CN117844772A - Amine dehydrogenase mutant, engineering bacterium and application thereof in synthesis of (R) -3-aminobutanol - Google Patents

Abstract

The invention discloses an amine dehydrogenase mutant, engineering bacteria and application thereof in synthesizing (R) -3-aminobutanol, wherein the amine dehydrogenase mutant is obtained by carrying out single mutation or multiple mutation on amino acids at 69 th, 262 th, 147 th and 135 th of an amino acid sequence shown in SEQ ID No. 1. The enzyme activity of the amine dehydrogenase mutant provided by the invention is obviously improved from 0U/mg before mutation to 55.3U/g, and the defect of lower enzyme activity of the amine dehydrogenase is effectively overcome. Compared with the parent, the amine dehydrogenase mutant provided by the invention has better catalytic activity. Under the optimal system, the yield of the product of 120mM substrate reaches 75.4%, and the ee value reaches 99.9%, thus providing a certain basis for industrial production of (R) -3-aminobutanol.

Description

Amine dehydrogenase mutant, engineering bacterium and application thereof in synthesis of (R) -3-aminobutanol

Field of the art

The invention belongs to the technical field of biochemical engineering, and relates to a leum dehydrogenase derived from Geobacillus thermophilus (Geobacillus kaustophilus, GBlys), a mutant with amine dehydrogenase activity obtained through molecular modification, a recombinant expression vector and a recombinant expression transformant containing the mutant gene, and application of the mutant or the recombinant expression transformant as a catalyst in (R) -3-aminobutanol synthesis.

(II) background art

The (R) -3-aminobutanol is an important medical intermediate, has important application, is a key intermediate for synthesizing a chiral six-membered ring of an anti-AIDS drug Du Lute, and is also an important intermediate for synthesizing a penem antibiotic and an anti-tumor drug 4-methylcyclophosphoramide. Wherein Du Lute is a second generation integrase inhibitor approved by the U.S. FDA for treating HIV in 2013, has higher drug resistance barrier and better clinical effect compared with the first generation integrase inhibitor, and has slightly higher safety compared with the three-in-one oral drug, atriplan.

(R) -3-aminobutanol of formula:

currently, the synthesis of (R) -3-aminobutanol is mainly achieved by chemical methods. The chemical synthesis method mainly comprises chiral raw material synthesis method, chemical induction method, chiral resolution method, preparation chromatography and the like, and the chemical synthesis methods have the defects of complex procedures, serious pollution, harsh reaction conditions, low regio/enantioselectivity and the like.

Compared with chemical methods, the biocatalysis method has the advantages of mild reaction conditions, environmental friendliness and the like, and has been widely used in the industrial production of chiral chemicals such as medical intermediates, fine chemicals and the like. In recent years, aminotransferase (catalysts.2018; 8 (7): 254), lyase (Angewandte Chemie (international.). 2004; 43:788-824), imine reductase (Organic process research & development.2019; 23:1262-1268), amine dehydrogenase (Angewandte Chemie (international.). 2012; 51:3969-3972) and the like have been developed successively for the synthesis of chiral amines. The involvement of ammonia donors in the reaction process of transaminases results in a limited donor range, as enzymes of different configurations require different configurations of ammonia donors. Ammonia donors, the affinity of ammonia donors for enzymes, and coenzymes are limiting factors in the preparation of chiral amines using aminotransferases. In addition, the removal of by-products is also a concern in the transaminase reaction, further limiting the use of transaminases. Lyase is also used in the preparation of chiral amines, such as L-aspartic acid, alanine synthesis. However, lyase enzymes are not able to mediate the formation of C-N bonds in certain reactions such as the synthesis of L-phenylalanine (Angewandte Chemie (International.). 2004, 43:788-824). In addition, the optimum pH of the lyase is too different from that of the cascade enzyme, which severely limits the wide application. Imine reductase has low activity, an undefined catalytic mechanism, inability to perform efficient semi-rational design, and a small substrate range, and is difficult to apply to industrial production (Angewandte Chemie (international.). 2018; 57:1863-1868).

Compared with other biocatalysis methods, the method for synthesizing chiral amine by asymmetric reductive amination of ketone and ammonia catalyzed by amine dehydrogenase not only uses cheap inorganic ammonia as an amino donor, but also uses water as a reaction byproduct, so that the method has the advantages of high atom economy, high optical purity of products, mild reaction conditions, environment friendliness, simple product post-treatment and the like, has good application prospect in synthesis of various chiral amines, and is expected to be applied to industrial production.

