CN113512571A

CN113512571A - Method for synthesizing L-pipecolic acid by ornithine cyclodeaminase catalysis

Info

Publication number: CN113512571A
Application number: CN202110788366.XA
Authority: CN
Inventors: 范文超; 高书良; 杨海锋; 黎肖平
Original assignee: Zhejiang Huarui Biotechnology Co ltd
Current assignee: Zhejiang Huarui Biotechnology Co ltd
Priority date: 2021-07-13
Filing date: 2021-07-13
Publication date: 2021-10-19
Anticipated expiration: 2041-07-13
Also published as: CN113512571B

Abstract

The invention provides a method for preparing L-pipecolic acid by utilizing ornithine cyclodeaminase, which comprises the following steps: l-lysine is used as a substrate, and ornithine cyclodeaminase SEQ ID NO 1, 3 or 5 is used for catalyzing cyclodeamination reaction to obtain the L-pipecolic acid.

Description

Method for synthesizing L-pipecolic acid by ornithine cyclodeaminase catalysis

Technical Field

The invention belongs to the technical field of biocatalysis, and particularly relates to a method for preparing L-pipecolic acid by utilizing ornithine cyclodeaminase catalysis.

Background

L-pipecolic acid (CAS3105-95-1, PA for short) is one of imino acids, exists in most plants in a free state, is particularly found in leguminous plants at a high concentration, is used as an important rigid cyclic nonprotein amino acid, can limit the conformation of a polypeptide, can be used as a multifunctional skeleton in a synthesis library of different compounds, can be widely used for synthesizing a plurality of chiral drugs, and is a widely applied medical intermediate. For example, Ropivacaine (Ropivacaine), Thioridazine (Thioridazine), an immunosuppressant Rapamycin (Rapamycin), and the antitumor antibiotic Sandramycin (Sandramycin) of the new generation are all prepared from pipecolic acid or derivatives thereof as main raw materials.

Due to the biological activity and wide application prospect of the piperidine acid and the derivatives thereof, a plurality of new synthetic methods and new technologies are developed around the preparation of the piperidine acid and the derivatives thereof. The current preparation methods mainly comprise biochemical catalysis, chemical asymmetric synthesis and photocatalytic synthesis. Because L-pipecolic acid is a chiral molecule and asymmetric chiral synthesis is difficult, the preparation of the L-pipecolic acid with high optical purity by using an enzyme catalysis method with high selectivity is always a research hotspot.

Patent document CN1529756A reports that L-lysine is directly deaminated and cyclized to L-pipecolic acid by using enzymes such as Ornithine Cyclodeaminase (OCD) derived from Agrobacterium tumefaciens (Agrobacterium tumefaciens C58), iminopeptidase pipA derived from Streptomyces pristinaespiralis ATCC25486, and lysine cyclodeaminase (rapL) derived from Streptomyces hygroscopicus. Patent document CN109402099A further reports that Lysine Cyclodeaminase (LCD) derived from Streptomyces pristinaespiralis ATCC25486 and its mutant can convert L-Lysine into L-pipecolic acid, demonstrating that the enzyme-catalyzed method converts L-Lysine into L-pipecolic acid is a green process with application prospects.

However, experiments prove that the actual catalytic reaction efficiency when the enzyme reported in the patent literature is used for L-lysine conversion is far from different, obviously low and far from the enzyme activity/catalytic efficiency requirements of industrial application.

Disclosure of Invention

In order to explore the industrial feasibility of preparing L-pipecolic acid by an enzyme-catalyzed L-lysine conversion method, the inventor conducts extensive screening on enzymes such as Ornithine Cyclodeaminase (OCD), Lysine Cyclodeaminase (LCD) and imino peptidase pipA from a plurality of microorganisms, and finally screens enzyme varieties with obviously improved enzyme activity (at least one time higher) than the enzymes in the prior art, including the ornithine cyclodeaminase from Streptomyces melanospongiensis (NCBI accession number WP-093467660.1, with the amino acid sequence of SEQ ID NO: 1); based on this enzyme, we also mutated it in order to increase its enzymatic activity. Specifically, the present invention includes the following technical solutions.

