CN120574328A - Directed immobilized dual enzyme for synthesizing glutathione, preparation method and application - Google Patents

Abstract

The present invention discloses a directed immobilized dual enzyme for glutathione synthesis, as well as its preparation method and application. The dual enzyme is prepared by modifying polyphosphate kinase (PPK) with a short peptide SpyTag and glutathione bifunctional synthetase (GshF) with a short peptide SpyCatcher. The enzyme then utilizes affinity with agarose resin and specific SpyCatcher/SpyTag binding to improve the enzyme activity recovery rate of the immobilized dual enzyme. The immobilization method is simple, the reaction conditions are mild, and the enzyme is easy to operate, thereby improving the thermal stability of the prepared immobilized enzyme.

Description

Directional immobilized double enzyme for synthesizing glutathione, preparation method and application

Field of the art

The invention belongs to the technical field of bioengineering, and particularly relates to a directional immobilized double-enzyme of glutathione bifunctional enzyme and polyphosphate kinase, a preparation method and application thereof.

(II) background art

Multienzyme catalysis has advantages of system development, simplified process, controllable process, etc., and is widely used in biological manufacturing of medicines and nutritional chemicals (Science, 2019,366,1255-1259). However, the free enzyme has poor stability, is difficult to recycle and is unfavorable for downstream separation processes. The enzyme immobilization technology can improve the stability of the enzyme, and simultaneously maintain the characteristics of high efficiency and specificity of enzyme catalysis. In addition, the immobilized enzyme can be recycled, which is favorable for separating products, simplifies the separation process, prolongs the service life of the enzyme catalyst and reduces the production cost (appl. Environ. Microb.,2024, 90:e01574-23).

The glutathione bifunctional synthetase (Glutathione bifunctional enzymes, gshF) is coupled with the polyphosphate kinase (Polyphosphate kinase, PPK) to synthesize the glutathione, and the method has the advantages of simple reaction system components, easy operation, high yield and the like, and is one of the most effective methods for synthesizing the glutathione at present. At present, glutathione bifunctional synthetases and polyphosphate kinases have been reported to be immobilized less. The enzyme immobilization methods commonly used include physical adsorption, covalent bonding, crosslinking, and embedding. However, the immobilized enzyme is randomly combined with the carrier, so that the recovery rate of the immobilized enzyme is low. The directional enzyme immobilization is that the enzyme is combined with the carrier in a certain orientation and specific combination mode, so that the enzyme can be immobilized on the carrier in an orderly mode, the steric hindrance is reduced, the mass transfer resistance caused by accumulation in the immobilization is avoided, the diffusion speed of a substrate and a product can be accelerated, the multi-enzyme synergistic catalysis is facilitated, and a high-efficiency cascade reaction system is constructed.

Therefore, the directional immobilization method capable of combining the multienzyme with the carrier specifically is developed, so that the reduction of enzyme activity can be avoided, the multienzyme immobilization efficiency is improved, the efficient directional immobilization is realized, meanwhile, the stability of the immobilized multienzyme is enhanced, and the method has important application value in the field of immobilized multienzyme.

(III) summary of the invention

The invention aims to provide a directional immobilized double enzyme for synthesizing glutathione, a preparation method and application, the invention respectively uses short peptide SpyCatcher and short peptide SpyTag to modify glutathione double function enzyme and polyphosphate kinase and then uses agarose resin to perform directional immobilization, the multi-enzyme immobilization efficiency is improved, the efficient directional immobilization is realized, the stability of immobilized multi-enzyme is enhanced, and the problems of low multi-enzyme immobilization efficiency, poor stability and the like are solved.

The technical scheme adopted by the invention is as follows:

The invention provides a directional immobilized double enzyme for synthesizing glutathione, which is obtained by co-immobilizing glutathione bifunctional synthetase (GshF) and polyphosphate kinase (PPK) in a phosphate buffer solution with pH of 7-10 and containing imidazole at 20-30 ℃ respectively through fusion expression of connecting peptide, short peptide SpyCatcher and short peptide SpyTag as active components and agarose resin as a carrier.

Further, the connecting peptide comprises [ EAAAK ] ₃、[Gly]₈、[Gly]₆、[GGGGS]₃, preferably [ GGGGS ] ₃, and the amino acid sequence is shown as SEQ ID NO. 4.