In 2012, the Bommarius group of the university of georgia, usa, first advanced naturally occurring leucine dehydrogenases to artificial amine dehydrogenases by protein engineering techniques (Angewandte Chemie (international.). 2012, 51:3969-3972). Hereafter, the development of amine dehydrogenases has begun to receive extensive attention from industry and academia. In recent years, many researchers have reported amine dehydrogenases of various origins. For example, bommarius and Li are modified for substrate spectrum expansion, the created amine dehydrogenase substrate spectrum can be expanded to benzyl acetone, but the catalytic activity is extremely low (adv. Synth. Catalyst. 2013,355,1780-1786;ACS Catal.2015,5,1119-1122), and the actual application requirements cannot be met.

Therefore, it is important to develop highly active, highly stable amine dehydrogenases to meet the biosynthesis of chiral amino alcohol compounds.

(III) summary of the invention

The invention aims to provide an amine dehydrogenase mutant with improved activity, engineering bacteria and application thereof in synthesizing (R) -3-aminobutanol, and solves the problem of low activity of the amine dehydrogenase.

The technical scheme adopted by the invention is as follows:

the invention provides an amine dehydrogenase mutant with improved activity, which is obtained by single mutation or multiple mutation of amino acid 69, 262, 147 and 135 of an amino acid sequence shown in SEQ ID No. 1.

The amine dehydrogenase mutant is a mutant with enhanced activity, which is obtained by performing site-directed saturation mutation based on an amine dehydrogenase (marked as GkGBLeuDH and having an amino acid sequence shown as SEQ ID No. 1) derived from Geobacillus thermophilus (G.kaustophilus).

Further, it is preferable that the amine dehydrogenase mutant is one in which the amino acid sequence shown in SEQ ID No.1 is mutated to: (1) The lysine at position 69 is mutated to serine, designated GKGBLeuDH _K69S The amino acid sequence is shown as SEQ ID No.2, and the nucleotide sequence of the coding gene is shown as SEQ ID No. 7; (2) Lysine at position 69 was mutated to serine, while asparagine at position 262 was mutated to leucine, designated GkGBAmDH _K69S/N262L The amino acid sequence is shown as SEQ ID No.3, and the nucleotide sequence of the coding gene is shown as SEQ ID No. 8; (3) Lysine at position 69 is mutated to serine, asparagine at position 262 is mutated to leucine, and proline at position 147 is mutated to valine, designated GkGBAmDH _{K69S/N262L/P147V} The amino acid sequence is shown as SEQ ID No.4, and the nucleotide sequence of the coding gene is shown as SEQ ID No. 9; (4) Lysine at position 69 is mutated to serine, asparagine at position 262 is mutated to leucine, proline at position 147 is mutated to valine, and threonine at position 135 is mutated to glycine, designated GkGBAmDH _{K69S/N262L/P147V/T135G} The amino acid sequence is shown as SEQ ID No.5, and the nucleotide sequence of the coding gene is shown as SEQ ID No. 10.

The invention also provides a coding gene of the amine dehydrogenase mutant, a recombinant expression vector containing the coding gene and recombinant genetic engineering bacteria prepared by transforming the recombinant expression vector.

The recombinant expression vector may be constructed by ligating nucleic acids encoding the amine dehydrogenase mutant genes of the present invention to various suitable vectors by conventional methods in the art. The vector may be various conventional vectors in the art, such as a commercially available plasmid, cosmid, phage, or viral vector, etc., as long as the recombinant expression vector can normally replicate in the corresponding expression host and express the amine dehydrogenase mutant. The amine dehydrogenase mutant gene may be operably linked downstream of suitable regulatory sequences in the vector to achieve constitutive or inducible expression of the amine dehydrogenase mutant. The vector is preferably a plasmid, more preferably plasmid pET-28a. The host cell is a variety of conventional host cells in the art, as long as the recombinant expression vector is capable of stably self-replicating and efficiently expressing the target protein after induction by an inducer. The invention is preferably used as a host cell, and more preferably E.coli BL21 (DE 3) is used for efficiently expressing the amine dehydrogenase mutant. The culture of the recombinant expression transformant is a method and culture conditions conventional in the art.