A method for preparing L-pipecolic acid through enzyme catalysis comprises the following steps: l-lysine is used as a substrate, and ornithine cyclodeaminase SEQ ID NO. 1, or (G69V, G145L) mutant SEQ ID NO. 3 or (G69N, V142H) mutant SEQ ID NO. 5 of the ornithine cyclodeaminase catalyzes cyclodeamination reaction to obtain the L-pipecolic acid.

Wherein, the ornithine cyclodeaminase mutant SEQ ID NO. 3 is a mutant of wild ornithine cyclodeaminase SEQ ID NO. 1 amino acid sequences G69V and G145L:

METTVLTRQHVAKIVQGKGLDLFMDRMIDRLDEAFRAESRWGITPARDGFVRGPQNTAVLEWMPHHQPVDSITIKTVAYTPTNPFTHQLPTIIGTMARYDDVTGRLLAVGDGILPTAVRTGAASAIASRLLAHPDSRVLGLVGALAQAVTQAHALSRVFRLDRILVHDIEPAHAESFAERVEFLGIDVEVASVAEIEAASDIICTVTSVGVGDGPVLHGERLRPHVHINAIGADLIGKYEVPLSVLKSAFVTPDHQGQALREGECQQLDQSELGPELPALCADPALAEGRREQPTVFDSTGFALEDHVAFDVLLELAEEAGIGDRVQLEHLPEDALNPYSFQ(SEQ ID NO:3)；

ornithine cyclodeaminase mutant SEQ ID NO. 5 is a mutant of SEQ ID NO. 1 amino acid sequence G69N, V142H:

METTVLTRQHVAKIVQGKGLDLFMDRMIDRLDEAFRAESRWGITPARDGFVRGPQNTAVLEWMPHHQPNDSITIKTVAYTPTNPFTHQLPTIIGTMARYDDVTGRLLAVGDGILPTAVRTGAASAIASRLLAHPDSRVLGLHGAGAQAVTQAHALSRVFRLDRILVHDIEPAHAESFAERVEFLGIDVEVASVAEIEAASDIICTVTSVGVGDGPVLHGERLRPHVHINAIGADLIGKYEVPLSVLKSAFVTPDHQGQALREGECQQLDQSELGPELPALCADPALAEGRREQPTVFDSTGFALEDHVAFDVLLELAEEAGIGDRVQLEHLPEDALNPYSFQ(SEQ ID NO:5)。

more preferably, the above method uses ornithine cyclodeaminase SEQ ID NO 3 as a biocatalyst to catalyze the reaction of L-pipecolic acid.

In an alternative embodiment, the ornithine cyclodeaminase SEQ ID NO 1 or a mutant thereof SEQ ID NO 3 or SEQ ID NO 5 may be in the form of an expression microorganism thereof.

The reaction system is preferably a buffer solution, and may be a phosphate buffer solution, for example, a buffer solution having a pH of 6.5 to 7.5, preferably a pH of 6.8 to 7.2, more preferably a pH of about 7.0.

The reaction temperature may be 25 to 45 ℃.

In a second aspect, the invention provides an ornithine cyclodeaminase, the amino acid sequence of which is SEQ ID NO. 3 or SEQ ID NO. 5.

In a third aspect, the present invention provides a gene encoding the ornithine cyclodeaminase as described above.

Preferably, the nucleotide sequence of the gene encoding ornithine cyclodeaminase SEQ ID NO 3 is SEQ ID NO 4.

A fourth aspect of the present invention provides a microorganism expressing the ornithine cyclodeaminase of SEQ ID NO. 3 or SEQ ID NO. 5.