Further, the amino acid sequence of the short peptide SpyCatcher is shown as SEQ ID NO.3, and the amino acid sequence of the short peptide SpyTag is shown as SEQ ID NO. 7.

Further, the amino acid sequence of the glutathione bifunctional synthetase is shown as SEQ ID NO.5, and the nucleotide sequence is shown as SEQ ID NO. 6.

Further, the amino acid sequence of the polyphosphate kinase is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO. 2.

Further, the final concentration of imidazole is 30-50 mM.

Further, the phosphate buffer is 1.0-2.0M phosphate buffer, preferably 2.0M phosphate buffer with pH of 9.0.

Further, the agarose resin includes GST agarose resin, ni-NTA agarose resin, ni-IDA agarose resin, preferably Ni-IDA agarose resin.

The invention also provides a preparation method of the directional immobilized double enzyme, which comprises the following steps:

(1) Sequentially connecting a polyphosphate kinase target gene PPK, a connecting peptide, a short peptide SpyTag coding gene and a histidine tag, inserting the obtained gene fragment PPK-linker-SpyTag-His6 into an expression vector, then converting into escherichia coli to construct recombinant bacteria, and inducing the expressed wet bacteria to be subjected to ultrasonic crushing to obtain a mixed solution of the polyphosphate kinase with the SpyTag short peptide and the histidine tag, namely a crude enzyme solution;

(2) The glutathione bifunctional synthetase target gene GshF, the connecting peptide and the short peptide SpyCatcher coding gene are connected in sequence, the obtained gene fragment GshF-linker-SpyCatcher is inserted into an expression vector, then the expression vector is transformed into escherichia coli to construct recombinant bacteria, and the mixed solution of the wet bacteria induced to be expressed is subjected to ultrasonic disruption, so that the crude enzyme solution of the glutathione bifunctional synthetase with SpyCatcher short peptide is obtained;

(3) Mixing the crude enzyme solution obtained in the steps (1) and (2) with agarose resin and phosphate buffer solution containing 30-50 mM imidazole and having the pH of 7-10, fixing at 20-30 ℃ for 100-200min (preferably 25 ℃ for 150 min), and obtaining the directional immobilized double enzyme after centrifugation and washing with clear water.

Furthermore, in the steps (1) and (2), the vectors are pET28a (+), and the E.coli BL21 (DE 3) is adopted.

Further, the crude enzyme solutions in the steps (1) and (2) are prepared by inoculating recombinant strains to LB plates containing 50 mug/mL kanamycin resistance, culturing for 12 hours at 37 ℃, picking single bacterial colonies, culturing for 12 hours at 37 ℃ and 180rpm, inoculating the culture solution to LB liquid culture medium containing 50 mug/mL kanamycin according to the inoculation amount of 1% of the volume concentration, shake flask culturing, culturing until OD ₆₀₀ reaches 0.6-0.8 at 37 ℃ and 180rpm, adding 0.1mmol/L IPTG, culturing for 12 hours at 28 ℃, centrifuging for 10 minutes at 8000rpm, collecting wet bacterial colonies, preparing a polyphosphate kinase bacterial suspension (preferably 3g/L in bacterial concentration) and a glutathione bifunctional synthetase bacterial suspension (preferably 9g/L in bacterial concentration) by using 50mM Tris-HCl (pH 7) buffer respectively, performing ultrasonic crushing, wherein the crushing power is 300W, the total working time is 10 minutes, and collecting the mixed solution with the short-label of the short-label and the crude enzyme solution of the glutathione with the crude enzyme can be obtained.

The invention also provides an application of the directional immobilized double enzyme in synthesizing glutathione, which comprises the steps of adding L-cysteine, L-glutamic acid, glycine, mgSO ₄, ATP (adenosine triphosphate) and sodium hexametaphosphate into 100mM Tris-HCl buffer solution (pH 8.0) to form a reaction system, regulating the pH of the reaction system to 8.0 by using 2mol/L sodium hydroxide, and stirring and reacting for 10 hours under the condition of introducing nitrogen at 25 ℃ to obtain glutathione.