The invention also provides an application of the amine dehydrogenase mutant in catalyzing the synthesis of (R) -3-aminobutanol by using 4-hydroxy-2-butanone, wherein the application method comprises the following steps: wet bacterial body obtained by fermenting recombinant genetic engineering bacteria containing amine dehydrogenase mutant coding genes is used as a catalyst, glucose dehydrogenase is used as coenzyme, 4-hydroxy-2-butanone is used as a substrate, glucose is used as an auxiliary substrate, and NAD is used ⁺ As cofactor, buffer with pH 8.5-10.0 (preferably NH with pH9.0, 1M) ₄ Cl/NH ₃ ·H ₂ O buffer) is used as a reaction medium to form a reaction system, the conversion reaction is carried out at the temperature of 20-45 ℃ and the rotation speed of 1000-1500rpm (preferably 40 ℃ and 1100 rpm), and after the reaction is finished, the reaction solution is separated and purified to obtain the (R) -3-aminobutanol.

Further, it is preferable that the amount of the catalyst used in the reaction system is 10 to 100g/L (preferably 50 to 100g/L, most preferably 50 g/L) based on the weight of the wet microorganism, the final concentration of the substrate is 30 to 200mM (preferably 30 to 150mM, more preferably 30 to 50 mM), the final concentration of the cofactor is 0.1 to 2mM (preferably 2 mM), the final concentration of the glucose is 100 to 300mM (preferably 200 mM), and the final concentration of the glucose dehydrogenase is 1 to 5g/L (preferably 2 g/L). The specific enzyme activity of the freeze-dried enzyme powder of the glucose dehydrogenase is 56.12U/mg.

Further, the catalyst is prepared as follows:

inoculating recombinant genetic engineering bacteria containing an amine dehydrogenase mutant encoding gene into LB solid medium containing 50 mug/mL kanamycin, and culturing at 37 ℃ overnight; picking single colony in LB liquid medium containing 50 mug/mL kanamycin, shaking and culturing for 8-10h at 37 ℃ with 180rpm to obtain seed liquid; inoculating the seed solution into LB liquid medium containing 50 mug/mL kanamycin according to the inoculum size of 1-2% (v/v), shaking the culture medium at 37 ℃ and 180rpm, and obtaining the OD of the culture solution ₆₀₀ When the concentration reaches 0.6-0.8, isopropyl-beta-D-thiogalactoside (IPTG) with the final concentration of 0.1-0.5 mmol/L is added as an inducer, after the induction is carried out for 12 hours at 28 ℃, the culture solution is centrifuged, and the precipitate is collected, thus obtaining the catalyst.

LB liquid medium composition: 10g/L peptone, 5g/L yeast powder, 10g/L NaCl, water as solvent and natural pH. The LB solid culture medium is prepared by adding 20g/L agar powder into the LB liquid culture medium.

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

(1) The enzyme activity of the amine dehydrogenase mutant provided by the invention is obviously improved from 0U/mg before mutation to 55.3U/g, and the defect of lower enzyme activity of the amine dehydrogenase is effectively overcome.

(2) The amine dehydrogenase mutant is used as a catalyst for catalyzing 4-hydroxy-2-butanone to synthesize (R) -3-aminobutanol, adopts a one-pot formula, starts the reaction after adding a substrate and enzyme, and directly obtains the final product (R) -3-aminobutanol, and has low industrial cost. Meanwhile, the initial raw material is 4-hydroxy-2-butanone, the amino donor is inorganic ammonia, and the price is low; the catalyst can be prepared in large quantity by constructing escherichia coli genetic engineering bacteria and then fermenting, and is relatively easy to obtain and low in cost.

(3) Compared with the parent, the amine dehydrogenase mutant provided by the invention has better catalytic activity. Under the optimal system, the product yield of 120mM substrate reaches 77.4%, and the ee value reaches 99.9%, thus providing an important basis for efficiently preparing (R) -3-aminobutanol.

(IV) description of the drawings

FIG. 1 is a reaction scheme of the biological preparation method of (R) -3-aminobutanol.

FIG. 2 is a graph of the HPLC results of R-3-aminobutanol standard.

FIG. 3 is a graph of HPLC results for S-3-aminobutanol standard.

FIG. 4 is a graph showing the HPLC result of the reaction for 24 hours in the reaction system 3 of example 4.