The microorganism can be selected from Bacillus subtilis, Lactobacillus brevis, Escherichia coli, and Corynebacterium glutamicum. Preferably, the microorganism is escherichia coli BL21(DE 3).

Obviously, the microorganisms described above can be used as biocatalysts for the production of L-pipecolic acid.

The ornithine cyclodeaminase SEQ ID NO. 1 and the ornithine cyclodeaminase mutants SEQ ID NO. 3 and 4 provided by the invention can promote L-lysine to generate cyclodeamination reaction to obtain L-pipecolic acid, and a new way is provided for industrialization of producing the L-pipecolic acid by a biological catalysis method.

Detailed Description

L-pipecolic acid is also called L-2-pipecolic acid, L-pipecolic acid or L-2-pipecolic acid. In order to find an enzyme which can be industrially exploited and applied for the conversion of L-lysine into L-pipecolic acid, the inventors have conducted extensive analyses of enzymes derived from microorganisms having a function of cyclodeaminase of amino acids with high stereoselectivity, and have found that Lysine Cyclodeaminase (LCD) and Ornithine Cyclodeaminase (OCD) are the main enzymes. Through comparison experiments, the enzyme activity of the ornithine cyclodeaminase (NCBI accession number WP _093467660.1) from streptomyces nigrosporus is higher; the amino acid number (342aa) is slightly smaller than that of the LCD and OCD in the prior art, and the research and the modification of the molecular structure of the enzyme are also facilitated.

As used herein, the terms "ornithine cyclodeaminase mutant", "mutant ornithine cyclodeaminase", "mutant OCD" and "mutant enzyme" mean the same and all refer to a mutant ornithine cyclodeaminase. For the sake of brevity, sometimes for the sake of convenience of description, the wild-type ornithine cyclodeaminase and its mutant may be collectively referred to as "ornithine cyclodeaminase" in the present invention as long as they are not confused with the wild-type enzyme SEQ ID NO: 1. Wherein the amino acid sequence of the wild-type ornithine cyclodeaminase is SEQ ID NO: 1:

METTVLTRQHVAKIVQGKGLDLFMDRMIDRLDEAFRAESRWGITPARDGFVRGPQNTAVLEWMPHHQPGDSITIKTVAYTPTNPFTHQLPTIIGTMARYDDVTGRLLAVGDGILPTAVRTGAASAIASRLLAHPDSRVLGLVGAGAQAVTQAHALSRVFRLDRILVHDIEPAHAESFAERVEFLGIDVEVASVAEIEAASDIICTVTSVGVGDGPVLHGERLRPHVHINAIGADLIGKYEVPLSVLKSAFVTPDHQGQALREGECQQLDQSELGPELPALCADPALAEGRREQPTVFDSTGFALEDHVAFDVLLELAEEAGIGDRVQLEHLPEDALNPYSFQ(SEQ ID NO:1)。

interestingly, the mutation sites in the two mutants with significantly improved enzyme activities of the present invention both comprise the 69 th G, and the 145 th G of the other mutation site of SEQ ID NO. 3 is also very close to the 142 th V of the other mutation site of SEQ ID NO. 5, which suggests that the sites near the 69 th and the 142 rd and 145 th sites may be in the active center of the enzyme, so it is necessary to further perform simulation and calculation of the three-dimensional structure of the enzyme by bioinformatics technology in order to further improve the ornithine cyclodeaminase and apply it to production practice, which is the content of subsequent research.

Since the number of the amino acids of the ornithine cyclodeaminase and the mutant thereof selected by the present invention is only 342 and the structure is clear, the encoding genes thereof, expression cassettes and plasmids containing the genes, and transformants containing the plasmids can be easily obtained by those skilled in the art.

These genes, expression cassettes, plasmids, and transformants can be obtained by genetic engineering construction means well known to those skilled in the art.