Further, L-cysteine is added to the reaction system at a final concentration of 40 to 80mM (preferably 60 mM), L-glutamic acid is added at a final concentration of 60 to 100mM (preferably 80 mM), glycine is added at a final concentration of 80 to 120mM (preferably 100 mM), mgSO ₄ is added at a final concentration of 40 to 80mM (preferably 60 mM), ATP is added at a final concentration of 1 to 3mM (preferably 2 mM), sodium hexametaphosphate is added at a final concentration of 20 to 50mM (preferably 35 mM), and the immobilized enzyme is added at a directional concentration of 10 to 30g/L (preferably 20 g/L).

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

According to the invention, the PPK is modified by the SpyTag short peptide, the GSH bifunctional synthetase GshF is modified by the SpyCatcher short peptide, and the directional immobilized double enzyme is prepared by utilizing the technical scheme of affinity with agarose resin and SpyCatcher/SpyTag specific combination, so that the multi-enzyme immobilization efficiency and the enzyme activity recovery rate and stability of the immobilized double enzyme are improved.

The immobilization method is simple, mild in reaction condition and easy to operate.

(IV) description of the drawings

FIG. 1 is a schematic illustration of the preparation of a directionally immobilized double enzyme.

FIG. 2 is a comparison of the recovery of immobilized enzyme activity in the orientation with that in the random immobilization.

FIG. 3 is a comparison of the yield of glutathione catalyzed by the immobilized enzyme directed to the random immobilized enzyme.

(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:

Protein loading = (initial total enzyme solution protein amount-residual total protein amount)/initial total enzyme solution protein amount x 100%.

Definition of enzyme Activity the amount of enzyme required to produce 1. Mu. Mol of glutathione per minute at 35℃under 100mM Tris-HCl (pH 8.0) was one viability unit (1U).

Specific enzyme activity is defined as the number of units of enzyme activity per gram of enzyme protein. Generally expressed in U/g.

Enzyme activity recovery = total enzyme activity of immobilized enzyme/total free enzyme activity for immobilization x 100%.

LB liquid culture medium is composed of 10g/L peptone, 5g/L yeast extract and 10g/L NaCl, and the solvent is water.

The LB solid medium is prepared by adding 20g/L agar into LB liquid medium.

EXAMPLE 1 preparation of modified PPK crude enzyme solution

1. Acquisition of Gene fragments

(1) Target gene PPK

The PPK of polyphosphate kinase from Cytophaga hutchinsonii ATCC 33406 is synthesized artificially, and cloned to the T7 tag site TCCGAATTC of pET28a (+) and transformed into E.coli BL21 (DE 3) before CACCATTAG site, and the E.coli BL21/pET28a (+) -PPK is obtained, wherein the PPK amino acid sequence is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO. 2.

SEQ ID NO.1

MATDFSKLSKYVETLRVKPKQSIDLKKDFDTDYDHKMLTKEEGEELLNLGISKLSEIQEKLYASGTKSVLIVFQAMDAAGKDGTVKHIMTGLNPQGVKVTSFKVPSKIELSHDYLWRHYVALPATGEIGIFNRSHYENVLVTRVHPEYLLSEQTSGVTAIEQVNQKFWDKRFQQINNFEQHISENGTIVLKFFLHVSKKEQKKRFIERIELDTKNWKFSTGDLKERAHWKDYRNAYEDMLANTSTKQAPWFVIPADDKWFTRLLIAEIICTELEKLNLTFPTVSLEQKAELEKAKAELVAEKSSD.

(2) Short peptide SpyTag

SpyTag is derived from Streptococcus pyogenes (Streptococcus pyogenes), and the amino acid sequence of JQ478411.1 is shown as SEQ ID NO. 7.

SEQ ID NO.7

MGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTW ISDGQVKDFYLYPGKYTFVETAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHILE.

2. Construction of recombinant genetically engineered bacteria

(1) Recombinant plasmid pET28a (+) -PPK-linker-SpyTag-His6

PCR amplification was performed using E.coli BL21/pET28a (+) -PPK as a template and PPK-SPYT and PPK-SPYT-R as primers, and the recombinant plasmid pET28a (+) -PPK- [ GGS ] ₃ -SpyTag-His6 was obtained by sequentially connecting the PCR amplification product, the flexible connecting peptide [ GGGGS ] ₃ (the amino acid sequence is shown as SEQ ID NO. 4), the short peptide SpyTag gene sequence and the histidine tag to the plasmid pET28a (+) -PPK (the end of the PPK sequence, after AGCGAT and before CATTAG) using a one-step cloning kit (ClonExpress Ultra One Step Cloning Kit, purchased from Nanjinouzan Biotechnology Co., ltd.). Recombinant plasmid pET28a (+) -PPK-His6 was constructed simultaneously as a control.