(fifth) detailed description of the invention

The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:

the term "AxxB" in the present examples means that amino acid a at position xx is changed to amino acid B, e.g. "K69S" means that lysine K at position 69 is mutated to serine S, and so on.

LB liquid medium composition: 10g/L peptone, 5g/L yeast powder, 10g/L NaCl, water as solvent and natural pH. The LB solid culture medium is prepared by adding 20g/L agar powder into the LB liquid culture medium.

The specific enzyme activity of the Glucose Dehydrogenase (GDH) freeze-dried powder in the embodiment of the invention is 56.12U/mg. Wherein the enzyme activity is defined as: conversion of 1mmol of NAD per unit time, i.e. 1min ⁺ The amount of lyophilized enzyme powder required to produce 1mmol NADH.

EXAMPLE 1 construction of the parent strain of recombinant E.coli with amine dehydrogenase

Amine dehydrogenase gene (amino acid sequence shown in SEQ ID NO.1, nucleotide sequence shown in SEQ ID NO. 5) derived from Geobacillus thermophilus (G.kaustophilus, NCBI accession number GAD 13035.1) was selected for total gene synthesis, and after ligation to TATATAACCAT of plasmid pET-28a (NcoI) and before CTCGAG (XhoI cleavage site), E.coli BL21 (DE 3) competent cells were transferred, and LB plates containing kanamycin (50. Mu.g/mL) were plated, and after overnight incubation at 37℃positive transformants were picked and identified for sequencing.

The positive monoclonal is inoculated into 5mL LB liquid medium containing 50 mug/mL kanamycin, cultured overnight at 37 ℃, plasmids are extracted, after re-verification, the recombinant expression vector is transferred into escherichia coli BL21 (DE 3) strain to obtain recombinant strain E.coli BL21 (DE 3)/pET 28a-GkGBLeuDH (namely parent strain), and cultured overnight at 37 ℃ on LB flat plate containing kanamycin (50 mug/mL). The monoclonal is selected and inoculated into 10mL of LB liquid medium containing 50 mug/mL kanamycin, cultured overnight at 37 ℃, centrifuged at 8000rpm for 10min at 4 ℃, and the wet thalli are collected and stored for standby at-80 ℃.

SEQ ID NO.1

1 mmelfqymek ydyeqvlfcq dkesglkaii vihdttlgpa lggtrmwmyn seeealedal

61 rlargmtykn aaaglnlggg ktviigdprk dkneamfraf grfiqglngr yitaedvgtt

121 vadmdiiyqe tdyvtgispe fgssgnpspa taygvyrgmk aaakeafgsd slegkvvavq

181 gvgnvayhlc rhlheegakl ivtdinkeav araveefgak avdpndiygv ecdifapcal

241 ggiindqtip qlkakviags annqlkeprh gdmihemgiv yapdyvinag gvinvadely

301 gynreramkk ieqiydniek vfaiakrdni ptyvaadrma eerietmrka rsqflqnghh

361 ilsrrrarhh hhhh。

Example 2: construction of amine dehydrogenase mutant and preparation of recombinant expression transformant

1. Screening of mutant mutation sites of amine dehydrogenase

Plasmid pET28a-GkGBLeuDH in the parent strain of example 1 is extracted, sequence comparison is carried out through ESPrip 3.0, meanwhile, the structure of GkGBLeuDH protein (the amino acid sequence is shown as SEQ ID NO. 1) is modeled by utilizing an alpha Fold2 (alpha Fold2 TIB Server (biodesign. Ac. Cn)) website, hotSpot wizard is used for prediction, and 44, 69, 115, 135, 147, 262 and 292 alleles in structural hot spots and functional hot spots in a prediction result are subjected to saturation mutation.

2. Construction of saturated mutation library

Using the recombinant expression plasmid pET28a-GkGBLeuDH obtained in example 1 as a DNA template, a saturated mutation primer (Table 1) was designed, and amplified by PCR to obtain a mutation library.

TABLE 1 saturation mutagenesis primer

Adding thick fonts in tableThe degenerate codons represented contained 32 codon combinations covering all 20 amino acid residues, n=a/C/G/T, k=g/T.

