阅读说明：本技术 一种级联反应制备d-脂肪族氨基酸的方法 (Method for preparing D-aliphatic amino acid by cascade reaction ) 是由 倪晔 刘亚菲 许国超 韩瑞枝 于 2019-11-15 设计创作，主要内容包括：本发明公开了一种级联反应制备D-脂肪族氨基酸的方法。本发明以氨基酸序列如SEQ ID NO.2所示的D-N-氨甲酰水解酶作为催化剂,在海因酶过程级联反应中制备D-脂肪族氨基酸。本发明提供了一种D-N-氨甲酰水解酶可作为催化剂应用于海因酶过程制备光学纯D-脂肪族氨基酸,其可溶性好,催化效率高(转化率>99.9％)、立体选择性强(e.e.>99.9％)、适用的反应条件温和、环境友好。本发明的D-N-氨甲酰水解酶催化效果佳,底物适用性广,具有很好的应用开发前景。(The invention discloses a method for preparing D-aliphatic amino acid by cascade reaction. The invention takes D-N-carbamyl hydrolase with an amino acid sequence shown as SEQ ID NO.2 as a catalyst to prepare D-aliphatic amino acid in a hydantoinase process cascade reaction. The invention provides a D-N-carbamyl hydrolase which can be used as a catalyst for preparing optically pure D-aliphatic amino acid in a hydantoinase process, and has the advantages of good solubility, high catalytic efficiency (the conversion rate is more than 99.9%), strong stereoselectivity (e.e. > 99.9%), mild reaction conditions and environmental friendliness. The D-N-carbamyl hydrolase of the invention has good catalytic effect, wide substrate applicability and good application and development prospect.)

1. A method for preparing D-aliphatic amino acid by cascade reaction is characterized in that D-N-carbamyl hydrolase is used as a catalyst to prepare the D-aliphatic amino acid in the cascade reaction of a hydantoinase process, and the amino acid sequence of the D-N-carbamyl hydrolase is shown as SEQ ID NO. 2.

2. The method of claim 1, wherein the nucleotide sequence of the D-N-carbamoyl hydrolase is represented by SEQ ID No. 1.

3. The method of claim 1, wherein the reaction is carried out using L-methylthioethyl hydantoin as a substrate and D-N-carbamoyl hydrolase, hydantoin racemase and hydantoin enzyme as catalysts to produce D-methionine or other aliphatic amino acids.

4. The method according to claim 3, wherein the concentration of L-methylthioethyl hydantoin is 5 to 1000 mmol/L.

5. The method according to claim 3, wherein the amount of the D-N-carbamoylase added is 1 to 50 kU/L.

6. The method according to claim 3, wherein the amount of the hydantoin racemase added is 1 to 15 kU/L.

7. The method according to claim 3, wherein the amount of the hydantoinase added is 1 to 20 kU/L.

8. The method of claim 1, wherein the temperature of said hydantoinase process cascade is 20-40 ℃.

Technical Field

The invention relates to a method for preparing D-aliphatic amino acid by cascade reaction, belonging to the technical field of biological catalysis.

Background

D-amino acids are present in microorganisms (appl. Microbiol. Biotechnol.98, 5363-5374.), plants (origin. Life Eval. Biosph. 2002,32, 103-127.), and animals (Plant Soil,2012,354, 21-39.), and are used in a wide range of applications in the pharmaceutical, food and chemical industries. For example: d-serine has an activating effect on an excitatory receptor N-methyl-D-Asp, the latter has an important effect on the formation of memory (J.Cerebr.blood F.Met.,2014,34(12), 1928-1935), D-alanine can be used for synthesizing a sweetening agent Alitame, has high sweetness (2000 times of sucrose) and low calorific value, and is an important food additive; d-phenylglycine can be used as a synthetic beta-lactam antibiotic (appl. Microbiol. Biotechnol.99, 3341-3349.). D-valine was used as the core for the synthesis of penicillin. In recent years, demand for D-amino acids has rapidly increased in industries related to food additives, pharmaceutical intermediates, cosmetics, and the like. Global industry analytics predicts that the global D-amino acid market will exceed $ 37 billion in 2017 in D-amino acid market reports released in 2011. Among them, D-methionine has a wide range of actions as an important chiral amino acid, for example: d-methionine and gentamicin can prevent the occurrence of ototoxicity (Inorganica chimica acta,1998,273(1-2), 85-91); and 5-fluorouracil inhibit gastric cancer cell proliferation, induce gastric cancer cell apoptosis, etc. (J.Zhonghua gastrointestinal surgery, 2002, 5(3), 189-192.). Therefore, the research on the preparation process of the D-methionine is of great significance, and meanwhile, the efficient and green synthesis of the D-methionine is realized, so that the method is of great significance for meeting market demands and promoting the social and economic development.