In order to optimally express the ornithine cyclodeaminase SEQ ID NO. 1 and mutants SEQ ID NO. 3 and 5 thereof in a microbial host such as Escherichia coli which is most commonly used in genetic engineering, the expression gene is codon-optimized.

Codon optimization is one technique that can be used to maximize protein expression in an organism by increasing the translation efficiency of a gene of interest. Different organisms often show a special preference for one of several codons encoding the same amino acid due to mutation tendencies and natural selection. For example, in rapidly growing microorganisms such as E.coli, the optimized codons reflect the composition of their respective pools of genomic tRNA's. Thus, in a fast growing microorganism, the low frequency codons of an amino acid can be replaced with codons for the same amino acid but with a high frequency. Thus, expression of optimized DNA sequences is improved in fast growing microorganisms.

Through codon optimization, the coding gene of the wild ornithine cyclodeaminase SEQ ID NO. 1 can be SEQ ID NO. 2, and the coding gene of the ornithine cyclodeaminase mutant SEQ ID NO. 3 can be SEQ ID NO. 4.

When used as a biocatalyst for the production of L-pipecolic acid, the ornithine cyclodeaminase of the present invention may be in the form of an enzyme or in the form of a bacterial cell. The enzyme forms comprise free enzyme and immobilized enzyme, including purified enzyme, crude enzyme, fermentation liquor, enzyme immobilized by a carrier and the like; the form of the thallus comprises a viable thallus and a dead thallus.

As is well known in the field of biological catalysis, compared with a free enzyme method, the application of an immobilized enzyme technology has the advantages of simplified production process, improved production efficiency and the like. Meanwhile, the enzyme can be used for multiple times, and the stability of the enzyme is improved, so that the productivity of unit enzyme is effectively improved; and secondly, the immobilized enzyme is easily separated from the substrate and the product, the purification process is simplified, the yield is high, and the product quality is good.

As is readily understood by those skilled in the art, the bacterial cells themselves are a natural enzyme immobilized form, and can be used as an enzyme preparation for catalyzing reactions without carrying out crushing treatment, even extraction and purification treatment. Since the reaction substrate and the reaction product can easily pass through the cell membrane, which is a biological barrier of the cells, it is not necessary to subject the cells to a disruption treatment, which is economically advantageous.

On the other hand, compared with the catalysis of the separated enzyme, the invention can continuously and inexhaustibly provide the enzyme or the supply by utilizing the simple fermentation of the microorganism without further operations such as extraction, purification and separation of the enzyme, has obvious economic benefit and creates conditions for industrial application.

The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

In the examples, the addition, content and concentration of various substances are mentioned, wherein the percentages refer to mass percentages unless otherwise indicated.

Examples

Materials and methods

The whole gene synthesis, primer synthesis and sequencing in the examples were performed by Jinzhi Biotechnology, Inc., Suzhou.

The molecular biological experiments herein include plasmid construction, enzyme digestion, competent cell preparation, transformation, etc., which are mainly performed with reference to molecular cloning, a guide to experiments (third edition), J. SammBruk, D.W. Lassel (America), Huangpetang, et al, scientific Press, Beijing, 2002). For example, the competent cell transformation method and the competent cell preparation method are carried out with reference to chapter 1, page 96. The specific experimental conditions can be determined by simple experiments if necessary.

PCR amplification experiments were performed according to the reaction conditions or kit instructions provided by the supplier of the plasmid or DNA template. If necessary, it can be adjusted by simple experiments.

LB culture medium: 5g/L yeast extract, 10g/L tryptone, 10g/L sodium chloride. (20 g/L agar powder was additionally added to LB solid medium.)

TB culture medium: 24g/L yeast extract, 12g/L tryptone, 16.43g/L K₂HPO₄.3H₂O、2.31g/L KH₂PO₄5g/L of glycerol, and the pH value is 7.0-7.5. (20 g/L agar powder was additionally added to TB solid medium.)