PCR extension System (total volume 50. Mu.L): 25. Mu.L 2X Phanta Max buffer, 1. Mu.L dNTP Mix (10 mM each), 0.5. Mu.L template, 1. Mu.L (10. Mu.M) each of a pair of mutation primers, 0.5. Mu. L Phanta Max Super-Fidelity DNAPolymerase, and sterile distilled water to 50. Mu.L.

PCR reaction procedure: (1) denaturation at 95℃for 5min, (2) denaturation at 95℃for 30sec, (3) annealing at 59℃for 30sec, (4) elongation at 72℃for 4min for 30sec, steps (2) to (4) were carried out in total for 30 cycles, (5) elongation at 72℃for 10min, and (6) heat preservation at 16 ℃.

The PCR product was stored at 4 ℃. After the PCR product is verified by agarose gel electrophoresis analysis, the restriction enzyme DpnI is added to digest for 2-3 hours at 37 ℃. The digested product was transferred into E.coli BL21 (DE 3) competent cells and plated on LB plates containing 50. Mu.g/mL kanamycin resistance, and placed in a 37℃incubator for stationary culture for about 12 hours, thus obtaining a mutant library.

3. Screening of Single mutants

(1) Culture of Positive clones

Selecting monoclonal from the mutation library, inoculating to LB liquid medium, culturing in an incubator at 37 ℃ and 180rpm for 12h, transferring to a new LB liquid medium with an inoculum size of 1% of volume concentration, culturing at 37 ℃ and 180rpm for 2h, adding 0.1mM isopropyl thiogalactoside (IPTG), culturing at 28 ℃ and 180rpm for 12h, centrifuging, collecting wet thalli, namely amine dehydrogenase wet thalli, and carrying out research on catalytic synthesis of (R) -3-aminobutanol.

(2) Synthesis of (R) -3-aminobutanol

The final concentration composition of the 1mL reaction system is as follows: 4-hydroxy-2-butanone substrate 50mM,200mM glucose, 2.0mM NAD ⁺ 2mg/mL GDH and 100mg/mL wet cell of amine dehydrogenase, 1M ammonium chloride buffer (pH 10.0). After 24 hours of reaction at 40 ℃ and 1100rpm, the reaction solution is boiled for 5-10min and centrifuged. After the supernatant is subjected to derivatization reaction by using FDAA, sampling and measuring the peak area of the product (R) -3-aminobutanol by using liquid chromatography, and according to a standard curve prepared under the same condition by using a product standard substance, the peak outlet time of the product (R) -3-aminobutanol and a (S) -3-aminobutanol standard substance is shown in the accompanying drawings 2 and 3, calculating the yield of the product, and the result is shown in the table 2, and screening the recombinant bacteria with improved activity.

The derivatization reaction is as follows: 200. Mu.L of the centrifuged supernatant was added with 800. Mu.L of 6mM FDAA (solvent DMSO) and 50. Mu.L of 1M NaHCO was added thereto ₃ The solution was stirred and then reacted in a metal bath at 40℃and 300rpm for 1 hour, and after the reaction was completed, 50. Mu.L of 2M HCl was immediately added to terminate the reaction.

Yield = product concentration/substrate initial concentration 100%

The definition of enzyme activity is: under the above reaction conditions, the amount of enzyme required to catalyze the conversion of 1. Mu. Mol of substrate 4-hydroxy-2-butanone to (R) -3-aminobutanol per hour is one enzyme activity unit, denoted by U.

Liquid phase detection conditions: agilent 1260 Infinicity II, column C18 (Unitary, 5 μm. Times.250 mm. Times.4.6 mm). The detection wavelength is 340nm, the flow rate is 0.8mL/min, the sample injection amount is 10 mu L, and the column temperature is 30 ℃.

Table 2 single mutant enzyme activity table

Single mutants K69S, N262L, P V and T135G with activity were obtained by sequencing analysis and were screened to show 3.37, 3.01, 2.12 and 1.88U/G from the enzyme activity on 4-hydroxy-2-butanone. None of the individual mutations at positions 44, 115, 292 resulted in mutants with amine dehydrogenase activity. The single mutant K69S has the highest activity and is named GKGBLeuDH _K69S Amino acid sequences such asThe nucleotide sequence of the coding gene is shown as SEQ ID No.2 and SEQ ID No. 7. By a method comprising GKGBLeuDH _K69S Recombinant genetically engineered bacterium E.coli BL21 (DE 3) -pET28a-GkGBAmDH _K69S The following reactions were carried out for the dominant strains.

