CN114015665A - Engineered NADPH-dependent phenylglycine dehydrogenase and application thereof - Google Patents

Abstract

The invention belongs to the technical field of enzyme engineering, and particularly relates to an engineered NADPH-dependent phenylglycine dehydrogenase and application thereof, in order to develop the NADPH-dependent phenylglycine dehydrogenase with catalytic activity to phenylglyoxylic acid, the invention improves the catalytic activity of the glutamate dehydrogenase derived from Pseudomonas putida by nearly 40-72 times through substrate specificity modification, and develops and obtains a novel engineered NADPH-dependent phenylglycine dehydrogenase. The obtained NADPH-dependent phenylglycine dehydrogenase shows good catalytic efficiency in the preparation of L-phenylglycine by a biological catalysis method or a fermentation method, which shows that the NADPH-dependent phenylglycine dehydrogenase developed by the invention can efficiently synthesize chiral L-phenylglycine, lays a foundation for further developing an efficient biosynthesis process of L-phenylglycine, and has great application value.

Description

Engineered NADPH-dependent phenylglycine dehydrogenase and application thereof

Technical Field

The invention belongs to the technical field of enzyme engineering, and particularly relates to an engineered NADPH-dependent phenylglycine dehydrogenase and application thereof.

Background

L-phenylglycine is a high-value non-natural chiral amino acid, and can be used as an intermediate for synthesizing various medicaments, such as beta-lactam antibiotics, anticancer medicaments, namely taxol, antiplatelet medicaments, namely clopidogrel and the like. Meanwhile, the L-phenylglycine can also be used as a food additive, and partial research shows that the L-phenylglycine derivative has certain anti-diabetic activity. The L-phenylglycine has large consumption in domestic and international markets, and the development of the synthetic method of the L-phenylglycine has important significance.

The synthesis of L-phenylglycine is mainly divided into chemical synthesis and biological synthesis, wherein the biological synthesis can be divided into biological fermentation and biological catalysis. The chemical synthesis method has high catalytic speed, but has strict catalytic conditions, low stereoselectivity and is generally accompanied with the generation of byproducts. In contrast, the biosynthesis method based on the enzyme catalyst has the remarkable advantages of high stereoselectivity, mild reaction conditions, less environmental pollution and the like, and is an ideal process route for preparing the L-phenylglycine. The asymmetric amination reaction of phenylglyoxylic acid is an important way for generating L-phenylglycine by adopting a biological fermentation method or a biological catalysis method. While enzymes capable of catalyzing this reaction include two types: transaminases (Transaminases) and Amino acid dehydrogenases (Amino acid dehydrogenes). Among them, transaminases catalyzed transamination reactions are reversible, so the reaction system needs to provide an excess of organic amino donor to drive the reaction toward the product formation. In contrast, the preparation of L-phenylglycine by the reductive amination of phenylglyoxylic acid catalyzed by an amino acid dehydrogenase has two significant advantages: 1) due to its extremely small equilibrium constant (about 10-5M),keto acid substrates are capable of almost 100% conversion; 2) inorganic ammonium is used as an amino donor, so that byproducts which are difficult to separate are not generated, and the product is easy to purify and refine. At present, the amino acid dehydrogenases which have been reported to catalyze the reductive amination of phenylglyoxylic acid are all NADH-dependent, such as leucine dehydrogenase. The amino acid dehydrogenases have better catalytic efficiency in the process of synthesizing the L-phenylglycine by a biological catalysis method. However, these NADH-dependent amino acid dehydrogenases are not ideal alternatives for the construction of the fermentative biosynthetic pathway for L-phenylglycine. Because of intracellular NADH/NAD under aerobic conditions, which are customary in industrial amino acid fermentations⁺The ratio is NADPH/NADP only⁺The ratio of 1/10 is about, which results in poor catalytic effect in the process of L-phenylglycine artificial intracellular fermentation synthesis. Therefore, the development of NADPH-dependent amino acid dehydrogenase capable of catalyzing reductive amination of phenylglyoxylic acid, namely NADPH-dependent phenylglycine dehydrogenase, has important value for constructing a biosynthesis process of L-phenylglycine, particularly an artificial intracellular fermentation synthesis pathway of L-phenylglycine.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention obtains the NADPH-dependent phenylglycine dehydrogenase which can efficiently catalyze phenylglyoxylate to reduce, amine and synthesize L-phenylglycine by modifying the substrate specificity of glutamate dehydrogenase. The engineered NADPH dependent phenylglycine dehydrogenase can be used for preparing chiral L-phenylglycine by a biological catalysis method and a fermentation method, and has high practical application value.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention provides an engineered NADPH-dependent phenylglycine dehydrogenase which is a mutant formed by combined mutation of threonine 196, threonine 121 and leucine 123 in an amino acid sequence of glutamate dehydrogenase shown in SEQ ID NO. 1.

The invention firstly rationally designs the active pocket of glutamate dehydrogenase derived from Pseudomonas putida, and obtains a mutant T196A with remarkably improved catalytic activity to a target substrate phenylglyoxylate. On the basis of the above, a combinatorial mutation library of the amino acid residues at positions 121 and 123 was constructed. And further screening the mutant library by a high-throughput screening technology to obtain a plurality of mutants with the catalytic activity of the p-phenylglyoxylate further improved compared with that of T196A, namely the mutants are the engineered NADPH-dependent phenylglycine dehydrogenase.

As a preferred embodiment of the present invention, the engineered NADPH-dependent phenylglycine dehydrogenase comprises:

the amino acid sequence of the mutant is shown as SEQ ID NO.2, wherein the 196 th threonine of the amino acid sequence of the glutamate dehydrogenase shown as SEQ ID NO.1 is mutated into alanine, the 121 th threonine is mutated into isoleucine, and the 123 th leucine is mutated into aspartic acid;

the amino acid sequence of the mutant is shown as SEQ ID NO.3, wherein the 196 th threonine of the amino acid sequence of the glutamate dehydrogenase shown as SEQ ID NO.1 is mutated into alanine, the 121 th threonine is mutated into isoleucine, and the 123 th leucine is mutated into histidine;

the amino acid sequence of the mutant is shown in SEQ ID NO.4, wherein the 196 th threonine of the amino acid sequence of the glutamate dehydrogenase shown in SEQ ID NO.1 is mutated into alanine, the 121 th threonine is mutated into isoleucine, and the 123 th leucine is mutated into asparagine;

the amino acid sequence of the mutant is shown as SEQ ID NO.5, wherein the 196 th threonine of the amino acid sequence of the glutamate dehydrogenase shown as SEQ ID NO.1 is mutated into alanine, the 121 th threonine is mutated into isoleucine, and the 123 th leucine is mutated into serine;

the amino acid sequence of the mutant is shown in SEQ ID NO.6, wherein the 196 th threonine of the amino acid sequence of the glutamate dehydrogenase shown in SEQ ID NO.1 is mutated into alanine, the 121 th threonine is mutated into leucine, and the 123 th leucine is mutated into asparagine;

the amino acid sequence of the mutant is shown as SEQ ID NO.7, wherein the 196 th threonine of the amino acid sequence of the glutamate dehydrogenase shown as SEQ ID NO.1 is mutated into alanine, the 121 th threonine is mutated into leucine, and the 123 th leucine is mutated into serine;

the amino acid sequence of the mutant is shown in SEQ ID NO.8, wherein the 196 th threonine of the amino acid sequence of the glutamate dehydrogenase shown in SEQ ID NO.1 is mutated into alanine, the 121 th threonine is mutated into leucine, and the 123 th leucine is mutated into tyrosine;

the amino acid sequence of the mutant is shown in SEQ ID NO.9, wherein the 196 th threonine of the amino acid sequence of the glutamate dehydrogenase shown in SEQ ID NO.1 is mutated into alanine, the 121 th threonine is mutated into leucine, and the 123 th leucine is mutated into histidine.

The invention also provides an expression vector containing the engineered NADPH-dependent phenylglycine dehydrogenase.

Preferably, the expression vector is pET-28a (+) comprising the engineered NADPH-dependent phenylglycine dehydrogenase, or pTrc99a comprising the engineered NADPH-dependent phenylglycine dehydrogenase.

The invention also provides a recombinant engineering bacterium containing the engineered NADPH-dependent phenylglycine dehydrogenase.

Preferably, the recombinant engineered bacterium is escherichia coli BL21(DE3) introduced with the engineered NADPH-dependent phenylglycine dehydrogenase-containing expression vector; or E.coli BW25113 comprising the engineered NADPH-dependent phenylglycine dehydrogenase expression vector.

The invention also provides application of the engineered NADPH-dependent phenylglycine dehydrogenase in preparation of L-phenylglycine.

Preferably, the preparation of the L-phenylglycine comprises the biocatalytic preparation of the L-phenylglycine and the fermentative preparation of the L-phenylglycine.

Further, the preparation of the L-phenylglycine by the biocatalytic method comprises the following steps:

s1, constructing recombinant engineering bacteria for expressing the engineered NADPH-dependent phenylglycine dehydrogenase in claim 1 or 2, and preparing an enzyme solution after culturing;

s2, adding the enzyme solution obtained in the step S1 into a mixed system containing substrate phenylglyoxylic acid, an amino donor and reduced coenzyme, and carrying out reductive amination reaction to obtain the L-phenylglycine.

Further, the enzyme solution comprises a resting cell suspension of recombinant engineering bacteria or a crude enzyme solution of broken cells. Of course, the present invention is also applicable to a pure enzyme obtained by purifying the crude enzyme solution.

Furthermore, the temperature of the reductive amination reaction is 15-50 ℃, and the pH value of the reaction system is 5-10.

Further, the reduced coenzyme is reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH).

Further, the mixed system in step S2 further includes a coenzyme regeneration system. The coenzyme regeneration system comprises glucose dehydrogenase as coenzyme regeneration enzyme, glucose as coenzyme regeneration substrate, NADPH and NADP⁺The glucose dehydrogenase coenzyme regeneration system of (1); alcohol dehydrogenase as coenzyme regenerating enzyme, isopropanol as substrate, NADPH and NADP⁺The alcohol dehydrogenase coenzyme regeneration system of (1); uses formate dehydrogenase as coenzyme regenerating enzyme, uses formate as substrate for regenerating coenzyme, and contains NADPH and NADP⁺The system for regenerating a formate dehydrogenase coenzyme of (1).