PPK-SPYT(SEQ ID NO.8):

GGTGGTAGCGCCCACATTGTGATGGTTGATGCATATAAACCGACCAAACATCACCACCATCACCATTAG;

PPK-SPYT-R(SEQ ID NO.9):

ACAATGTGGGCGCTACCACCACCACCGCTACCACCACCACCATCGCTGCTTTTTTCTGC。

The PCR amplification system was (50. Mu.L) 1. Mu.L of template DNA, 2X Phanta Max Buffer. Mu.L, 1. Mu.L of dNTPs Mix, 1. Mu.L of each of the upstream and downstream primers, 1. Mu.L of PHANTA DNA Polymerase, and a total volume of ddH ₂ O were supplemented to 50. Mu.L.

The PCR amplification procedure (1) 10min of pre-denaturation at 95 ℃, (2) 30s of denaturation at 95 ℃, (3) 30s of annealing at 60 ℃, (4) 5min of extension at 72 ℃, (2) - (4) 35 cycles, and (5) 10min of complete extension at 72 ℃, and 16 ℃ for preservation. After the PCR product was analyzed to be positive by 0.9% agarose gel electrophoresis, 1. Mu.L of the endonuclease Dpn I was added to the PCR reaction solution and digested at 37℃for 2 hours to remove the template plasmid DNA.

(2) Recombinant genetically engineered bacterium

The plasmid was transferred into competent E.coli BL21 (DE 3) strain, and the bacterial liquid was spread on LB solid medium plates containing 50. Mu.g/mL kanamycin, and cultured upside down in a 37℃incubator for 16 hours. Screening recombinant strain E.coli BL21/pET28a (+) -PPK- [ GGGGS ] ₃ -SpyTag-His 6 single colony. The same method transfers pET28a (+) -PPK-His6 into E.coli BL21 (DE 3) strain, and single colony of E.coli BL21/pET28a (+) -PPK-His6 is screened as a control.

3. Preparation of crude enzyme solution

(1) The recombinant strain in step2 was inoculated into LB plate containing 50. Mu.g/mL kanamycin resistance, cultured at 37℃for 12 hours, and single colony was picked up in 10mL LB liquid medium containing 50. Mu.g/mL kanamycin, cultured at 37℃and 180rpm for 12 hours.

(2) Inoculating the culture solution in the step (1) to LB liquid culture medium containing 50 mug/mL kanamycin according to the inoculation amount with the volume concentration of 1%, culturing in a shaking bottle, adding 0.1mmol/L IPTG when the OD ₆₀₀ reaches 0.6-0.8 under the conditions of 37 ℃ and 180rpm, culturing for 12h at 28 ℃, centrifuging for 10min under the conditions of 8000rpm, and collecting wet thalli.

(3) Preparing bacterial suspension with bacterial concentration of 3g/L by using 50mM Tris-HCl (pH 8.0) buffer solution for wet bacterial cells, performing ultrasonic crushing, wherein the crushing power is 300W, the total working time is 10min, and collecting the crushing mixed solution to obtain PPK-linker-SpyTag-His6 crude enzyme solution, namely modified PPK crude enzyme solution, and the specific enzyme activity is 2034.67U/g.

In the same way, a crude enzyme solution of E.coli BL21/pET28a (+) -PPK-His6 is prepared and is marked as PPK crude enzyme solution, and the specific enzyme activity is 2158.29U/g.

4. Detection of crude enzyme liquid enzyme activity

Enzyme activity detection System substrate mixture A buffer solution of 50mM Tris-HCl (pH 8.0) was used to prepare solutions of 20mM MgSO ₄、10mM Poly P₆ (sodium hexametaphosphate) and 5mM ADP (adenosine diphosphate) respectively, and mixed in a volume ratio of 1:1:1, and the pH was adjusted again to 8.0. 400. Mu.L of the above crude enzyme solution (protein concentration 0.0625 g/L) was added to 600. Mu.L of the substrate mixture, reacted at 35℃and 600rpm for 10 minutes, and the reaction was terminated by adding 200. Mu.L of 2M hydrochloric acid, and the ATP yield was detected by High Performance Liquid Chromatography (HPLC).

