 25 microliter of sample solution is sucked, 10 microliter of 1M sodium bicarbonate and 40 microliter of 36.7mM2, 4-dinitrofluorobenzene solution in acetone are added, the mixture is incubated at 60 ℃ for 30 minutes, and then taken out and 20 microliter of 1M hydrochloric acid is added immediately to terminate the reaction), and the derivatized solution is subjected to HPLC to determine peak area of product, and yield and ee value are calculated.
The reaction process is as follows:
the specific yield and the product steric configuration result are as follows:
has the advantages that:
AT has wide source, is easy to carry out heterologous expression, and does not need to additionally add expensive cofactors; high activity, high stereoselectivity and the like;
2. cheap and reproducible substrate and cofactor are used, other cofactor regeneration systems are not required to be additionally added, the unfavorable chemical equilibrium is shifted to the synthesis direction of the product, and the inhibition effect of 2-ketoglutarate on transaminase is weakened.
3. Under mild reaction conditions (pH 8.0-8.5, 30 ℃), a one-pot two-step sequential synthesis process is adopted to prepare 2-keto acid from L-amino acid and perform two-step reaction with 5 times of equivalent of ammonia or methylamine to gradually realize the synthesis of chiral amine with high added value. From the results, it can be seen that the substrate is almost completely converted into the desired product, the HPLC yield of the respective target compound is high (> 92.7%) and high stereoselectivity is achieved (> 99% ee value for the product).
Claims (7)
1.A method for screening for a compound capable of converting an L-amino acid into a 2-ketolase, comprising the steps of:
scoring the sequences in the final database with each subfamily HMM profile to obtain sequences with E values below 0.001;
15 ATs belonging to classes I and II were obtained.
2. The method of claim 1, wherein the obtaining of the sequences in the final database comprises:
the sequences of the unaddressed AspAT/ARAT and AlaAT from bacteria and archaea in the UniprotKB database were downloaded as data sets, and then redundant sequences were removed from the data sets to obtain the final database sequences.
3. The method according to claim 2, wherein the redundant sequence is removed by removing a sequence having an amino acid number of > 200 and a similarity of < 90% to the sequence AspAT/ARAT or AlaAT.
4. The method according to claim 1, wherein the HMM subfamily graph building method comprises:
retrieving the sequences of AspAT/ARAT and AlaAT that have been confirmed by the study from Swiss-Port;
redundancy was removed from these sequences and HMM subfamily maps were created using the HMMER software package.
5. The method of claim 1, wherein the AT is used in a method of synthesizing a chiral unnatural amino acid, comprising:
oxidizing the L-amino acid in an AT-based cascade reaction system to form 2-keto acid to obtain 2-keto acid reaction liquid;
0.3mg of StDAPDH W121L/H227I or PfNMAADH, 0.2mg of GDH, 1.3 equivalents of glucose and 5 equivalents of amine were added to the 2-keto acid reaction mixture, and the reaction was carried out at pH 8.0 to 8.5.
6. The method according to claim 5, wherein the reaction system conditions are: 30 ℃, 200rpm, 1mL reaction volume.
7. The method of claim 5, wherein the reaction system components comprise: 0.2M phosphate buffer (pH7.5), 30mM L-amino acid, 1mM 2-oxoglutarate, 0.25mg/ml ATs, 0.1mg/ml LGOX, 0.1mg/ml catalase and 20. mu.M PLP.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2072622A1 (en) * | 2006-10-12 | 2009-06-24 | Kaneka Corporation | Method for production of l-amino acid |
| CN104630171A (en) * | 2013-11-08 | 2015-05-20 | 中国科学院天津工业生物技术研究所 | New (R)-transaminase from Fusarium oxysporum and application thereof |
| US20160289715A1 (en) * | 2012-12-13 | 2016-10-06 | Industry Academic Cooperation Foundation, Yonsei University | Method for producing optically active amine compounds by deracemization |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2072622A1 (en) * | 2006-10-12 | 2009-06-24 | Kaneka Corporation | Method for production of l-amino acid |
| US20160289715A1 (en) * | 2012-12-13 | 2016-10-06 | Industry Academic Cooperation Foundation, Yonsei University | Method for producing optically active amine compounds by deracemization |
| CN104630171A (en) * | 2013-11-08 | 2015-05-20 | 中国科学院天津工业生物技术研究所 | New (R)-transaminase from Fusarium oxysporum and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| TAO LI等: "Exploration of Transaminase Diversity for the Oxidative Conversion of Natural Amino Acids into 2‑Ketoacids and High-Value Chemicals", ACS CATAL., no. 10, pages 7950 * |
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