Specifically, the coenzyme regeneration system is an alcohol dehydrogenase regeneration system, and the amino acid sequence of the alcohol dehydrogenase is shown as SEQ ID No. 10.

Further, the fermentation method for preparing the L-phenylglycine comprises the following steps:

s1, constructing recombinant engineering bacteria for expressing the engineered NADPH-dependent phenylglycine dehydrogenase of claim 1 or 2, inoculating the recombinant engineering bacteria into a seed culture medium, culturing for a period of time, and transferring the seed solution into a fermentation culture medium containing a phenylglyoxylate substrate and inorganic ammonium;

and S2, fermenting to make the concentration of the bacteria reach a certain degree, adding an inducer, and continuously fermenting and culturing to prepare the L-phenylglycine.

Further, the temperature of fermentation culture is 25-40 deg.C, and the pH value of culture medium is 6-8.

Still further, the inorganic ammonium includes, but is not limited to, ammonium sulfate, ammonium chloride, diammonium phosphate, monoammonium phosphate, or aqueous ammonia.

Compared with the prior art, the invention has the beneficial effects that:

in order to develop the NADPH-dependent phenylglycine dehydrogenase with catalytic activity to the phenylglyoxylic acid, the invention improves the catalytic activity of the glutamate dehydrogenase derived from Pseudomonas putida by nearly 40-72 times through carrying out substrate specificity transformation on the glutamate dehydrogenase, and develops and obtains the novel engineered NADPH-dependent phenylglycine dehydrogenase. Researches show that the obtained NADPH-dependent phenylglycine dehydrogenase has good catalytic efficiency in the preparation of L-phenylglycine by a biological catalytic method, the substrate conversion rate is more than 99%, the product concentration is as high as 30.18g/L, the ee value is more than 99%, and the catalytic efficiency is obviously higher than that of a wild type; meanwhile, the good catalytic efficiency is also shown in the preparation of L-phenylglycine by a fermentation method, the substrate conversion rate reaches 95%, the product concentration reaches 3.12g/L, and the ee value is more than 99%, which indicates that the NADPH-dependent phenylglycine dehydrogenase developed by the invention can efficiently utilize intracellular NADPH to synthesize chiral L-phenylglycine. Therefore, the NADPH-dependent phenylglycine dehydrogenase with catalytic activity on the phenylglyoxylic acid is successfully developed and obtained, a foundation is laid for further developing an efficient biosynthesis process of the L-phenylglycine, and the method has a huge industrial application value.

Drawings

FIG. 1 shows the results of enzyme activity measurement of a mutant glutamate dehydrogenase derived from Pseudomonas putida;

FIG. 2 is a reaction scheme for the preparation of L-phenylglycine catalyzed by an engineered NADPH-dependent phenylglycine dehydrogenase;

FIG. 3 is a high performance liquid chromatography detection (achiral analysis) of the product L-phenylglycine of example 3 together with a standard sample of the substrate phenylglyoxylic acid;

FIG. 4 is a pre-column derivatization HPLC (chiral analysis) of the reaction solution (10h) in example 3;

in FIG. 4, A is an analytical spectrum of L-phenylglycine and D-phenylglycine standard samples, and B is an analytical spectrum of the reaction solution.

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The experimental procedures in the following examples were carried out by conventional methods unless otherwise specified, and the test materials used in the following examples were commercially available by conventional methods unless otherwise specified.

The experimental methods in the present invention are conventional methods unless otherwise specified, and the gene cloning procedures can be specifically described in molecular cloning protocols, compiled by J. Sambruka et al.

Reagents used for upstream genetic engineering: DNA polymerase (2X Phanta Max Master Mix), Dpn I enzyme, recombinant cloning kit, plasmid extraction kit and gel recovery kit were purchased from Nanjing Novozam Biotech Ltd; primer synthesis and gene sequencing are completed by the Oncology Catayi Hixi Biotechnology (Guangzhou). The method of using the above reagent is referred to the commercial specification.

The expression vector of the invention is pET-28a (+) or pTrc99a, and the used host is Escherichia coli BL21(DE3) or Escherichia coli BW25113 which are purchased from Novagen company in Germany and are preserved in the laboratory.

Reagents used in the downstream catalytic conversion process: phenylglyoxalic acid, D-phenylglycine, L-phenylglycine, NADP⁺Both NADPH and Sigma-Aldrich; other commonly used reagents are available from the national pharmaceutical group chemical agents, ltd. The three-letter or one-letter expression of Amino Acids used in the present text uses the Amino acid code specified by IUPAC (Nomenculture of. alpha. -Amino Acids, Recommendations 1974, biochem. J.149: 1-16, 1975).

In the following examples, the concentrations of the product L-phenylglycine and the substrate phenylglyoxylic acid were determined by High Performance Liquid Chromatography (HPLC). The HPLC analysis method comprises the following steps: the type of the chromatographic column: the total weight of Athena C18,

5 μm, 4.6X 250 mm. Mobile phase: 50mM phosphate buffer pH 7.5: acetonitrile 0.9: 0.1. detection wavelength: 220 nm. Flow rate: 1 mL/min. Column temperature: at 30 ℃.

In the following examples, chiral analysis (ee value) of the product was performed by pre-column derivatization hplc, and the specific analysis method was:

(1) chromatographic conditions are as follows: the type of the chromatographic column: the total weight of Athena C18,

5 μm, 4.6X 250 mm; mobile phase: 50mM sodium acetate solution: methanol 0.5: 0.5. detection wavelength: 338 nm; flow rate: 1 mL/min; column temperature: at 30 ℃.

(2) Derivatization reagent: 0.03g of o-phthalaldehyde and 0.1g of N-acetyl-L-cysteine are respectively weighed, dissolved with 400uL of ethanol, added with 4mL of 0.2mol/L boric acid buffer solution (pH 9.8), shaken to be fully dissolved, and stored in a refrigerator at 4 ℃ for later use.

(3) Derivatization reaction and determination: adding 100 mu L of sample into 150 mu L of derivatization reagent in the step (2), uniformly mixing, and then keeping the temperature at 25 ℃ for 5 min; add 1mL ddH₂O was diluted and 20. mu.L was applied after passing through the membrane.

Example 1 rational design of Pseudomonas putida-derived glutamate dehydrogenase

(1) Site-directed mutagenesis

Through homologous model analysis, an activity pocket of glutamate dehydrogenase (PpGluDH, the sequence of which is shown in SEQ ID NO.1 and SEQ ID NO.11) derived from Pseudomonas putida is selected to be enlarged, and the steric hindrance between the glutamate dehydrogenase and a large side chain substrate phenylglyoxylic acid is reduced. Specific primers (see table 1) were designed to site-specifically mutate the Lys93, Ala167, Thr196, Arg208, Val378 and Ser381 amino acid residues in the PpGluDH active pocket to alanine (Ala) or glycine (Gly) with smaller side chains.

The amino acid sequence of PpGluDH (SEQ ID NO: 1) is:

MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPAGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV。

the nucleotide sequence of PpGluDH (SEQ ID NO: 11) is:

atgtctaccatgatcgaatctgtcgacaatttccttgcacgcctgaagcagcgtgacccaggccagccggaattccaccaggcggtggaagaggtgctgcgcaccctgtggccattccttgaagccaacccccactatctgcagtccggcatcctcgagcgcatggtcgagcccgagcgcgccgtacttttccgcgtttcttgggtcgatgaccagggcaaggtgcaggtcaaccgcggctaccgcattcagatgagcagtgccattggcccgtacaagggcgggctgcgcttccacccgtcggtgaacctcagcgtgctgaagttcctggcgttcgagcaggtgttcaagaactcgctgacctcgctgcccatgggcggcggcaagggcggctcggactttgacccgaaaggcaagagcgacgccgaagtgatgcgcttctgccaggcgttcatgagcgagctgtatcgccacatcggtgctgactgcgacgtaccggccggtgacatcggtgtgggggcccgcgaaatcggcttcatgttcggccagtacaagcgcctggccaaccagttcacctcggtgttgaccggtaagggcatgacctatggcggcagcctgatccgcccggaagccaccggctatggctgcgtttacttcgccgaagaaatgctcaagcgtcaggacaagcgtatcgacggccgccgcgtggcggtgtccggttcgggcaacgtcgcccagtatgctgcgcgtaaggtgatggacctgggcggcaaggtgatctcgctgtctgactctgaaggtaccttgtatgcagaagccgggctgaccgacgcccagtgggacgccttgatggagctgaaaaacgtcaagcgcggacgtatcagcgagctggccgggcaattcggcctggagttccgcaagggccagaccccgtggagcctgccgtgcgacatcgccctgccatgcgccacccagaacgagctgggcgccgaagatgcccgcaccttgctgcgtaacggctgtatctgtgtggccgaaggcgccaacatgccgactaccctcgaggctgtggatatcttcctggacgccggcattctgtacgccccgggcaaggcctccaatgcgggcggcgtagccgtgtcgggcctggaaatgtcgcagaacgccatgcgcctgctgtggacggccggtgaagtggacagcaagctgcacaacatcatgcagtcgattcaccatgcatgcgtgcactacggtgaagaggctgatggccggatcaactacgtcaagggggcgaacatcgcgggcttcgtgaaagtggccgatgcgatgctggctcagggcgtggtctga。

TABLE 1 PpGluDH site-directed mutagenesis primer

Note: underlined codons encode mutated amino acids

Six mutants, K93A, A167G, T196A, R208A, V378A and S381A, were constructed by introducing mutations using the Quickchange technology (An effective one-step site-directed and site-preservation mutagenesis protocol [ J ]. nucleic acids Research,2004,32(14): e 115). The specific mutation method is as follows:

the PCR reaction system and reaction conditions were as follows:

PCR amplification System:

PCR amplification conditions: pre-denaturation: 2min at 95 ℃; denaturation: 15s at 95 ℃; annealing: 15s at 56 ℃; extension: 3min at 72 ℃; circulating for 30 times; and (3) post-extension: 8min at 72 ℃; storing at 4 ℃.