The enzyme activity detection conditions were that the ATP yield was detected using a high performance liquid chromatograph (Agilent 1260), a C18 column (4.6Xmmol/L, 5 μm), 50mM dipotassium hydrogen phosphate as the mobile phase (potassium dihydrogen phosphate adjusted pH=7.0, as prepared), 10. Mu.L of sample volume, 0.8mL/min flow rate, column temperature 40℃and 254nm wavelength.

EXAMPLE 2 preparation of modified GshF crude enzyme solution

1. Acquisition of the Gene fragment of interest

(1) Gene GshF of interest

The glutathione bifunctional synthetase GshF from Streptomyces AGALACTIAE V ATCC BAA-611 is artificially synthesized, cloned to the T7 tag site CGCGGATCC of pET28a (+) and then converted to E.coli BL21 (DE 3) before GCACTCGAG to obtain E.coli BL21/pET28a (+) -GshF, wherein the GshF amino acid sequence is shown as SEQ ID NO.5, and the nucleotide sequence is shown as SEQ ID NO. 6.

SEQ ID NO.5

MTLNQLLQKLEATSPILQANFGIERESLRVDRQGQLVHTPHPSCLGARSFHPYIQTDFCEFQMELITPVAKSTTEARRFLGAITDVAGRSIATDEVLWPLSMPPRLKAEEIQVAQLENDFERHYRNYLAEKYGTKLQAISGIHYNMELGKDLVEALFQESGQTDMIAFKNALYLKLAQNYLRYRWVITYLFGASPIAEQGFFDQEVPEPVRSFRNSDHGYVNKEEIQVSFVSLEDYVSAIETYIEQGDLNAEKEFYSAVRFRGQKVNRSFLDKGITYLEFRNFDLNPFERIGISQTTMDTVHLLILAFLWLDSPENVDQALAQGHALNEKIALSHPLEPLPSEAKTQDIVTALDQLVQHFGLGDYHQDLVKQVKAAFADPNQTL SAQLLPYIKDKSLAEFALNKALAYHDYDWTAHYALKGYEEMELSTQMLLFDAIQKGIHFEILDEQDQFLKLWHQDHVEYVKNGNMTSKDNYVVPLAMANKTVTKKILADAGFPVPSGDEFTSLEEGLAYYPLIKDKQIVVKPKSTNFGLGISIFQEPASLDNYQKALEIAFAEDTSVLVEEFIPGTEYRFFILDGRCEAVLLRVAANVIGDGKHTIRELVAQKNANPLRGRDHRSPLEIIELGDIEQLMLAQQGYTPDDILPEGKKVNLRRNSNISTGGDSIDITETMDSSYQELAAAMATSMGAWACGVDLIIPDETQIATKENPHCTCIELNFNPSMYMHTYCAEGPGQAITTKILDKLFPEIVAGQT.

(2) Short peptide Spycatcher

SpyCatcher is derived from Streptococcus pyogenes (Streptococcus pyogenes), and the amino acid sequence of JQ478411.1 is shown as SEQ ID NO. 3.

SEQ ID NO.3

AHIVMVDAYKPTPKK。

2. Construction of recombinant genetically engineered bacteria

(1) Recombinant plasmid pET28a (+) -GshF- [ GGGGS ] ₃ -SpyCatcher

PCR amplification was performed using E.coli BL21/pET28a (+) -GshF as template and GshF-SPYC-F, gshF-SPYC-R as primer. The PCR amplification product, the flexible connecting peptide [ GGGGS ] ₃ (the amino acid sequence is shown in SEQ ID NO. 4), the short peptide SpyCatcher gene sequence and the histidine tag are sequentially connected to a plasmid pET28a (+) -GshF (GshF sequence end, CAGACC and CACTAA before) by adopting a one-step cloning kit (ClonExpress Ultra One Step Cloning Kit, purchased from Nanjinouzan Biotechnology Co., ltd.) to construct a recombinant plasmid pET28a (+) -GshF- [ GGGGS ] ₃ -SpyCatcher, and pET28a (+) -GshF is used as a control.