4. Double mutation

Combined mutation of single point mutation with increased vigor to obtain plasmid pET28a-GkGBAmDH with mutation of 69 th lysine into serine _K69S The primer of 262 th amino acid mutation in table 1 is used as template, the 262 th asparagine of amino acid sequence is mutated into leucine through whole plasmid PCR, then the Dpn I enzyme degradation is carried out to remove DNA template, the mutated plasmid is transformed into E.coli BL21 (DE 3), thus obtaining recombinant genetically engineered bacterium E.coli BL21 (DE 3) -pET28a-GkGBAmDH _K69S/N262L The corresponding amine dehydrogenase mutant is a double mutant in which the 69 th lysine is mutated to serine and the 262 th asparagine is mutated to leucine, and is named GkGBAmDH _K69S/N262L (the amino acid sequence is shown as SEQ ID NO.3, and the nucleotide sequence is shown as SEQ ID NO. 8). The specific enzyme activity of the mutant wet thalli for catalyzing 4-hydroxy-2-butanone to synthesize (R) -3-amino butanol is 18.6U/g by adopting a single mutant enzyme activity detection method and conditions for detection.

In the same way, gkGBAmDH is obtained _K69S/P147V 、GkGBAmDH _K69S/T135G The results are shown in Table 3.

Table 3 Combined double mutant enzyme activity table

5. Three mutants

Recombinant expression plasmid pET28a-GkGBAmDH _K69S/N262L The primer of 147 th amino acid mutation in table 1 is used as template, the 147 th proline of amino acid sequence is mutated into valine through whole plasmid PCR, then the Dpn I enzyme degradation is carried out to remove DNA template, the mutated plasmid is transformed into E.coli BL21 (DE 3), thus obtaining the genome genetic engineering bacterium E.coli BL21 (DE 3) -pET28a-GkGBAmDH _{K69S/N262L/P147V} The corresponding mutant was No. 68Three mutants in which lysine was mutated to serine, asparagine 262 to leucine and proline 147 to valine were designated GkGBAmDH _{K69S/N262L/P147V} (the amino acid sequence is shown as SEQ ID NO.4, and the nucleotide sequence is shown as SEQ ID NO. 9). The specific enzyme activity of the wet mutant strain catalyzing the synthesis of (R) -3-aminobutanol by using the single mutant enzyme activity detection method and conditions is 34.7U/g.

Under the same conditions, gkGBAmDH is prepared _{K69S/N262L/P147N} 、GkGBAmDH _{K69S/N262L/T135G} The results are shown in Table 4.

Table 4 Combined three mutant enzyme activity table

6. Four mutant

Recombinant expression plasmid pET28a-GkGBAmDH _{K69S/N262L/P147V} The 135 th threonine of the amino acid sequence is mutated into glycine by full plasmid PCR by using a primer mutated at the 135 th amino acid in the table 1 as a template, then a DNA template is removed by Dpn I enzyme degradation, and the mutated plasmid is transformed into E.coli BL21 (DE 3), thus obtaining the genome genetic engineering bacterium E.coli BL21 (DE 3) -pET28a-GkGBAmDH _{K69S/N262L/P147V/T135G} The corresponding mutant is a four mutant with lysine 69 mutated to serine, asparagine 262 mutated to leucine, proline 147 mutated to valine and threonine 135 mutated to glycine, and is named GKBBAMDH _{K69S/N262L/P147V/T135G} (the amino acid sequence is shown as SEQ ID NO.5, and the nucleotide sequence is shown as SEQ ID NO. 10). The specific enzyme activity of the wet mutant thallus for catalyzing the synthesis of (R) -3-aminobutanol by using the single mutant enzyme activity detection method and conditions is 55.3U/g.

Under the same conditions, gkGBAmDH is obtained _{K69S/N262L/P147V/T135C} 、GkGBAmDH _{K69S/N262L/P147V/T135V} The results are shown in Table 5.