After PCR amplification is finished, digesting an amplification product for 3 hours by using Dpn I digestive enzyme to remove a template plasmid, converting the digestion product into E.coli BL21(DE3) competent cells, coating a plate, selecting a single colony to be cultured in an LB liquid culture medium, and sequencing to verify mutation correctness. And (5) preserving the positive mutant which is verified to be error-free at-80 ℃ for later use.

(2) Culture of cells and preparation of crude enzyme solution

After streaking and activating the preserved engineering bacteria (K93A, A167G, T196A, R208A, V378A and S381A), selecting a single colony to be inoculated into 5mL LB liquid culture medium containing 50 mu g/mL kanamycin, shaking and culturing for 12h at 37 ℃, collecting bacterial liquid, transferring the bacterial liquid into 50mL fresh LB liquid culture medium containing 50 mu g/mL kanamycin according to the inoculum size of 2%, shaking and culturing at 37 ℃ until OD is achieved₆₀₀When the concentration reached about 0.6, IPTG was added to a final concentration of 0.5mM, and the cells were induced at 18 ℃ for 16 hours.

After the culture was completed, the culture was centrifuged at 10000rpm for 10min, and the supernatant was discarded to collect the cells. The collected cells were washed twice with 50mM phosphate buffer (pH7.5), and then suspended in 50mM phosphate buffer (pH7.5) and disrupted by 400W sonication for 10 min. The cell disruption solution was centrifuged at 12000g at 4 ℃ for 10min to remove the precipitate, and the supernatant was obtained as a crude enzyme solution.

(3) Determination of enzyme Activity

The standard enzyme activity detection system (1mL) is as follows: appropriate amount of enzyme (crude enzyme solution), 10mM phenylglyoxylic acid, 0.12mM NADPH, 500mM NH₄ ⁺((NH₄)₂SO₄) The reaction medium was 0.1M phosphate buffer (pH 7.5). The reaction is triggered by adding enzyme solution (crude enzyme solution), and then detecting OD within 1min with spectrophotometer₃₄₀Change at nm.

Wherein the definition of enzyme activity is as follows: under standard reaction conditions, 1. mu. mol NADPH was consumed per minute.

According to the above enzyme activity determination method, the enzyme activity of the fermentation broth per unit volume of 6 mutant strains was determined. As shown in figure 1, the catalytic activity of T196A is significantly improved compared with the wild type, and the unit fermentation broth activity reaches 100.24U/L, which is 24 times that of the wild type.

Example 2 semi-rational modification of T196A

(1) Construction of enzyme libraries

A primer (Table 2) was synthesized, and a linearized vector fragment was obtained by PCR using a recombinant plasmid (pET28a-PpGluDH-T196A) containing a T196A mutant gene (PpGluDH-T196A, the nucleotide sequence of which differs from that of PpGluDH in that the nucleotide at position 586-588 was changed from ACC to GCA) as a template, and using T121X/L123X-F and P-Aid-R as primer pairs, and then using T121X/L123X-R and P-Aid-F as primer pairs. The PCR system and conditions were as in step (1) of example 1.

The subsequent Dpn I digestion, gel recovery, recombination and transformation operations were performed with reference to a recombinant Cloning Kit (Clonexpress II One Step Cloning Kit) using the instructions to construct a combinatorial saturated mutation library of the 121 and 123 amino acid residues.

TABLE 2 primers for semi-rational engineering of T196A

Note: underlined codons encode mutated amino acids

(2) Screening of mutant pools

200. mu.L of LB medium (containing 50mg/L kanamycin) was added to a sterilized 96-well plate, and a single colony was picked up into a 96-well deep-well plate using a sterilized tip. The deep well plate was then incubated at 37 ℃ for 8h with shaking at 200rpm, and was referred to as a primary plate. In another sterilized 96-well plate, 400. mu.L of LB medium (containing 50mg/L of kanamycin) was added as a secondary plate, 50. mu.L of the bacterial solution was taken out of the primary plate and transferred to the secondary plate, and the primary plate was placed in a refrigerator at-80 ℃ for long-term storage with 20% of glycerol added thereto. The secondary plate was placed at 37 ℃ for shake culture for 3 hours, and IPTG was added to a final concentration of 0.5mM, 200rpm, and shake culture was carried out at 18 ℃ for 20 hours.

The secondary plate was centrifuged at 4000rpm at 4 ℃ for 15min to collect the cells. Adding cell-breaking solution [ 10mM phosphate buffer (pH7.5), 750mg/L lysozyme, 10mg/LDNase I ], and shaking in a shaking table at 37 deg.C for 1 hr. The cell debris was then removed by centrifugation at 4000rpm at 4 ℃ for 15min and placed on ice until use. mu.L of the supernatant was added to 180uL of mix solution [ 50mM phosphate buffer (pH7.5), 1mM NADPH, 10mM phenylglyoxylic acid, 100mM NH ]₄ ⁺20uL of the reaction solution was allowed to react at 37 ℃ and 200rpm for 1 hour, and then added to a 96-well plate to which 180uL of a chromogenic solution [ 50mM Tris-HCl buffer (pH 8.0), 300. mu.M nitroblue tetrazolium chloride, 100. mu.M phenazine methosulfate and 0.02% gelatin ] were added. Mutants with a lower purple color than the control (T196A) were selected as potential positive mutants for rescreening. The method for measuring the enzyme activity of the rescreened enzyme is shown in step (3) of example 1.

8 mutants (shown in table 3) with enzyme activity remarkably improved compared with that of T196A are obtained through re-screening, and the enzyme activity of fermentation liquor in unit volume reaches 168.64-301.05U/L, which is 1.68-3.01 times of that of T196A and 40-72 times of that of wild type. The mutants are the engineered NADPH-dependent phenylglycine dehydrogenase, and the amino acid sequences of the mutants are shown in SEQ ID NO. 2-9. The sequence alignment shows that the obtained 8 positive mutants are all mutants formed by combined mutation of the 196 th threonine, the 121 th threonine and the 123 th leucine of the amino acid sequence (SEQ ID NO.1) of the wild-type glutamate dehydrogenase. The specific mutations were as follows:

the T196A/T121I/L123D mutant is a mutant formed by mutating threonine at position 196 to alanine, threonine at position 121 to isoleucine and leucine at position 123 to aspartic acid of wild type glutamate dehydrogenase;

the T196A/T121I/L123H 2 mutant is a mutant formed by mutating threonine at position 196 to alanine, threonine at position 121 to isoleucine and leucine at position 123 to histidine of wild type glutamate dehydrogenase;

the T196A/T121I/L123N 3 mutant is a mutant formed by mutating threonine at position 196 to alanine, threonine at position 121 to isoleucine and leucine at position 123 to asparagine of wild type glutamate dehydrogenase;

the T196A/T121I/L123S mutant is a mutant formed by mutating threonine at position 196 to alanine, threonine at position 121 to isoleucine and leucine at position 123 to serine of wild type glutamate dehydrogenase;

the T196A/T121L/L123N mutant is a mutant formed by mutating threonine at position 196 to alanine, threonine at position 121 to leucine and leucine at position 123 to asparagine of wild type glutamate dehydrogenase;

the T196A/T121L/L123S mutant is a mutant formed by mutating threonine at position 196 to alanine, threonine at position 121 to leucine and leucine at position 123 to serine of wild type glutamate dehydrogenase;

the T196A/T121L/L123Y mutant is a mutant formed by mutating threonine at position 196 to alanine, threonine at position 121 to leucine and leucine at position 123 to tyrosine of wild type glutamate dehydrogenase;

the T196A/T121L/L123H mutant is a mutant formed by mutating threonine at position 196 to alanine, threonine at position 121 to leucine and leucine at position 123 to histidine of wild type glutamate dehydrogenase.

TABLE 3 Positive mutants obtained by semi-rational modification of T196A

^a)The mutant has the fold improvement of the unit fermentation broth enzyme activity (4.2U/L) relative to the wild type.

The amino acid sequence of each mutant is:

SEQ ID NO.2：T196A/T121I/L123D

MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLISDPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPAGDIGVGAREIGFMFGQYKRLANQFTSVLAGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV。

SEQ ID NO.3：T196A/T121I/L123H

MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLISHPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPAGDIGVGAREIGFMFGQYKRLANQFTSVLAGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV。

SEQ ID NO.4：T196A/T121I/L123N

MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLISNPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPAGDIGVGAREIGFMFGQYKRLANQFTSVLAGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV。

SEQ ID NO.5：T196A/T121I/L123S

MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLISSPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPAGDIGVGAREIGFMFGQYKRLANQFTSVLAGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV。

SEQ ID NO.6：T196A/T121L/L123N

MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLLSNPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPAGDIGVGAREIGFMFGQYKRLANQFTSVLAGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV。

SEQ ID NO.7：T196A/T121L/L123S

MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLLSSPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPAGDIGVGAREIGFMFGQYKRLANQFTSVLAGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV。

SEQ ID NO.8：T196A/T121L/L123Y

MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLLSYPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPAGDIGVGAREIGFMFGQYKRLANQFTSVLAGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV。

SEQ ID NO.9：T196A/T121L/L123H

MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLLSHPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPAGDIGVGAREIGFMFGQYKRLANQFTSVLAGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV。

example 3 application of engineered NADPH-dependent phenylglycine dehydrogenase in preparation of L-phenylglycine by biocatalysis

The reaction formula for preparing L-phenylglycine by a biological catalysis method is shown in figure 2, and the specific preparation method comprises the following steps:

first, recombinant engineered bacteria of NADPH-dependent phenylglycine dehydrogenase (T196A/T121I/L123D, SEQ ID NO.2) and alcohol dehydrogenase (CbADH-6M, SEQ ID NO.10, coenzyme-regenerating enzyme) were cultured according to the method of step (2) in example 1, and cells were collected by centrifugation and disrupted by sonication to prepare a crude enzyme solution. Wherein the amino acid sequence of the CbADH-6M is as follows:

MKGFAMLGINKLGWIEKERPVAGPYDAIVRPLAVSPCTSDIHTVFEGALGDRKNMILGHEAVGEVVEVGSEVKDFKPGDRVIVPCTTPDWRSLEVQAGFQQHSNGMLAGWKFSNFKDGVFGEYFHVNDADMNLAILPKDMPLENAVMITDMMTTGFHGAELADIQMGSSVVVIGIGAVGLMAIAGAKLRGAGRIIAVGSRPICVEAAKFYGATDILNYKNGDIVDQVMKLTNGKGVDRVIMAGGGSETLEQAVRMVKPGGIISNINYHGSGDALLIPRVEWGCGMAHKTIKGGLCPGGRLRAEMLRDMVVYNRVDLSKLVTHVYHGFDHIEEALLLMKDKPKDLIKAVVIL。

then, the reaction was carried out in a system of 10mL containing 200mM of phenylglyoxylic acid, 250mM of isopropyl alcohol (coenzyme-regenerating substrate), 125mM of NH₄)₂SO₄(amino Donor) with 0.2mM NADP⁺(reduced coenzyme), the concentration of phenylglycine dehydrogenase cells (dry weight of crude enzyme solution) was 5.0g/L, and the concentration of alcohol dehydrogenase cells (dry weight of crude enzyme solution) was 1.25 g/L. Controlling the reaction temperature to be 30 ℃ by water bath, and reactingThe pH value is controlled to be 7.5 by dropwise adding ammonia water, the generated concentration of the product L-phenylglycine and the residual concentration of the substrate phenylglyoxylic acid are detected by achiral HPLC after 10 hours of reaction, and meanwhile, the ee value of the L-phenylglycine is detected by pre-column derivatization high performance liquid chromatography.