HPLC detection conditions of L-pipecolic acid:

a detection instrument:	agilent 1200 type high performance liquid chromatograph (Agilent SB-Aq column)
		Mobile phase A:	3% sodium dihydrogen phosphate (15g to 500ml water)
A: b (acetonitrile)	95：5
		Flow rate of flow	0.5mL/min
Column oven	30℃
		Detection wavelength	210nm
Time of detection	15min
		Sample volume	10μl
L-pipecolic acid retention time	6.324min

Determination of Activity of Ornithine Cyclodeaminase (OCD)

Reaction system: 50mM L-lysine, 150mM, pH7.0 potassium phosphate buffer, 0.1% triton X-100, 20mM FeSO₄The concentration of the bacteria is 10 v/v%;

reaction conditions are as follows: 37 ℃ and 150 rpm;

sampling time: and (5) 30 h.

Definition of enzyme activity unit: the amount of enzyme required to catalyze the production of 1. mu. mol (. mu. mol) of L-pipecolic acid from the substrate L-lysine per hour at pH7.0 at 37 ℃ is defined as 1 unit (U).

Example 1: construction of recombinant Escherichia coli expressing wild-type ornithine cyclodeaminase

For wild-type ornithine cyclodeaminase SEQ ID NO:1 derived from Streptomyces melanosporaneciens, an optimized codon sequence SEQ ID NO:2 suitable for Escherichia coli expression was designed, and a gold-only intelligence was requested to perform whole gene synthesis, and restriction enzyme sites Nde I and XhoI were designed at both ends of the gene, and subcloned into pSH plasmid (containing NdeI/XhoI sites) constructed by Wai Biotechnology Limited, Zhejiang, to obtain recombinant plasmid pSH-OCD. The recombinant plasmid pSH-OCD was transformed into expression host E.coli BL21(DE3) to obtain recombinant E.coli expressing wild-type Ornithine Cyclodeaminase (OCD), which is abbreviated as Ocd.

Example 2: construction of error-prone PCR and random mutation libraries

2 is used as a template, a primer pair Ocd-Nde1-F/Ocd-Xho1-R is designed, and an error-prone PCR technology is applied to construct a random mutant library.

Forward primer Ocd-Nde 1-F: 5'-ATATACATATGGAAACCACCGTGC-3' the flow of the air in the air conditioner,

reverse primer Ocd-Xho 1-R: 5'-GTCGACTTACTGAAAGCTATACGGG-3' are provided.

The 50 μ L error-prone PCR reaction system included: 50ng of plasmid template pSH-OCD, 5. mu.L of DMSO,30pmol of a pair of primers Ocd-Nde1-F and Ocd-Xho1-R, 1 XTaq buffer, 0.2mM dGTP, 0.2mM dATP, 1mM dCTP, 1mM dTTP, 7mM MgCl₂，(0mM、0.05mM、0.1mM、0.15mM、0.2mM)MnCl₂2.5 units of Taq enzyme (Thermo Fisher Scientific).

The PCR reaction conditions are as follows: 5min at 95 ℃; 94 ℃ for 30s, 60 ℃ for 30s, 72 ℃ for 2 min/kbp; 30 cycles; 10min at 72 ℃. Gel recovery of 1kb random mutant fragment as large primer, Megaprimer PCR with KOD-plus DNA polymerase: 5min at 94 ℃; 10s at 98 ℃, 30s at 60 ℃, 2min/kbp at 68 ℃ and 25 cycles; 10min at 68 ℃.

DpnI digestion of plasmid template, electrotransformation of E.coli BL21(DE3) to yield more than 10⁴Random mutant pools of individual clones.