At present, the synthesis of D-amino acid mainly comprises enzymatic conversion separation and asymmetric conversion. In the field of enzymatic conversion and separation preparation, there have been many reports (WO2003044206), and the preparation process generally includes chemically modifying DL-methionine (such as inoculating acetyl, carbamoyl, phenylacetyl, etc.), and using corresponding enzyme to convert and separate to obtain D-methionine. The enzymatic resolution method has complex process and harsh operating conditions; in the aspect of asymmetric transformation research, Noda et al (Tetrahedron: Asymmetry,2002,13(24), 2649-. Kim et al (US20060173211) reported that L-methionine as a raw material was reacted with S-2, 2' -binaphthol derivative and triethylamine to convert 91.6% of L-methionine into D-methionine, the reaction time was long, about 48 hours was required, and the optical purity of the product was not high enough. Liao benren, etc. (synthesis and process technology, 2013, 35(6), 558-. Substituted salicylaldehyde is used as a racemization catalyst, and a chiral organic acid auxiliary agent is added to realize the asymmetric transformation of L-methionine to prepare D-methionine, wherein the once-through yield is more than 80%. The methods generally have the problems of low yield of final products, complex extraction of intermediates, low optical purity of products and the like. It is therefore of crucial importance to find suitable processes for the preparation of D-methionine and other aliphatic amino acids.

The hydantoinase process is a classical multi-enzyme cascade comprising: hydantoin racemases, D/L-hydantoinases and D/L-carbamoyl hydrolases. Wherein, the hydantoin racemase is responsible for the racemization of L-or D-hydantoin, D/L-hydantoin enzyme catalytic hydrolysis D-or L-hydantoin produces D-or L-carbamoylamino acid, obtain D-or L-amino acid through D-or L-N-carbamoylase finally, the theoretical yield of reaction is high, there is no intermediate extraction and purification step. The dynamic kinetic resolution reaction can realize 100% of theoretical yield and high enantioselectivity, and has great application potential in the production and preparation of chiral amino acid. The currently reported method utilizing the hydantoinase process can realize the complete conversion of 300mmol/L L-methylthioethyl hydantoin to generate corresponding D-methionine (appl. environ. Microbiol.,2007,73(5), 1525-.

Disclosure of Invention

To solve the above problems, the present invention provides a D-N-carbamoyl hydrolase having good solubility, exhibiting high stereoselectivity to a carbamoyl amino acid substrate in D configuration, and having high catalytic activity to D-N-carbamoyl methionine and other aliphatic amino acids. The compound is applied to the preparation of D-aliphatic amino acid by the hydantoinase cascade reaction, has high catalytic efficiency (the conversion rate is more than 99.9 percent), strong stereoselectivity (e.e. >99.9 percent), mild reaction conditions and environmental friendliness.

The first purpose of the invention is to provide a method for preparing D-aliphatic amino acid by cascade reaction, which takes D-N-carbamyl hydrolase as a catalyst to prepare the D-aliphatic amino acid in the course of hydantoinase cascade reaction, wherein the amino acid sequence of the D-N-carbamyl hydrolase is shown as SEQ ID NO. 2.

Furthermore, the nucleotide sequence of the D-N-carbamyl hydrolase is shown as SEQ ID NO. 1.

Further, the method specifically comprises the step of reacting L-methylthioethyl hydantoin serving as a substrate with D-N-carbamoyl hydrolase, hydantoin racemase and hydantoin enzyme serving as catalysts to generate D-methionine or other aliphatic amino acids.

Furthermore, the concentration of the L-methylthio ethyl hydantoin is 5-1000 mmol/L.

Furthermore, the addition amount of the D-N-carbamyl hydrolase is 1-50 kU/L.

Furthermore, the addition amount of the hydantoin racemase is 1-15 kU/L.

Furthermore, the addition amount of the hydantoinase is 1-20 kU/L.

Further, the temperature of the hydantoin enzyme process cascade reaction is 20-40 ℃.

In the invention, a D-N-carbamoylase gene with a nucleotide sequence shown as SEQ ID NO.1 is constructed on a pET28a vector, and is introduced into recombinant Escherichia coli BL21(DE3) to obtain recombinant bacteria BL21(DE3)/pET28 a-CahyuC. Culturing the recombinant bacteria, and inducing to obtain the recombinant D-N-carbamyl hydrolase.

The invention has the beneficial effects that:

the invention provides a D-N-carbamyl hydrolase which can be used as a catalyst for preparing optically pure D-aliphatic amino acid in a hydantoinase process, and has the advantages of good solubility, high catalytic efficiency (the conversion rate is more than 99.9%), strong stereoselectivity (e.e. > 99.9%), mild reaction conditions and environmental friendliness. The D-N-carbamyl hydrolase of the invention has good catalytic effect, wide substrate applicability and good application and development prospect.

Drawings

FIG. 1 is the PCR amplification electrophoresis pattern of gene CahyuC. M, Marker; 1, gene CahyuC

FIG. 2 is a physical map of pET28a-CahyuC recombinant plasmid.

FIG. 3 is a protein electrophoresis of recombinant D-N-carbamoylase. M, Marker; lanes 1, 2 and 3 are the supernatant and precipitate after induction by recombinant genetically engineered bacterium BL21(DE3)/pET28a-CahyuC, respectively, and the electrophoretogram after purification.

FIG. 4 is a liquid phase assay of the selectivity of D-N-carbamoylase.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.