The statistical results of the data after the reaction are as follows: the concentration of produced L-phenylglycine is 30.18g/L, the conversion rate is more than 99.0%, and the ee value is more than 99.0%. Wherein, the peak appearance of the product L-phenylglycine and the substrate phenylglyoxylic acid in HPLC detection is shown in figure 2; the peak appearance of L-phenylglycine and D-phenylglycine in the detection of pre-column derivatization high performance liquid chromatography is shown in FIG. 3.

Example 4 application of engineered NADPH-dependent phenylglycine dehydrogenase to preparation of L-phenylglycine by fermentation

The gene of engineered NADPH dependent phenylglycine dehydrogenase (T196A/T121I/L123D, SEQ ID No.2) was cloned into pTrc99a vector and transformed into e.coli BW25113 strain. A single colony was inoculated into 3mL of LB medium (containing 100. mu.g/mL of ampicillin) and shake-cultured overnight at 37 ℃. The overnight-cultured bacterial liquid was inoculated to 4mL of a culture containing 100. mu.g/mL ampicillin and 4g/LNH in an amount of 1%₄Cultured with shaking Cl and 3.0g/L substrate phenylglyoxylic acid in TB liquid medium (pH7.5) at 37 ℃ to OD₆₀₀Reaching about 1.0, adding 0.25mM IPTG (inducer), and inducing and culturing at 30 ℃ for 48 h. Detecting the generation concentration of the product L-phenylglycine and the residual concentration of the substrate phenylglyoxylic acid in the fermentation liquid by using achiral HPLC, and detecting the ee value of the L-phenylglycine by using pre-column derivatization high performance liquid chromatography.

The statistical results of the data after the reaction are as follows: the L-phenylglycine was produced at a concentration of 3.12g/L, a conversion of about 95.0% and an ee value of > 99.0%.

Comparative example 1 application of glutamate dehydrogenase wild type to biocatalytic preparation of L-phenylglycine

First, the wild type glutamate dehydrogenase (SEQ ID NO.1) and the engineered bacteria of alcohol dehydrogenase (CbADH-6M, SEQ ID NO.10) were cultured according to the method of step (2) in example 1, and the cells were collected by centrifugation and disrupted by sonication to prepare a crude enzyme solution.

Then catalytically reactingThe system is 10mL, and contains 200mM phenylglyoxylic acid, 250mM isopropanol, 125mM (NH)₄)₂SO₄With 0.2mM NADP⁺The concentration of phenylglycine dehydrogenase cells (dry weight of crude enzyme solution) was 5.0g/L, and the concentration of alcohol dehydrogenase cells (dry weight of crude enzyme solution) was 1.25 g/L. Controlling the reaction temperature to be 30 ℃ through water bath, controlling the pH to be 7.5 in the reaction process through dropwise adding ammonia water, detecting the generation concentration of the L-phenylglycine and the residual concentration of a substrate phenylglyoxylic acid by using achiral HPLC after reacting for 10 hours, and simultaneously detecting the ee value of the L-phenylglycine by using pre-column derivatization high performance liquid chromatography.

The statistical results of the data after the reaction are as follows: the L-phenylglycine was produced at a concentration of 3.62g/L, a conversion of about 12.0% and an ee value of > 99.0%.

Comparative example 2 fermentation preparation of L-phenylglycine by glutamate dehydrogenase wild type

The gene for glutamate dehydrogenase wild type (SEQ ID No.1) was cloned into pTrc99a vector and transformed into e.coli BW25113 strain. A single colony was inoculated into 3mL of LB medium (containing 100. mu.g/mL of ampicillin) and shake-cultured overnight at 37 ℃. The overnight-cultured bacterial liquid was inoculated to 4mL of a culture containing 100. mu.g/mL ampicillin and 4g/LNH in an amount of 1%₄In TB liquid medium (pH7.5) containing Cl and 3.0g/L of the substrate phenylglyoxylic acid, the mixture was shake-cultured at 37 ℃ until OD was reached₆₀₀Reaching about 1.0, adding 0.25mM IPTG, and carrying out induction culture at 30 ℃ for 48 h. Detecting the generation concentration of the product L-phenylglycine and the residual concentration of the substrate phenylglyoxylic acid in the fermentation liquid by using achiral HPLC, and detecting the ee value of the L-phenylglycine by using pre-column derivatization high performance liquid chromatography.

The statistical results of the data after the reaction are as follows: the L-phenylglycine was produced at a concentration of 1.01g/L, a conversion of about 30.5% and an ee value of > 99.0%.

In conclusion, compared with the wild type, the NADPH-dependent phenylglycine dehydrogenase developed by the invention has the advantage that the catalytic activity of the target substrate phenylglyoxylate is improved by nearly 40-72 times. The NADPH-dependent phenylglycine dehydrogenase shows good catalytic efficiency in the preparation of L-phenylglycine by a biological catalytic method, the substrate conversion rate is more than 99%, the product generation concentration is as high as 30.18g/L, the ee value is more than 99%, and the catalytic efficiency is obviously higher than that of wild-type glutamate dehydrogenase; meanwhile, the NADPH-dependent phenylglycine dehydrogenase developed by the invention is applied to the preparation of L-phenylglycine by a fermentation method, the substrate conversion rate reaches 95%, the product concentration reaches 3.12g/L, the ee value is more than 99%, the catalytic efficiency is also obviously superior to that of wild-type glutamate dehydrogenase, and the NADPH-dependent phenylglycine dehydrogenase can efficiently utilize intracellular NADPH to synthesize chiral L-phenylglycine.

The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.

Sequence listing

<110> Zhongshan university

<120> engineered NADPH-dependent phenylglycine dehydrogenase and application thereof

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 449

<212> PRT

<213> wild type glutamate dehydrogenase (Artificial Sequence)

<400> 1

Met Ser Thr Met Ile Glu Ser Val Asp Asn Phe Leu Ala Arg Leu Lys

1 5 10 15

Gln Arg Asp Pro Gly Gln Pro Glu Phe His Gln Ala Val Glu Glu Val

20 25 30

Leu Arg Thr Leu Trp Pro Phe Leu Glu Ala Asn Pro His Tyr Leu Gln

35 40 45

Ser Gly Ile Leu Glu Arg Met Val Glu Pro Glu Arg Ala Val Leu Phe

50 55 60

Arg Val Ser Trp Val Asp Asp Gln Gly Lys Val Gln Val Asn Arg Gly

65 70 75 80

Tyr Arg Ile Gln Met Ser Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu

85 90 95

Arg Phe His Pro Ser Val Asn Leu Ser Val Leu Lys Phe Leu Ala Phe

100 105 110

Glu Gln Val Phe Lys Asn Ser Leu Thr Ser Leu Pro Met Gly Gly Gly

115 120 125

Lys Gly Gly Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val

130 135 140

Met Arg Phe Cys Gln Ala Phe Met Ser Glu Leu Tyr Arg His Ile Gly

145 150 155 160

Ala Asp Cys Asp Val Pro Ala Gly Asp Ile Gly Val Gly Ala Arg Glu

165 170 175

Ile Gly Phe Met Phe Gly Gln Tyr Lys Arg Leu Ala Asn Gln Phe Thr

180 185 190

Ser Val Leu Thr Gly Lys Gly Met Thr Tyr Gly Gly Ser Leu Ile Arg

195 200 205

Pro Glu Ala Thr Gly Tyr Gly Cys Val Tyr Phe Ala Glu Glu Met Leu

210 215 220

Lys Arg Gln Asp Lys Arg Ile Asp Gly Arg Arg Val Ala Val Ser Gly

225 230 235 240

Ser Gly Asn Val Ala Gln Tyr Ala Ala Arg Lys Val Met Asp Leu Gly

245 250 255

Gly Lys Val Ile Ser Leu Ser Asp Ser Glu Gly Thr Leu Tyr Ala Glu

260 265 270

Ala Gly Leu Thr Asp Ala Gln Trp Asp Ala Leu Met Glu Leu Lys Asn

275 280 285

Val Lys Arg Gly Arg Ile Ser Glu Leu Ala Gly Gln Phe Gly Leu Glu

290 295 300

Phe Arg Lys Gly Gln Thr Pro Trp Ser Leu Pro Cys Asp Ile Ala Leu

305 310 315 320

Pro Cys Ala Thr Gln Asn Glu Leu Gly Ala Glu Asp Ala Arg Thr Leu

325 330 335

Leu Arg Asn Gly Cys Ile Cys Val Ala Glu Gly Ala Asn Met Pro Thr

340 345 350

Thr Leu Glu Ala Val Asp Ile Phe Leu Asp Ala Gly Ile Leu Tyr Ala

355 360 365

Pro Gly Lys Ala Ser Asn Ala Gly Gly Val Ala Val Ser Gly Leu Glu

370 375 380

Met Ser Gln Asn Ala Met Arg Leu Leu Trp Thr Ala Gly Glu Val Asp

385 390 395 400

Ser Lys Leu His Asn Ile Met Gln Ser Ile His His Ala Cys Val His

405 410 415

Tyr Gly Glu Glu Ala Asp Gly Arg Ile Asn Tyr Val Lys Gly Ala Asn

420 425 430

Ile Ala Gly Phe Val Lys Val Ala Asp Ala Met Leu Ala Gln Gly Val

435 440 445

Val

<210> 2

<211> 449

<212> PRT

<213> T196A/T121I/L123D(Artificial Sequence)