Example 3: screening of pools of random mutants

Transformants in the mutant pool were selected and inoculated into a 96-well deep-well plate containing 700. mu.L of LB medium containing 100. mu.g/mL kanamycin, cultured at 37 ℃ for 6 hours, added with 0.1mM IPTG at the final concentration, cooled to 25 ℃ and cultured overnight. Centrifuging at 5000rpm for 10min, discarding supernatant, adding 200 μ L50 mM Tris-HCl (pH7.5), and resuspending the thallus for OCD enzyme activity determination.

The experimental scheme for determining the activity of the mutant OCD enzyme, namely the L-lysine conversion, is as follows:

reaction system: 400mM L-lysine, 150mM, pH7.0 potassium phosphate buffer, 0.1% triton X-100, 20mM FeSO₄Adjusting the pH value to 7.5, and carrying out bacterial concentration of 15 v/v%;

reaction conditions are as follows: at 37 ℃, 150rpm for 24 h;

termination conditions were as follows: 90 ℃ for 5 min.

After the reaction was terminated, the reaction mixture was centrifuged at 13000rpm for 5min, and the supernatant was collected and subjected to liquid phase detection.

Through screening, the enzyme activities of the two mutants are found to be obviously higher than that of a wild ornithine cyclodeaminase expression strain Ocd, and the two mutant strains are numbered Ocd-1322 and Ocd-4500 respectively.

The mutant strain is entrusted with Jinzhi to carry out genome sequencing alignment, the gene sequence of the ornithine cyclodeaminase in the genome of the strain Ocd-4500 is SEQ ID NO. 4, the coded amino acid sequence is confirmed to be SEQ ID NO. 3, and the coded amino acid sequence is a (G69V, G145L) mutant. The amino acid sequence of ornithine cyclodeaminase expressed by the other mutant strain Ocd-1322 was SEQ ID NO. 5, which is a (G69N, V142H) mutant.

Example 4: validation of mutant enzymes

Plasmids pSH-Ocd-4500 and pSH-Ocd-1322, which express ornithine cyclodeaminase (G69V, G145L) mutant of SEQ ID NO:3 and (G69N, V142H) mutant of SEQ ID NO:5, were constructed according to the method of example 1.

Plasmids pSH-Ocd-1322 and pSH-Ocd-4500 containing Ocd-1322 and Ocd-4500 genes are transformed into BL21(DE3) competent cells respectively to obtain recombinant escherichia coli Ocd-1322 and Ocd-4500 respectively. Culturing overnight in kan + sakaguchi, selecting 10 single colonies, inoculating to test tube containing LB culture medium, culturing overnight, centrifuging to collect thallus, extracting plasmid, and sequencing gene to determine the correct mutation.

The enzyme activities of two mutant strains Ocd-1322 and Ocd-4500 were measured according to the method of example 3 and compared with that of strain Ocd. The results are shown in the following table.

Table 1: comparison of catalytic Activity of OCD enzymes catalyzing conversion of L-lysine in respective strains

Bacterial strains	OCD mutant amino acids	Amino acid sequence of enzyme	Relative activity fold
				Ocd	-	SEQ ID NO:1	1.00
Ocd-1322	G69N,V142H	SEQ ID NO:5	6.82
				Ocd-4500	G69V,G145L	SEQ ID NO:3	12.5

Specific activity of enzyme: the ratio of the fermentation activity (U/ml) of the wild enzyme to the thallus concentration OD600(OD/ml) was 100%.

The experimental results show that, from the thallus level, the enzyme activity of the mutant SEQ ID NO. 3 is 11 times higher than that of the wild enzyme SEQ ID NO. 1, and the other mutant SEQ ID NO. 5 is 5 times higher than that of the wild enzyme, so that the mutant has higher catalytic activity for catalyzing the L-lysine cyclodeamination reaction. The mutant SEQ ID NO 3 was examined with emphasis.