TABLE 5 four mutant enzyme activity tables

Example 3 preparation of an amine dehydrogenase recombinant genetically engineered Strain catalyst

The recombinant genetically engineered bacteria containing the amine dehydrogenase encoding gene of example 1 and the recombinant genetically engineered bacteria containing the amine dehydrogenase mutant encoding gene screened in example 2 were fermented and cultured:

E.coli BL21(DE3)-pET28a-GkGBLeuDH、

E.coli BL21(DE3)-pET28a-GkGBAmDH _K69S 、

E.coli BL21(DE3)-pET28a-GkGBAmDH _K69S/N262L 、

E.coli BL21(DE3)-pET28a-GkGBAmDH _{K69S/N262L/P147V} 、

E.coli BL21(DE3)-pET28a-GkGBAmDH _{K69S/N262L/P147V/T135G} 。

(1) Plate culture: inoculating the recombinant genetically engineered bacteria to LB culture medium containing 50 mug/ml kanamycin, and culturing at 37 ℃ for 12-14h to obtain thalli on a flat plate;

(2) Seed culture: inoculating the slant thallus to LB liquid medium containing 50 mug/ml kanamycin, culturing at 37 ℃ and 180rpm for 8-10h to obtain seed liquid;

(3) Fermentation culture: inoculating the seed solution into LB liquid medium containing 50 μg/ml kanamycin resistance at 1% by volume, culturing at 37deg.C and 180rpm to OD ₆₀₀ Adding 0.1mM isopropyl-beta-D-galactoside with final concentration, culturing at 28deg.C for 12 hr, centrifuging, collecting wet thallus, and respectively obtaining wet thallus GKGBLeuDH, GKGBLeuDH _K69S 、GKGBLeuDH _K69S/N262L 、GKGBLeuDH _{K69S/N262L/P147V} 、GKGBLeuDH _{K69S/N262L/P147V/T135G} Storing at-20deg.C for use.

EXAMPLE 4 Synthesis of (R) -3-aminobutanol by amine dehydrogenase mutant catalyzing 4-hydroxy-2-butanone

The wet cell prepared in example 3 was used as a catalyst, 4-hydroxy-2-butanone was used as a substrate, glucose was used as an auxiliary substrate, GDH was used as a coenzyme,in NAD ⁺ As cofactor, a buffer with a pH of 8.5 to 10.0 (preferably NH with ph=10.0) ₄ Cl/NH ₃ ·H ₂ O buffer) as a reaction medium to form a reaction system, and carrying out the following reactions:

(1) Reaction system 1: the substrate concentration was 30mM,100mM glucose, 1.0mM NAD ⁺ 2mg/mL GDH and 50g/L amine dehydrogenase wet cells, 1M ammonium chloride buffer (pH 9.0) constituted 1.0mL reaction system. After 24 hours of reaction at 40℃and 1100rpm, the mixture was boiled for 10 minutes and centrifuged, and the yield of the product (R) -3-aminobutanol was measured by the method of example 2.

Analysis shows that the wet thalli GkGBLeuDH is used as a catalyst, and no product is detected; wet bacterial strain GKGBLeuDH _K69S The catalyst is used, and the yield reaches 5.5%; wet bacterial strain GKGBLeuDH _K69S/N262L The yield is up to 35.3% as a catalyst; wet bacterial strain GKGBLeuDH _{K69S/N262L/P147V} The catalyst is used, and the yield reaches 52.1%; wet bacterial strain GKGBLeuDH _{K69S/N262L/P147V/T135G} The yield of the catalyst reaches 83.4 percent.

(2) Reaction system 2: the substrate concentration was 50mM,200mM glucose, 2.0mM NAD ⁺ 2mg/mL GDH and 50g/L amine dehydrogenase wet cell, 1M ammonium chloride buffer (pH 10.0) 1.0mL reaction system. After 24 hours of reaction at 40℃and 1100rpm, the mixture was boiled for 10 minutes and centrifuged, and the yield of the product (R) -3-aminobutanol was measured by the method of example 2. The wet bacterial GkGBLeuDH is used as a catalyst and the generation of the product is not detected through detection and analysis; wet bacterial strain GKGBLeuDH _K69S The yield is up to 4.3% as a catalyst; wet bacterial strain GKGBLeuDH _K69S/N262L The yield is up to 30.4% as a catalyst; wet bacterial strain GKGBLeuDH _{K69S/N262L/P147V} The yield is 48.1% as a catalyst; wet bacterial strain GKGBLeuDH _{K69S/N262LP147V/T135G} The yield is 68.3% as a catalyst; the reaction time was prolonged under the reaction system, and wet bacterial cells GKGBLeuDH were used during the reaction for 36 hours _{K69S/N262L/P147V/T135G} The yield of the catalyst reaches 79.3 percent.