<400> 2

Met Ser Thr Met Ile Glu Ser Val Asp Asn Phe Leu Ala Arg Leu Lys

1 5 10 15

Gln Arg Asp Pro Gly Gln Pro Glu Phe His Gln Ala Val Glu Glu Val

20 25 30

Leu Arg Thr Leu Trp Pro Phe Leu Glu Ala Asn Pro His Tyr Leu Gln

35 40 45

Ser Gly Ile Leu Glu Arg Met Val Glu Pro Glu Arg Ala Val Leu Phe

50 55 60

Arg Val Ser Trp Val Asp Asp Gln Gly Lys Val Gln Val Asn Arg Gly

65 70 75 80

Tyr Arg Ile Gln Met Ser Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu

85 90 95

Arg Phe His Pro Ser Val Asn Leu Ser Val Leu Lys Phe Leu Ala Phe

100 105 110

Glu Gln Val Phe Lys Asn Ser Leu Ile Ser Asp Pro Met Gly Gly Gly

115 120 125

Lys Gly Gly Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val

130 135 140

Met Arg Phe Cys Gln Ala Phe Met Ser Glu Leu Tyr Arg His Ile Gly

145 150 155 160

Ala Asp Cys Asp Val Pro Ala Gly Asp Ile Gly Val Gly Ala Arg Glu

165 170 175

Ile Gly Phe Met Phe Gly Gln Tyr Lys Arg Leu Ala Asn Gln Phe Thr

180 185 190

Ser Val Leu Ala Gly Lys Gly Met Thr Tyr Gly Gly Ser Leu Ile Arg

195 200 205

Pro Glu Ala Thr Gly Tyr Gly Cys Val Tyr Phe Ala Glu Glu Met Leu

210 215 220

Lys Arg Gln Asp Lys Arg Ile Asp Gly Arg Arg Val Ala Val Ser Gly

225 230 235 240

Ser Gly Asn Val Ala Gln Tyr Ala Ala Arg Lys Val Met Asp Leu Gly

245 250 255

Gly Lys Val Ile Ser Leu Ser Asp Ser Glu Gly Thr Leu Tyr Ala Glu

260 265 270

Ala Gly Leu Thr Asp Ala Gln Trp Asp Ala Leu Met Glu Leu Lys Asn

275 280 285

Val Lys Arg Gly Arg Ile Ser Glu Leu Ala Gly Gln Phe Gly Leu Glu

290 295 300

Phe Arg Lys Gly Gln Thr Pro Trp Ser Leu Pro Cys Asp Ile Ala Leu

305 310 315 320

Pro Cys Ala Thr Gln Asn Glu Leu Gly Ala Glu Asp Ala Arg Thr Leu

325 330 335

Leu Arg Asn Gly Cys Ile Cys Val Ala Glu Gly Ala Asn Met Pro Thr

340 345 350

Thr Leu Glu Ala Val Asp Ile Phe Leu Asp Ala Gly Ile Leu Tyr Ala

355 360 365

Pro Gly Lys Ala Ser Asn Ala Gly Gly Val Ala Val Ser Gly Leu Glu

370 375 380

Met Ser Gln Asn Ala Met Arg Leu Leu Trp Thr Ala Gly Glu Val Asp

385 390 395 400

Ser Lys Leu His Asn Ile Met Gln Ser Ile His His Ala Cys Val His

405 410 415

Tyr Gly Glu Glu Ala Asp Gly Arg Ile Asn Tyr Val Lys Gly Ala Asn

420 425 430

Ile Ala Gly Phe Val Lys Val Ala Asp Ala Met Leu Ala Gln Gly Val

435 440 445

Val

<210> 3

<211> 449

<212> PRT

<213> T196A/T121I/L123H(Artificial Sequence)

<400> 3

Met Ser Thr Met Ile Glu Ser Val Asp Asn Phe Leu Ala Arg Leu Lys

1 5 10 15

Gln Arg Asp Pro Gly Gln Pro Glu Phe His Gln Ala Val Glu Glu Val

20 25 30

Leu Arg Thr Leu Trp Pro Phe Leu Glu Ala Asn Pro His Tyr Leu Gln

35 40 45

Ser Gly Ile Leu Glu Arg Met Val Glu Pro Glu Arg Ala Val Leu Phe

50 55 60

Arg Val Ser Trp Val Asp Asp Gln Gly Lys Val Gln Val Asn Arg Gly

65 70 75 80

Tyr Arg Ile Gln Met Ser Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu

85 90 95

Arg Phe His Pro Ser Val Asn Leu Ser Val Leu Lys Phe Leu Ala Phe

100 105 110

Glu Gln Val Phe Lys Asn Ser Leu Ile Ser His Pro Met Gly Gly Gly

115 120 125

Lys Gly Gly Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val

130 135 140

Met Arg Phe Cys Gln Ala Phe Met Ser Glu Leu Tyr Arg His Ile Gly

145 150 155 160

Ala Asp Cys Asp Val Pro Ala Gly Asp Ile Gly Val Gly Ala Arg Glu

165 170 175

Ile Gly Phe Met Phe Gly Gln Tyr Lys Arg Leu Ala Asn Gln Phe Thr

180 185 190

Ser Val Leu Ala Gly Lys Gly Met Thr Tyr Gly Gly Ser Leu Ile Arg

195 200 205

Pro Glu Ala Thr Gly Tyr Gly Cys Val Tyr Phe Ala Glu Glu Met Leu

210 215 220

Lys Arg Gln Asp Lys Arg Ile Asp Gly Arg Arg Val Ala Val Ser Gly

225 230 235 240

Ser Gly Asn Val Ala Gln Tyr Ala Ala Arg Lys Val Met Asp Leu Gly

245 250 255

Gly Lys Val Ile Ser Leu Ser Asp Ser Glu Gly Thr Leu Tyr Ala Glu

260 265 270

Ala Gly Leu Thr Asp Ala Gln Trp Asp Ala Leu Met Glu Leu Lys Asn

275 280 285

Val Lys Arg Gly Arg Ile Ser Glu Leu Ala Gly Gln Phe Gly Leu Glu

290 295 300

Phe Arg Lys Gly Gln Thr Pro Trp Ser Leu Pro Cys Asp Ile Ala Leu

305 310 315 320

Pro Cys Ala Thr Gln Asn Glu Leu Gly Ala Glu Asp Ala Arg Thr Leu

325 330 335

Leu Arg Asn Gly Cys Ile Cys Val Ala Glu Gly Ala Asn Met Pro Thr

340 345 350

Thr Leu Glu Ala Val Asp Ile Phe Leu Asp Ala Gly Ile Leu Tyr Ala

355 360 365

Pro Gly Lys Ala Ser Asn Ala Gly Gly Val Ala Val Ser Gly Leu Glu

370 375 380

Met Ser Gln Asn Ala Met Arg Leu Leu Trp Thr Ala Gly Glu Val Asp

385 390 395 400

Ser Lys Leu His Asn Ile Met Gln Ser Ile His His Ala Cys Val His

405 410 415

Tyr Gly Glu Glu Ala Asp Gly Arg Ile Asn Tyr Val Lys Gly Ala Asn

420 425 430

Ile Ala Gly Phe Val Lys Val Ala Asp Ala Met Leu Ala Gln Gly Val

435 440 445

Val

<210> 4

<211> 449

<212> PRT

<213> T196A/T121I/L123N(Artificial Sequence)

<400> 4

Met Ser Thr Met Ile Glu Ser Val Asp Asn Phe Leu Ala Arg Leu Lys

1 5 10 15

Gln Arg Asp Pro Gly Gln Pro Glu Phe His Gln Ala Val Glu Glu Val

20 25 30

Leu Arg Thr Leu Trp Pro Phe Leu Glu Ala Asn Pro His Tyr Leu Gln

35 40 45

Ser Gly Ile Leu Glu Arg Met Val Glu Pro Glu Arg Ala Val Leu Phe

50 55 60

Arg Val Ser Trp Val Asp Asp Gln Gly Lys Val Gln Val Asn Arg Gly

65 70 75 80

Tyr Arg Ile Gln Met Ser Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu

85 90 95

Arg Phe His Pro Ser Val Asn Leu Ser Val Leu Lys Phe Leu Ala Phe

100 105 110

Glu Gln Val Phe Lys Asn Ser Leu Ile Ser Asn Pro Met Gly Gly Gly

115 120 125

Lys Gly Gly Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val

130 135 140

Met Arg Phe Cys Gln Ala Phe Met Ser Glu Leu Tyr Arg His Ile Gly

145 150 155 160

Ala Asp Cys Asp Val Pro Ala Gly Asp Ile Gly Val Gly Ala Arg Glu

165 170 175

Ile Gly Phe Met Phe Gly Gln Tyr Lys Arg Leu Ala Asn Gln Phe Thr

180 185 190

Ser Val Leu Ala Gly Lys Gly Met Thr Tyr Gly Gly Ser Leu Ile Arg

195 200 205

Pro Glu Ala Thr Gly Tyr Gly Cys Val Tyr Phe Ala Glu Glu Met Leu

210 215 220

Lys Arg Gln Asp Lys Arg Ile Asp Gly Arg Arg Val Ala Val Ser Gly

225 230 235 240

Ser Gly Asn Val Ala Gln Tyr Ala Ala Arg Lys Val Met Asp Leu Gly

245 250 255

Gly Lys Val Ile Ser Leu Ser Asp Ser Glu Gly Thr Leu Tyr Ala Glu

260 265 270

Ala Gly Leu Thr Asp Ala Gln Trp Asp Ala Leu Met Glu Leu Lys Asn

275 280 285

Val Lys Arg Gly Arg Ile Ser Glu Leu Ala Gly Gln Phe Gly Leu Glu

290 295 300

Phe Arg Lys Gly Gln Thr Pro Trp Ser Leu Pro Cys Asp Ile Ala Leu

305 310 315 320

Pro Cys Ala Thr Gln Asn Glu Leu Gly Ala Glu Asp Ala Arg Thr Leu

325 330 335

Leu Arg Asn Gly Cys Ile Cys Val Ala Glu Gly Ala Asn Met Pro Thr

340 345 350

Thr Leu Glu Ala Val Asp Ile Phe Leu Asp Ala Gly Ile Leu Tyr Ala

355 360 365

Pro Gly Lys Ala Ser Asn Ala Gly Gly Val Ala Val Ser Gly Leu Glu

370 375 380

Met Ser Gln Asn Ala Met Arg Leu Leu Trp Thr Ala Gly Glu Val Asp

385 390 395 400

Ser Lys Leu His Asn Ile Met Gln Ser Ile His His Ala Cys Val His

405 410 415

Tyr Gly Glu Glu Ala Asp Gly Arg Ile Asn Tyr Val Lys Gly Ala Asn

420 425 430

Ile Ala Gly Phe Val Lys Val Ala Asp Ala Met Leu Ala Gln Gly Val

435 440 445

Val

<210> 5

<211> 449

<212> PRT

<213> T196A/T121I/L123S(Artificial Sequence)