Example 5: preparation of L-pipecolic acid

Selecting a single clone from a plate of Ocd-4500 engineering strains, inoculating the single clone to 5ml of LB culture medium, and culturing overnight at 37 ℃; inoculating 1 v/v% into 1000ml shake flask containing 100ml TB medium, culturing for 4-6 hr until OD600 reaches 1.2-1.5, adding 0.1mM IPTG for induction, cooling to 25 deg.C, culturing for 10-16 hr, centrifuging to obtain thallus, and freezing at-80 deg.C for 24 hr.

The reaction system is 200mL, the substrate L-lysine is 50g/L, the enzyme adding amount is respectively 10 v/v% and 15 v/v% concentration, the temperature is 37 ℃, the rpm is 200rpm, the pH is controlled to be 7.0, the reaction is carried out for 96h, the L-pipecolic acid generation amount is measured, and the product generation rate is calculated. The results are shown in Table 2 below.

Table 2: investigation of mutant strain SEQ ID NO. 3 catalyzing conversion of L-lysine

The experiments show that the ornithine cyclodeaminase SEQ ID NO. 1 and the mutant SEQ ID NO. 3 and the mutant SEQ ID NO. 5 thereof can catalyze the conversion of L-lysine to obtain the L-pipecolic acid, wherein the mutant SEQ ID NO. 3 has higher catalytic efficiency and has development and application prospects.

Sequence listing

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<213> Artificial sequence ()

<400> 4

atggaaacca ccgtgctgac ccgccagcat gtggcgaaaa ttgtgcaggg caaaggcctg 60

gatctgttta tggatcgcat gattgatcgc ctggatgaag cgtttcgcgc ggaaagccgc 120

tggggcatta ccccggcgcg cgatggcttt gtgcgcggcc cgcagaacac cgcggtgctg 180

gaatggatgc cgcatcatca gccggtggat agcattacca ttaaaaccgt ggcgtatacc 240

ccgaccaacc cgtttaccca tcagctgccg accattattg gcaccatggc gcgctatgat 300

gatgtgaccg gccgcctgct ggcggtgggc gatggcattc tgccgaccgc ggtgcgcacc 360

ggcgcggcga gcgcgattgc gagccgcctg ctggcgcatc cggatagccg cgtgctgggc 420

ctggtgggcg cgctggcgca ggcggtgacc caggcgcatg cgctgagccg cgtgtttcgc 480

ctggatcgca ttctggtgca tgatattgaa ccggcgcatg cggaaagctt tgcggaacgc 540

gtggaatttc tgggcattga tgtggaagtg gcgagcgtgg cggaaattga agcggcgagc 600

gatattattt gcaccgtgac cagcgtgggc gtgggcgatg gcccggtgct gcatggcgaa 660

cgcctgcgcc cgcatgtgca tattaacgcg attggcgcgg atctgattgg caaatatgaa 720

gtgccgctga gcgtgctgaa aagcgcgttt gtgaccccgg atcatcaggg ccaggcgctg 780

cgcgaaggcg aatgccagca gctggatcag agcgaactgg gcccggaact gccggcgctg 840

tgcgcggatc cggcgctggc ggaaggccgc cgcgaacagc cgaccgtgtt tgatagcacc 900

ggctttgcgc tggaagatca tgtggcgttt gatgtgctgc tggaactggc ggaagaagcg 960

ggcattggcg atcgcgtgca gctggaacat ctgccggaag atgcgctgaa cccgtatagc 1020

tttcagtaa 1029

<210> 5

<211> 342

<212> PRT

<213> Artificial sequence ()