(3) Reaction system 3: substrate concentration was 120mM,200mM glucose, 2.0mM NAD ⁺ 2mg/mL GDH and 50g/L amine dehydrogenase wet cells, 1M ammonium chloride buffer (pH 10.0) constituted 1.0mL reaction system. 40 c,1100rpm, boiling for 10min after 24h reaction, centrifuging, detecting the yield of the product (R) -3-aminobutanol by the method of example 2, and obtaining a liquid chromatogram as shown in FIG. 4, wherein no S-type product is generated basically. Obtained by detection and analysis, and wet bacterial cells GKGBLeuDH _{K69S/N262L/P147V/T135G} The yield is up to 60.1% as a catalyst; under the reaction system, the mixture is reacted for 36 hours, and wet bacterial cells GKGBLeuDH are used _{K69S/N262LP147V/T135G} As a catalyst, the yield reaches 75.4%, and the ee value reaches 99.9%.

The invention is not limited by the specific literal description above. The invention is susceptible of various modifications within the scope of the claims, which modifications are all intended to be within the scope of the invention.

Claims (7)

1. An amine dehydrogenase mutant with improved activity, which is characterized in that the amine dehydrogenase mutant is obtained by single mutation or multiple mutation of amino acid 69, 262, 147 and 135 of an amino acid sequence shown in SEQ ID No. 1.

2. The amine dehydrogenase mutant of claim 1, wherein the amine dehydrogenase mutant is characterized by a mutation of the amino acid sequence set forth in SEQ ID No.1 to one of the following: (1) lysine at position 69 is mutated to serine; (2) Lysine at position 69 is mutated to serine, while asparagine at position 262 is mutated to leucine; (3) Lysine at position 69 is mutated to serine, asparagine at position 262 is mutated to leucine, and proline at position 147 is mutated to valine; (4) Lysine at position 69 is mutated to serine, asparagine at position 262 is mutated to leucine, proline at position 147 is mutated to valine, and threonine at position 135 is mutated to glycine.

3. A recombinant genetically engineered bacterium constructed from the gene encoding the amine dehydrogenase mutant of claim 1.

4. Use of an amine dehydrogenase mutant according to claim 1 for catalyzing the synthesis of (R) -3-aminobutanol from 4-hydroxy-2-butanone.

5. The application according to claim 4, wherein the method of application is: wet bacterial body obtained by fermenting recombinant genetic engineering bacteria containing amine dehydrogenase mutant coding genes is used as a catalyst, glucose dehydrogenase is used as coenzyme, 4-hydroxy-2-butanone is used as a substrate, glucose is used as an auxiliary substrate, and NAD is used ⁺ The cofactor is a reaction system formed by taking buffer solution with pH of 8.5-10.0 as a reaction medium, the conversion reaction is carried out at the temperature of 20-45 ℃ and the rotation speed of 1000-1500rpm, and after the reaction is finished, the reaction solution is separated and purified to obtain the (R) -3-aminobutanol.

6. The use according to claim 5, wherein the catalyst is used in an amount of 10 to 100g/L based on the weight of the wet microorganism, the final concentration of the substrate is 30 to 200mM, the final concentration of the cofactor is 0.1 to 2mM, the final concentration of the glucose is 100 to 300mM, and the final concentration of the glucose dehydrogenase is 1 to 5g/L.

7. The use according to claim 5, wherein the catalyst is prepared by the following method: inoculating recombinant genetic engineering bacteria containing an amine dehydrogenase mutant encoding gene into LB solid medium containing 50 mug/mL kanamycin, and culturing at 37 ℃ overnight; picking single colony in LB liquid medium containing 50 mug/mL kanamycin, shaking and culturing for 8-10h at 37 ℃ with 180rpm shaking table to obtain seed liquid; inoculating the seed solution into LB liquid culture medium containing 50 mug/mL kanamycin according to the inoculum size of 1-2% of the volume concentration, shaking the culture medium at 37 ℃ at 180rpm, and obtaining the OD of the culture solution ₆₀₀ When the concentration reaches 0.6-0.8, adding isopropyl-beta-D-thiogalactoside with the final concentration of 0.1-0.5 mmol/L as an inducer, and centrifuging the culture solution after 12h of induction at 28 ℃, and collecting the precipitate, thus obtaining the catalyst.