<400> 5

Met Ser Thr Met Ile Glu Ser Val Asp Asn Phe Leu Ala Arg Leu Lys

1 5 10 15

Gln Arg Asp Pro Gly Gln Pro Glu Phe His Gln Ala Val Glu Glu Val

20 25 30

Leu Arg Thr Leu Trp Pro Phe Leu Glu Ala Asn Pro His Tyr Leu Gln

35 40 45

Ser Gly Ile Leu Glu Arg Met Val Glu Pro Glu Arg Ala Val Leu Phe

50 55 60

Arg Val Ser Trp Val Asp Asp Gln Gly Lys Val Gln Val Asn Arg Gly

65 70 75 80

Tyr Arg Ile Gln Met Ser Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu

85 90 95

Arg Phe His Pro Ser Val Asn Leu Ser Val Leu Lys Phe Leu Ala Phe

100 105 110

Glu Gln Val Phe Lys Asn Ser Leu Ile Ser Ser Pro Met Gly Gly Gly

115 120 125

Lys Gly Gly Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val

130 135 140

Met Arg Phe Cys Gln Ala Phe Met Ser Glu Leu Tyr Arg His Ile Gly

145 150 155 160

Ala Asp Cys Asp Val Pro Ala Gly Asp Ile Gly Val Gly Ala Arg Glu

165 170 175

Ile Gly Phe Met Phe Gly Gln Tyr Lys Arg Leu Ala Asn Gln Phe Thr

180 185 190

Ser Val Leu Ala Gly Lys Gly Met Thr Tyr Gly Gly Ser Leu Ile Arg

195 200 205

Pro Glu Ala Thr Gly Tyr Gly Cys Val Tyr Phe Ala Glu Glu Met Leu

210 215 220

Lys Arg Gln Asp Lys Arg Ile Asp Gly Arg Arg Val Ala Val Ser Gly

225 230 235 240

Ser Gly Asn Val Ala Gln Tyr Ala Ala Arg Lys Val Met Asp Leu Gly

245 250 255

Gly Lys Val Ile Ser Leu Ser Asp Ser Glu Gly Thr Leu Tyr Ala Glu

260 265 270

Ala Gly Leu Thr Asp Ala Gln Trp Asp Ala Leu Met Glu Leu Lys Asn

275 280 285

Val Lys Arg Gly Arg Ile Ser Glu Leu Ala Gly Gln Phe Gly Leu Glu

290 295 300

Phe Arg Lys Gly Gln Thr Pro Trp Ser Leu Pro Cys Asp Ile Ala Leu

305 310 315 320

Pro Cys Ala Thr Gln Asn Glu Leu Gly Ala Glu Asp Ala Arg Thr Leu

325 330 335

Leu Arg Asn Gly Cys Ile Cys Val Ala Glu Gly Ala Asn Met Pro Thr

340 345 350

Thr Leu Glu Ala Val Asp Ile Phe Leu Asp Ala Gly Ile Leu Tyr Ala

355 360 365

Pro Gly Lys Ala Ser Asn Ala Gly Gly Val Ala Val Ser Gly Leu Glu

370 375 380

Met Ser Gln Asn Ala Met Arg Leu Leu Trp Thr Ala Gly Glu Val Asp

385 390 395 400

Ser Lys Leu His Asn Ile Met Gln Ser Ile His His Ala Cys Val His

405 410 415

Tyr Gly Glu Glu Ala Asp Gly Arg Ile Asn Tyr Val Lys Gly Ala Asn

420 425 430

Ile Ala Gly Phe Val Lys Val Ala Asp Ala Met Leu Ala Gln Gly Val

435 440 445

Val

<210> 6

<211> 449

<212> PRT

<213> T196A/T121L/L123N(Artificial Sequence)

<400> 6

Met Ser Thr Met Ile Glu Ser Val Asp Asn Phe Leu Ala Arg Leu Lys

1 5 10 15

Gln Arg Asp Pro Gly Gln Pro Glu Phe His Gln Ala Val Glu Glu Val

20 25 30

Leu Arg Thr Leu Trp Pro Phe Leu Glu Ala Asn Pro His Tyr Leu Gln

35 40 45

Ser Gly Ile Leu Glu Arg Met Val Glu Pro Glu Arg Ala Val Leu Phe

50 55 60

Arg Val Ser Trp Val Asp Asp Gln Gly Lys Val Gln Val Asn Arg Gly

65 70 75 80

Tyr Arg Ile Gln Met Ser Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu

85 90 95

Arg Phe His Pro Ser Val Asn Leu Ser Val Leu Lys Phe Leu Ala Phe

100 105 110

Glu Gln Val Phe Lys Asn Ser Leu Leu Ser Asn Pro Met Gly Gly Gly

115 120 125

Lys Gly Gly Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val

130 135 140

Met Arg Phe Cys Gln Ala Phe Met Ser Glu Leu Tyr Arg His Ile Gly

145 150 155 160

Ala Asp Cys Asp Val Pro Ala Gly Asp Ile Gly Val Gly Ala Arg Glu

165 170 175

Ile Gly Phe Met Phe Gly Gln Tyr Lys Arg Leu Ala Asn Gln Phe Thr

180 185 190

Ser Val Leu Ala Gly Lys Gly Met Thr Tyr Gly Gly Ser Leu Ile Arg

195 200 205

Pro Glu Ala Thr Gly Tyr Gly Cys Val Tyr Phe Ala Glu Glu Met Leu

210 215 220

Lys Arg Gln Asp Lys Arg Ile Asp Gly Arg Arg Val Ala Val Ser Gly

225 230 235 240

Ser Gly Asn Val Ala Gln Tyr Ala Ala Arg Lys Val Met Asp Leu Gly

245 250 255

Gly Lys Val Ile Ser Leu Ser Asp Ser Glu Gly Thr Leu Tyr Ala Glu

260 265 270

Ala Gly Leu Thr Asp Ala Gln Trp Asp Ala Leu Met Glu Leu Lys Asn

275 280 285

Val Lys Arg Gly Arg Ile Ser Glu Leu Ala Gly Gln Phe Gly Leu Glu

290 295 300

Phe Arg Lys Gly Gln Thr Pro Trp Ser Leu Pro Cys Asp Ile Ala Leu

305 310 315 320

Pro Cys Ala Thr Gln Asn Glu Leu Gly Ala Glu Asp Ala Arg Thr Leu

325 330 335

Leu Arg Asn Gly Cys Ile Cys Val Ala Glu Gly Ala Asn Met Pro Thr

340 345 350

Thr Leu Glu Ala Val Asp Ile Phe Leu Asp Ala Gly Ile Leu Tyr Ala

355 360 365

Pro Gly Lys Ala Ser Asn Ala Gly Gly Val Ala Val Ser Gly Leu Glu

370 375 380

Met Ser Gln Asn Ala Met Arg Leu Leu Trp Thr Ala Gly Glu Val Asp

385 390 395 400

Ser Lys Leu His Asn Ile Met Gln Ser Ile His His Ala Cys Val His

405 410 415

Tyr Gly Glu Glu Ala Asp Gly Arg Ile Asn Tyr Val Lys Gly Ala Asn

420 425 430

Ile Ala Gly Phe Val Lys Val Ala Asp Ala Met Leu Ala Gln Gly Val

435 440 445

Val

<210> 7

<211> 449

<212> PRT

<213> T196A/T121L/L123S(Artificial Sequence)