<400> 5

Met Glu Thr Thr Val Leu Thr Arg Gln His Val Ala Lys Ile Val Gln

1 5 10 15

Gly Lys Gly Leu Asp Leu Phe Met Asp Arg Met Ile Asp Arg Leu Asp

20 25 30

Glu Ala Phe Arg Ala Glu Ser Arg Trp Gly Ile Thr Pro Ala Arg Asp

35 40 45

Gly Phe Val Arg Gly Pro Gln Asn Thr Ala Val Leu Glu Trp Met Pro

50 55 60

His His Gln Pro Asn Asp Ser Ile Thr Ile Lys Thr Val Ala Tyr Thr

65 70 75 80

Pro Thr Asn Pro Phe Thr His Gln Leu Pro Thr Ile Ile Gly Thr Met

85 90 95

Ala Arg Tyr Asp Asp Val Thr Gly Arg Leu Leu Ala Val Gly Asp Gly

100 105 110

Ile Leu Pro Thr Ala Val Arg Thr Gly Ala Ala Ser Ala Ile Ala Ser

115 120 125

Arg Leu Leu Ala His Pro Asp Ser Arg Val Leu Gly Leu His Gly Ala

130 135 140

Gly Ala Gln Ala Val Thr Gln Ala His Ala Leu Ser Arg Val Phe Arg

145 150 155 160

Leu Asp Arg Ile Leu Val His Asp Ile Glu Pro Ala His Ala Glu Ser

165 170 175

Phe Ala Glu Arg Val Glu Phe Leu Gly Ile Asp Val Glu Val Ala Ser

180 185 190

Val Ala Glu Ile Glu Ala Ala Ser Asp Ile Ile Cys Thr Val Thr Ser

195 200 205

Val Gly Val Gly Asp Gly Pro Val Leu His Gly Glu Arg Leu Arg Pro

210 215 220

His Val His Ile Asn Ala Ile Gly Ala Asp Leu Ile Gly Lys Tyr Glu

225 230 235 240

Val Pro Leu Ser Val Leu Lys Ser Ala Phe Val Thr Pro Asp His Gln

245 250 255

Gly Gln Ala Leu Arg Glu Gly Glu Cys Gln Gln Leu Asp Gln Ser Glu

260 265 270

Leu Gly Pro Glu Leu Pro Ala Leu Cys Ala Asp Pro Ala Leu Ala Glu

275 280 285

Gly Arg Arg Glu Gln Pro Thr Val Phe Asp Ser Thr Gly Phe Ala Leu

290 295 300

Glu Asp His Val Ala Phe Asp Val Leu Leu Glu Leu Ala Glu Glu Ala

305 310 315 320

Gly Ile Gly Asp Arg Val Gln Leu Glu His Leu Pro Glu Asp Ala Leu

325 330 335

Asn Pro Tyr Ser Phe Gln

340

Claims

1. A method for preparing L-pipecolic acid through enzyme catalysis comprises the following steps:

l-lysine is used as a substrate, and ornithine cyclodeaminase SEQ ID NO. 1, or (G69V, G145L) mutant SEQ ID NO. 3 or (G69N, V142H) mutant SEQ ID NO. 5 of the ornithine cyclodeaminase catalyzes cyclodeamination reaction to obtain the L-pipecolic acid.

2. The process as claimed in claim 1, wherein ornithine cyclodeaminase SEQ ID NO 3 is used as catalyst.

3. The method of claim 1, wherein the ornithine cyclodeaminase SEQ ID No. 1 or mutant thereof SEQ ID No. 3 or SEQ ID No. 5 is in the form of its expressing microorganism.

4. The method of claim 1, wherein the reaction system is a buffer solution.

5. The method of claim 4, wherein the reaction system is phosphate buffered saline at pH 6.5-7.5.

6. An ornithine cyclodeaminase, characterized in that the amino acid sequence is SEQ ID NO. 3 or SEQ ID NO. 5.

7. A gene encoding the ornithine cyclodeaminase of claim 6.

8. The gene of claim 7, wherein the nucleotide sequence of the gene encoding ornithine cyclodeaminase SEQ ID NO 3 is SEQ ID NO 4.

9. A microorganism expressing the ornithine cyclodeaminase of SEQ ID NO 3 or SEQ ID NO 5 as set forth in claim 6.

10. The microorganism of claim 9, wherein the microorganism is escherichia coli.