GshF-SPYC-F(SEQ ID NO.10):

GGTGATGCACACATCCTGGAACATCACCATCACCATCACTAAG;

GshF-SPYC-R(SEQ ID NO.11):

GTGTATCAACCATTGCACCCATGGTCTGACCAGcaCAACGATTTC。

The PCR amplification system was (50. Mu.L) and the PCR amplification procedure was the same as in example 1, and will not be described here.

(2) Construction of recombinant genetically engineered bacteria

By the method of example 1, recombinant plasmid pET28a (+) -GshF- [ GGGGS ] ₃ -SpyCatcher was transferred into competent E.coli BL21 (DE 3) strain to obtain E.coli BL21/pET28a (+) -GshF- [ GGGGS ] ₃ -SpyCatcher.

3. Preparation of modified GshF crude enzyme solution

Preparing bacterial suspension with the bacterial concentration of 9g/L by adopting the method of the example 1, carrying out ultrasonic crushing, wherein the crushing power is 300W, and the total working time is 15min, so as to obtain GshF- [ GGGGS ] ₃ -SpyCatcher crude enzyme liquid, namely modified GshF crude enzyme liquid, and the specific enzyme activity is 48.52U/g. In the same way, a crude enzyme solution of recombinant E.coli BL21/pET28a (+) -GshF is prepared and is marked as GshF crude enzyme solution, and the specific activity is 53.97U/g.

4. Detection of enzyme Activity

The final concentration of the substrate mixture of the enzyme activity detection system is 20mM L-cysteine, 20mM L-glutamic acid, 20mM glycine, 20mM MgSO ₄ and 10mM ATP, and the solvent is 100mM Tris-HCl (pH 8.0) buffer solution, and the pH is adjusted to 8.0. mu.L of the above crude enzyme solution (protein concentration 4 g/L) was added to 500. Mu.L of the substrate mixture, reacted at 35℃and 600rpm for 10 minutes, and the reaction was terminated by adding 100. Mu.L of 2M hydrochloric acid, and the yield of GSH was measured by High Performance Liquid Chromatography (HPLC).

The enzyme activity was detected using a high performance liquid chromatograph (Agilent 1260), a C18 column (4.6Xmmol/L, 5 μm), 10mM sodium heptanesulfonate, 50mM potassium dihydrogen phosphate (pH 2.8) -methanol (95:5, V/V)), 10. Mu.L sample loading, a flow rate of 1mL/min, a column temperature of 30℃and GSH yield at a wavelength of 210 nm.

EXAMPLE 3 preparation of PPK and GshF directed immobilized double enzyme (one)

1. Preparation of directional immobilized double enzymes

10ML of each of the modified PPK crude enzyme solution and the modified GshF crude enzyme solution prepared in examples 1 and 2, 10g of Ni-IDA agarose resin, and a phosphate buffer solution (pH 9.0) containing 30mM imidazole and having a concentration of 1.5M were added into 50mL of the reaction system, respectively, to make up 50mL. The pellet was washed with ultrapure water by fixing at 25℃and 180rpm for 120min and centrifuging at 6000rpm for 10min to obtain 9.8g of the directionally immobilized double enzyme.

2. Detection of enzyme recovery

Preparation of substrate mixture in the immobilized double enzyme PPK enzyme Activity detection System As in example 1, 20mg of immobilized double enzyme, 6mL of substrate mixture, 6mL of 50mM Tris-HCl buffer (pH 8.0) were added to a 50mL reaction flask, reacted at 35℃and 600rpm for 10min, and the reaction was terminated by adding 200. Mu.L of 2M hydrochloric acid, and the yield of ATP was detected by High Performance Liquid Chromatography (HPLC).

Preparation of substrate mixture in the immobilized double enzyme GshF enzyme activity detection System As in example 2, 20mg of immobilized double enzyme, 5mL of substrate mixture, 5mL of 100mM Tris-HCl buffer (pH 8.0) were added to a 50mL reaction flask, reacted at 35℃and 600rpm for 10min, and the reaction was terminated by adding 1mL of 2M hydrochloric acid, and the yield of GSH was detected by High Performance Liquid Chromatography (HPLC).