<400> 7

Met Ser Thr Met Ile Glu Ser Val Asp Asn Phe Leu Ala Arg Leu Lys

1 5 10 15

Gln Arg Asp Pro Gly Gln Pro Glu Phe His Gln Ala Val Glu Glu Val

20 25 30

Leu Arg Thr Leu Trp Pro Phe Leu Glu Ala Asn Pro His Tyr Leu Gln

35 40 45

Ser Gly Ile Leu Glu Arg Met Val Glu Pro Glu Arg Ala Val Leu Phe

50 55 60

Arg Val Ser Trp Val Asp Asp Gln Gly Lys Val Gln Val Asn Arg Gly

65 70 75 80

Tyr Arg Ile Gln Met Ser Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu

85 90 95

Arg Phe His Pro Ser Val Asn Leu Ser Val Leu Lys Phe Leu Ala Phe

100 105 110

Glu Gln Val Phe Lys Asn Ser Leu Leu Ser Ser Pro Met Gly Gly Gly

115 120 125

Lys Gly Gly Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val

130 135 140

Met Arg Phe Cys Gln Ala Phe Met Ser Glu Leu Tyr Arg His Ile Gly

145 150 155 160

Ala Asp Cys Asp Val Pro Ala Gly Asp Ile Gly Val Gly Ala Arg Glu

165 170 175

Ile Gly Phe Met Phe Gly Gln Tyr Lys Arg Leu Ala Asn Gln Phe Thr

180 185 190

Ser Val Leu Ala Gly Lys Gly Met Thr Tyr Gly Gly Ser Leu Ile Arg

195 200 205

Pro Glu Ala Thr Gly Tyr Gly Cys Val Tyr Phe Ala Glu Glu Met Leu

210 215 220

Lys Arg Gln Asp Lys Arg Ile Asp Gly Arg Arg Val Ala Val Ser Gly

225 230 235 240

Ser Gly Asn Val Ala Gln Tyr Ala Ala Arg Lys Val Met Asp Leu Gly

245 250 255

Gly Lys Val Ile Ser Leu Ser Asp Ser Glu Gly Thr Leu Tyr Ala Glu

260 265 270

Ala Gly Leu Thr Asp Ala Gln Trp Asp Ala Leu Met Glu Leu Lys Asn

275 280 285

Val Lys Arg Gly Arg Ile Ser Glu Leu Ala Gly Gln Phe Gly Leu Glu

290 295 300

Phe Arg Lys Gly Gln Thr Pro Trp Ser Leu Pro Cys Asp Ile Ala Leu

305 310 315 320

Pro Cys Ala Thr Gln Asn Glu Leu Gly Ala Glu Asp Ala Arg Thr Leu

325 330 335

Leu Arg Asn Gly Cys Ile Cys Val Ala Glu Gly Ala Asn Met Pro Thr

340 345 350

Thr Leu Glu Ala Val Asp Ile Phe Leu Asp Ala Gly Ile Leu Tyr Ala

355 360 365

Pro Gly Lys Ala Ser Asn Ala Gly Gly Val Ala Val Ser Gly Leu Glu

370 375 380

Met Ser Gln Asn Ala Met Arg Leu Leu Trp Thr Ala Gly Glu Val Asp

385 390 395 400

Ser Lys Leu His Asn Ile Met Gln Ser Ile His His Ala Cys Val His

405 410 415

Tyr Gly Glu Glu Ala Asp Gly Arg Ile Asn Tyr Val Lys Gly Ala Asn

420 425 430

Ile Ala Gly Phe Val Lys Val Ala Asp Ala Met Leu Ala Gln Gly Val

435 440 445

Val

<210> 8

<211> 449

<212> PRT

<213> T196A/T121L/L123Y(Artificial Sequence)

<400> 8

Met Ser Thr Met Ile Glu Ser Val Asp Asn Phe Leu Ala Arg Leu Lys

1 5 10 15

Gln Arg Asp Pro Gly Gln Pro Glu Phe His Gln Ala Val Glu Glu Val

20 25 30

Leu Arg Thr Leu Trp Pro Phe Leu Glu Ala Asn Pro His Tyr Leu Gln

35 40 45

Ser Gly Ile Leu Glu Arg Met Val Glu Pro Glu Arg Ala Val Leu Phe

50 55 60

Arg Val Ser Trp Val Asp Asp Gln Gly Lys Val Gln Val Asn Arg Gly

65 70 75 80

Tyr Arg Ile Gln Met Ser Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu

85 90 95

Arg Phe His Pro Ser Val Asn Leu Ser Val Leu Lys Phe Leu Ala Phe

100 105 110

Glu Gln Val Phe Lys Asn Ser Leu Leu Ser Tyr Pro Met Gly Gly Gly

115 120 125

Lys Gly Gly Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val

130 135 140

Met Arg Phe Cys Gln Ala Phe Met Ser Glu Leu Tyr Arg His Ile Gly

145 150 155 160

Ala Asp Cys Asp Val Pro Ala Gly Asp Ile Gly Val Gly Ala Arg Glu

165 170 175

Ile Gly Phe Met Phe Gly Gln Tyr Lys Arg Leu Ala Asn Gln Phe Thr

180 185 190

Ser Val Leu Ala Gly Lys Gly Met Thr Tyr Gly Gly Ser Leu Ile Arg

195 200 205

Pro Glu Ala Thr Gly Tyr Gly Cys Val Tyr Phe Ala Glu Glu Met Leu

210 215 220

Lys Arg Gln Asp Lys Arg Ile Asp Gly Arg Arg Val Ala Val Ser Gly

225 230 235 240

Ser Gly Asn Val Ala Gln Tyr Ala Ala Arg Lys Val Met Asp Leu Gly

245 250 255

Gly Lys Val Ile Ser Leu Ser Asp Ser Glu Gly Thr Leu Tyr Ala Glu

260 265 270

Ala Gly Leu Thr Asp Ala Gln Trp Asp Ala Leu Met Glu Leu Lys Asn

275 280 285

Val Lys Arg Gly Arg Ile Ser Glu Leu Ala Gly Gln Phe Gly Leu Glu

290 295 300

Phe Arg Lys Gly Gln Thr Pro Trp Ser Leu Pro Cys Asp Ile Ala Leu

305 310 315 320

Pro Cys Ala Thr Gln Asn Glu Leu Gly Ala Glu Asp Ala Arg Thr Leu

325 330 335

Leu Arg Asn Gly Cys Ile Cys Val Ala Glu Gly Ala Asn Met Pro Thr

340 345 350

Thr Leu Glu Ala Val Asp Ile Phe Leu Asp Ala Gly Ile Leu Tyr Ala

355 360 365

Pro Gly Lys Ala Ser Asn Ala Gly Gly Val Ala Val Ser Gly Leu Glu

370 375 380

Met Ser Gln Asn Ala Met Arg Leu Leu Trp Thr Ala Gly Glu Val Asp

385 390 395 400

Ser Lys Leu His Asn Ile Met Gln Ser Ile His His Ala Cys Val His

405 410 415

Tyr Gly Glu Glu Ala Asp Gly Arg Ile Asn Tyr Val Lys Gly Ala Asn

420 425 430

Ile Ala Gly Phe Val Lys Val Ala Asp Ala Met Leu Ala Gln Gly Val

435 440 445

Val

<210> 9

<211> 449

<212> PRT

<213> T196A/T121L/L123H(Artificial Sequence)

<400> 9

Met Ser Thr Met Ile Glu Ser Val Asp Asn Phe Leu Ala Arg Leu Lys

1 5 10 15

Gln Arg Asp Pro Gly Gln Pro Glu Phe His Gln Ala Val Glu Glu Val

20 25 30

Leu Arg Thr Leu Trp Pro Phe Leu Glu Ala Asn Pro His Tyr Leu Gln

35 40 45

Ser Gly Ile Leu Glu Arg Met Val Glu Pro Glu Arg Ala Val Leu Phe

50 55 60

Arg Val Ser Trp Val Asp Asp Gln Gly Lys Val Gln Val Asn Arg Gly

65 70 75 80

Tyr Arg Ile Gln Met Ser Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu

85 90 95

Arg Phe His Pro Ser Val Asn Leu Ser Val Leu Lys Phe Leu Ala Phe

100 105 110

Glu Gln Val Phe Lys Asn Ser Leu Leu Ser His Pro Met Gly Gly Gly

115 120 125

Lys Gly Gly Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val

130 135 140

Met Arg Phe Cys Gln Ala Phe Met Ser Glu Leu Tyr Arg His Ile Gly

145 150 155 160

Ala Asp Cys Asp Val Pro Ala Gly Asp Ile Gly Val Gly Ala Arg Glu

165 170 175

Ile Gly Phe Met Phe Gly Gln Tyr Lys Arg Leu Ala Asn Gln Phe Thr

180 185 190

Ser Val Leu Ala Gly Lys Gly Met Thr Tyr Gly Gly Ser Leu Ile Arg

195 200 205

Pro Glu Ala Thr Gly Tyr Gly Cys Val Tyr Phe Ala Glu Glu Met Leu

210 215 220

Lys Arg Gln Asp Lys Arg Ile Asp Gly Arg Arg Val Ala Val Ser Gly

225 230 235 240

Ser Gly Asn Val Ala Gln Tyr Ala Ala Arg Lys Val Met Asp Leu Gly

245 250 255

Gly Lys Val Ile Ser Leu Ser Asp Ser Glu Gly Thr Leu Tyr Ala Glu

260 265 270

Ala Gly Leu Thr Asp Ala Gln Trp Asp Ala Leu Met Glu Leu Lys Asn

275 280 285

Val Lys Arg Gly Arg Ile Ser Glu Leu Ala Gly Gln Phe Gly Leu Glu

290 295 300

Phe Arg Lys Gly Gln Thr Pro Trp Ser Leu Pro Cys Asp Ile Ala Leu

305 310 315 320

Pro Cys Ala Thr Gln Asn Glu Leu Gly Ala Glu Asp Ala Arg Thr Leu

325 330 335

Leu Arg Asn Gly Cys Ile Cys Val Ala Glu Gly Ala Asn Met Pro Thr

340 345 350

Thr Leu Glu Ala Val Asp Ile Phe Leu Asp Ala Gly Ile Leu Tyr Ala

355 360 365

Pro Gly Lys Ala Ser Asn Ala Gly Gly Val Ala Val Ser Gly Leu Glu

370 375 380

Met Ser Gln Asn Ala Met Arg Leu Leu Trp Thr Ala Gly Glu Val Asp

385 390 395 400

Ser Lys Leu His Asn Ile Met Gln Ser Ile His His Ala Cys Val His

405 410 415

Tyr Gly Glu Glu Ala Asp Gly Arg Ile Asn Tyr Val Lys Gly Ala Asn

420 425 430

Ile Ala Gly Phe Val Lys Val Ala Asp Ala Met Leu Ala Gln Gly Val

435 440 445

Val

<210> 10

<211> 351

<212> PRT

<213> CbADH-6M(Artificial Sequence)