The enzyme activity detection conditions are the same as those in examples 1 and 2, and are not described in detail here.

The enzyme activity recovery rates are shown in FIG. 2, and the enzyme activity recovery rates of GshF and PPK in the directional immobilized double enzyme are 90.58% and 86.62%, respectively.

EXAMPLE 4 PPK and GshF preparation of directed immobilized double enzyme (II)

The enzyme activity recovery rates of GshF and PPK in the immobilized double enzyme prepared in this example were 84.28% and 73.67%, respectively, as determined by the same operation except that 1.5M in the phosphate buffer solution (pH 9.0) of example 3 was changed to 2.0M.

EXAMPLE 5 PPK and GshF preparation of directed immobilized double enzyme (III)

The recovery rates of enzyme activities of GshF and PPK in the directionally immobilized double enzyme prepared in this example were 85.56% and 79.13%, respectively, by changing pH 9.0 in the 1.5M phosphate buffer solution (pH 9.0) of example 3 to pH 8.0.

Comparative example 1 preparation of oriented immobilized double enzymes

The PPK and GshF modified short peptides in example 1 and example 2 were replaced with each other, namely, modified enzymes GshF-linker-SpyTag-His6 and PPK- [ GGGGS ] ₃ -SpyCatcher were constructed, and modified PPK crude enzyme solutions and modified GshF crude enzyme solutions were prepared by the same procedures as in examples 1 and 2, so that 9.8g of a directional immobilized double enzyme was obtained.

The recovery rates of GshF and PPK enzyme activities in the directionally immobilized double enzyme were 28.6% and 36.1% respectively, as measured by the method of example 3.

Comparative example 2 random immobilized double enzyme preparation

The modified PPK crude enzyme solution and the modified GshF crude enzyme solution in example 3 were replaced with PPK crude enzyme solution and GshF crude enzyme solution, respectively, and the other operations were the same, to obtain 9.8g of a random immobilized double enzyme.

According to the measurement, the recovery rates of GshF% and PPK enzyme activities in the random immobilized double enzymes are 35.5% and 31.17%, respectively.

EXAMPLE 6 PPK and GshF preparation of two enzymes immobilized in a directed manner (IV)

The enzyme activity recovery rates of GshF and PPK in the directional immobilized double enzyme prepared in this example were 65.11% and 62.59%, respectively, by changing the immobilization time of 120min at 25℃and 180rpm in example 3 to 60 min.

EXAMPLE 7 PPK and GshF preparation of directed immobilized double enzyme (five)

The modified PPK crude enzyme liquid and the modified GshF crude enzyme liquid prepared in the embodiment 1 and the embodiment 2 are respectively added into 50mL of a reaction system in the embodiment 3, 10mL of each of the modified PPK crude enzyme liquid and the modified GshF crude enzyme liquid are changed into 20mL of modified PPK crude enzyme liquid and 5mL of modified GshF crude enzyme liquid, and the recovery rates of the enzyme activities of GshF and PPK in the directional immobilized double enzyme prepared in the embodiment are 57.12% and 72.85% respectively.

Example 8 thermal stability test

The modified PPK crude enzyme liquid and the modified GshF crude enzyme liquid prepared in examples 1 and 2, the directional immobilized double enzyme prepared in example 3 and the random immobilized double enzyme prepared in comparative example 2 are respectively placed under the condition of 25 ℃ for incubation for 160 hours, the enzyme activity is measured by sampling at regular intervals of 12 hours, the percentage of residual enzyme activity (defined as 100% of the initial enzyme activity) is calculated, and the half life of the enzyme under the condition of 25 ℃ is calculated.

The results are shown in Table 1, with a free GshF half-life of 28.42h and a free PPK half-life of 20.31h at 25℃and a GshF half-life of 78.51h and a PPK half-life of 70.32h in the case of the random immobilized double enzyme, and a GshF half-life of 99.79h and a PPK half-life of 92.15h in the case of the oriented immobilized double enzyme. Compared with free enzyme and random immobilized double enzyme, the immobilized double enzyme prepared by the directional immobilization strategy has obviously improved thermal stability.