<400> 10

Met Lys Gly Phe Ala Met Leu Gly Ile Asn Lys Leu Gly Trp Ile Glu

1 5 10 15

Lys Glu Arg Pro Val Ala Gly Pro Tyr Asp Ala Ile Val Arg Pro Leu

20 25 30

Ala Val Ser Pro Cys Thr Ser Asp Ile His Thr Val Phe Glu Gly Ala

35 40 45

Leu Gly Asp Arg Lys Asn Met Ile Leu Gly His Glu Ala Val Gly Glu

50 55 60

Val Val Glu Val Gly Ser Glu Val Lys Asp Phe Lys Pro Gly Asp Arg

65 70 75 80

Val Ile Val Pro Cys Thr Thr Pro Asp Trp Arg Ser Leu Glu Val Gln

85 90 95

Ala Gly Phe Gln Gln His Ser Asn Gly Met Leu Ala Gly Trp Lys Phe

100 105 110

Ser Asn Phe Lys Asp Gly Val Phe Gly Glu Tyr Phe His Val Asn Asp

115 120 125

Ala Asp Met Asn Leu Ala Ile Leu Pro Lys Asp Met Pro Leu Glu Asn

130 135 140

Ala Val Met Ile Thr Asp Met Met Thr Thr Gly Phe His Gly Ala Glu

145 150 155 160

Leu Ala Asp Ile Gln Met Gly Ser Ser Val Val Val Ile Gly Ile Gly

165 170 175

Ala Val Gly Leu Met Ala Ile Ala Gly Ala Lys Leu Arg Gly Ala Gly

180 185 190

Arg Ile Ile Ala Val Gly Ser Arg Pro Ile Cys Val Glu Ala Ala Lys

195 200 205

Phe Tyr Gly Ala Thr Asp Ile Leu Asn Tyr Lys Asn Gly Asp Ile Val

210 215 220

Asp Gln Val Met Lys Leu Thr Asn Gly Lys Gly Val Asp Arg Val Ile

225 230 235 240

Met Ala Gly Gly Gly Ser Glu Thr Leu Glu Gln Ala Val Arg Met Val

245 250 255

Lys Pro Gly Gly Ile Ile Ser Asn Ile Asn Tyr His Gly Ser Gly Asp

260 265 270

Ala Leu Leu Ile Pro Arg Val Glu Trp Gly Cys Gly Met Ala His Lys

275 280 285

Thr Ile Lys Gly Gly Leu Cys Pro Gly Gly Arg Leu Arg Ala Glu Met

290 295 300

Leu Arg Asp Met Val Val Tyr Asn Arg Val Asp Leu Ser Lys Leu Val

305 310 315 320

Thr His Val Tyr His Gly Phe Asp His Ile Glu Glu Ala Leu Leu Leu

325 330 335

Met Lys Asp Lys Pro Lys Asp Leu Ile Lys Ala Val Val Ile Leu

340 345 350

<210> 11

<211> 1350

<212> DNA/RNA

<213> PpGluDH(Artificial Sequence)

<400> 11

atgtctacca tgatcgaatc tgtcgacaat ttccttgcac gcctgaagca gcgtgaccca 60

ggccagccgg aattccacca ggcggtggaa gaggtgctgc gcaccctgtg gccattcctt 120

gaagccaacc cccactatct gcagtccggc atcctcgagc gcatggtcga gcccgagcgc 180

gccgtacttt tccgcgtttc ttgggtcgat gaccagggca aggtgcaggt caaccgcggc 240

taccgcattc agatgagcag tgccattggc ccgtacaagg gcgggctgcg cttccacccg 300

tcggtgaacc tcagcgtgct gaagttcctg gcgttcgagc aggtgttcaa gaactcgctg 360

acctcgctgc ccatgggcgg cggcaagggc ggctcggact ttgacccgaa aggcaagagc 420

gacgccgaag tgatgcgctt ctgccaggcg ttcatgagcg agctgtatcg ccacatcggt 480

gctgactgcg acgtaccggc cggtgacatc ggtgtggggg cccgcgaaat cggcttcatg 540

ttcggccagt acaagcgcct ggccaaccag ttcacctcgg tgttgaccgg taagggcatg 600

acctatggcg gcagcctgat ccgcccggaa gccaccggct atggctgcgt ttacttcgcc 660

gaagaaatgc tcaagcgtca ggacaagcgt atcgacggcc gccgcgtggc ggtgtccggt 720

tcgggcaacg tcgcccagta tgctgcgcgt aaggtgatgg acctgggcgg caaggtgatc 780

tcgctgtctg actctgaagg taccttgtat gcagaagccg ggctgaccga cgcccagtgg 840

gacgccttga tggagctgaa aaacgtcaag cgcggacgta tcagcgagct ggccgggcaa 900

ttcggcctgg agttccgcaa gggccagacc ccgtggagcc tgccgtgcga catcgccctg 960

ccatgcgcca cccagaacga gctgggcgcc gaagatgccc gcaccttgct gcgtaacggc 1020

tgtatctgtg tggccgaagg cgccaacatg ccgactaccc tcgaggctgt ggatatcttc 1080

ctggacgccg gcattctgta cgccccgggc aaggcctcca atgcgggcgg cgtagccgtg 1140

tcgggcctgg aaatgtcgca gaacgccatg cgcctgctgt ggacggccgg tgaagtggac 1200

agcaagctgc acaacatcat gcagtcgatt caccatgcat gcgtgcacta cggtgaagag 1260

gctgatggcc ggatcaacta cgtcaagggg gcgaacatcg cgggcttcgt gaaagtggcc 1320

gatgcgatgc tggctcaggg cgtggtctga 1350

Claims (10)

1. An engineered NADPH-dependent phenylglycine dehydrogenase which is a mutant formed by combined mutation of threonine 196, threonine 121 and leucine 123 in an amino acid sequence of glutamate dehydrogenase shown in SEQ ID No. 1.

2. The engineered NADPH-dependent phenylglycine dehydrogenase of claim 1, wherein the engineered NADPH-dependent phenylglycine dehydrogenase comprises:

the amino acid sequence of the mutant is shown as SEQ ID NO.2, wherein the 196 th threonine of the amino acid sequence of the glutamate dehydrogenase shown as SEQ ID NO.1 is mutated into alanine, the 121 th threonine is mutated into isoleucine, and the 123 th leucine is mutated into aspartic acid;

the amino acid sequence of the mutant is shown as SEQ ID NO.3, wherein the 196 th threonine of the amino acid sequence of the glutamate dehydrogenase shown as SEQ ID NO.1 is mutated into alanine, the 121 th threonine is mutated into isoleucine, and the 123 th leucine is mutated into histidine;

the amino acid sequence of the mutant is shown in SEQ ID NO.4, wherein the 196 th threonine of the amino acid sequence of the glutamate dehydrogenase shown in SEQ ID NO.1 is mutated into alanine, the 121 th threonine is mutated into isoleucine, and the 123 th leucine is mutated into asparagine;

the amino acid sequence of the mutant is shown as SEQ ID NO.5, wherein the 196 th threonine of the amino acid sequence of the glutamate dehydrogenase shown as SEQ ID NO.1 is mutated into alanine, the 121 th threonine is mutated into isoleucine, and the 123 th leucine is mutated into serine;

the amino acid sequence of the mutant is shown in SEQ ID NO.6, wherein the 196 th threonine of the amino acid sequence of the glutamate dehydrogenase shown in SEQ ID NO.1 is mutated into alanine, the 121 th threonine is mutated into leucine, and the 123 th leucine is mutated into asparagine;

the amino acid sequence of the mutant is shown as SEQ ID NO.7, wherein the 196 th threonine of the amino acid sequence of the glutamate dehydrogenase shown as SEQ ID NO.1 is mutated into alanine, the 121 th threonine is mutated into leucine, and the 123 th leucine is mutated into serine;

the amino acid sequence of the mutant is shown in SEQ ID NO.8, wherein the 196 th threonine of the amino acid sequence of the glutamate dehydrogenase shown in SEQ ID NO.1 is mutated into alanine, the 121 th threonine is mutated into leucine, and the 123 th leucine is mutated into tyrosine;

the amino acid sequence of the mutant is shown in SEQ ID NO.9, wherein the 196 th threonine of the amino acid sequence of the glutamate dehydrogenase shown in SEQ ID NO.1 is mutated into alanine, the 121 th threonine is mutated into leucine, and the 123 th leucine is mutated into histidine.

3. An expression vector comprising the engineered NADPH-dependent phenylglycine dehydrogenase of claim 1 or 2.

4. A recombinant engineered bacterium comprising the engineered NADPH-dependent phenylglycine dehydrogenase of claim 1 or 2.

5. Use of an engineered NADPH-dependent phenylglycine dehydrogenase comprising the enzyme of claim 1 or 2 for the preparation of L-phenylglycine.

6. The use of the engineered NADPH-dependent phenylglycine dehydrogenase in the preparation of L-phenylglycine according to claim 5, wherein the preparation of L-phenylglycine comprises biocatalytic and fermentative preparation of L-phenylglycine.

7. The use of the engineered NADPH-dependent phenylglycine dehydrogenase in the preparation of L-phenylglycine according to claim 5, wherein the biocatalytic preparation of L-phenylglycine comprises the steps of:

s1, constructing recombinant engineering bacteria for expressing the engineered NADPH-dependent phenylglycine dehydrogenase in claim 1 or 2, and preparing an enzyme solution after culturing;

s2, adding the enzyme solution obtained in the step S1 into a mixed system containing substrate phenylglyoxylic acid, an amino donor and reduced coenzyme, and carrying out reductive amination reaction to obtain the L-phenylglycine.

8. The use of the engineered NADPH-dependent phenylglycine dehydrogenase in the preparation of L-phenylglycine according to claim 7, wherein the mixing system in step S2 further comprises a coenzyme regeneration system.

9. The use of the engineered NADPH-dependent phenylglycine dehydrogenase in the preparation of L-phenylglycine according to claim 7, wherein the coenzyme regeneration system comprises an alcohol dehydrogenase as a coenzyme-regenerating enzyme, isopropanol as a coenzyme-regenerating substrate, NADPH and NADP⁺The alcohol dehydrogenase coenzyme regeneration system of (1), the amino acid sequence of the alcohol dehydrogenaseShown as SEQ ID NO. 10.

10. The use of the engineered NADPH-dependent phenylglycine dehydrogenase in the preparation of L-phenylglycine according to claim 5, wherein the fermentative preparation of L-phenylglycine comprises the following steps:

s1, constructing recombinant engineering bacteria for expressing the engineered NADPH-dependent phenylglycine dehydrogenase of claim 1 or 2, inoculating the recombinant engineering bacteria into a seed culture medium, culturing for a period of time, and transferring the seed solution into a fermentation culture medium containing a phenylglyoxylate substrate and inorganic ammonium;

and S2, fermenting to make the concentration of the bacteria reach a certain degree, adding an inducer, and continuously fermenting and culturing to prepare the L-phenylglycine.

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