TABLE 1 thermal stability

Example 9 directed immobilized double enzyme catalyzed Synthesis of glutathione

50ML of the reaction system was subjected to final concentration by adding 60mM L-cysteine, 80mM L-glutamic acid, 100mM glycine, 60mM MgSO ₄, 2mM ATP, 35mM sodium hexametaphosphate and 1g of the immobilized enzyme obtained in example 3, respectively, to 50mL of 100mM Tris-HCl buffer (pH 8.0), adjusting pH of the reaction system to 8.0 with 2mol/L sodium hydroxide, stirring and reacting at 25℃under nitrogen atmosphere for 10 hours, sampling every 2 hours, and detecting glutathione content by High Performance Liquid Chromatography (HPLC). The control was a randomly immobilized double enzyme of comparative example 2.

GSH detection conditions are the same as those in example 2, and will not be described in detail here.

The result shows that as shown in figure 3, the directional immobilized enzyme catalyzes and synthesizes the glutathione, the reaction lasts for 8 hours, and the yield of the glutathione reaches 15.82g/L.

Comparative example 3 Synthesis of glutathione by random immobilized double enzyme catalysis

The procedure was the same except that 1g of the immobilized enzyme obtained in example 3 was added in example 9, and 1g of the immobilized double enzyme prepared in comparative example 2 was replaced. And (3) randomly immobilizing double enzymes to catalyze and synthesize glutathione, wherein the reaction is carried out for 8 hours, and the yield of the glutathione is 11.21g/L.

Claims (10)

1. The directional immobilized double enzyme for synthesizing glutathione is characterized in that the directional immobilized double enzyme is obtained by respectively fusing and expressing enzyme proteins of glutathione double-function synthetase and polyphosphate kinase through connecting peptide, short peptide SpyCatcher and short peptide SpyTag as active components, taking agarose resin as a carrier, and co-immobilizing in phosphate buffer solution containing imidazole and having pH of 7-10 at 20-30 ℃.

2. The directionally-immobilized dual enzyme of claim 1, wherein the linking peptide comprises [ EAAAK ] ₃、[Gly]₈、[Gly]₆、[GGGGS]₃.

3. The directional immobilized double enzyme as claimed in claim 1, wherein the amino acid sequence of the short peptide SpyCatcher is shown in SEQ ID NO.3, and the amino acid sequence of the short peptide SpyTag is shown in SEQ ID NO. 7.

4. The directional immobilized dual enzyme of claim 1, wherein the glutathione bifunctional synthetase has an amino acid sequence shown in SEQ ID NO.5, and the polyphosphate kinase has an amino acid sequence shown in SEQ ID NO. 1.

5. The directional immobilized dual enzyme of claim 1, wherein the final concentration of imidazole is 30-50 mM, and the phosphate buffer is 1.5M phosphate buffer with pH of 9.0.

6. A method of preparing the directionally immobilized double enzyme of claim 1, comprising the steps of:

(1) Sequentially connecting a polyphosphate kinase target gene PPK, a connecting peptide, a short peptide SpyTag coding gene and a histidine tag, inserting the obtained gene fragment PPK-linker-SpyTag-His6 into an expression vector, then converting into escherichia coli to construct recombinant bacteria, and inducing the expressed wet bacteria to be subjected to ultrasonic crushing to obtain a mixed solution of the polyphosphate kinase with the SpyTag short peptide and the histidine tag, namely a crude enzyme solution;

(2) The glutathione bifunctional synthetase target gene GshF, the connecting peptide and the short peptide SpyCatcher coding gene are connected in sequence, the obtained gene fragment GshF-linker-SpyCatcher is inserted into an expression vector, then the expression vector is transformed into escherichia coli to construct recombinant bacteria, and the mixed solution of the wet bacteria induced to be expressed is subjected to ultrasonic disruption, so that the crude enzyme solution of the glutathione bifunctional synthetase with SpyCatcher short peptide is obtained;

(3) Mixing the crude enzyme solution obtained in the steps (1) and (2) with agarose resin and phosphate buffer solution containing 30-50 mM imidazole and having the pH of 7-10, fixing at 20-30 ℃ for 100-200min, and obtaining the directional immobilized double enzyme after centrifugation and washing with clear water.

7. The method of claim 6, wherein the vectors in steps (1) and (2) are pET28a (+), and E.coli is E.coli BL21 (DE 3).

8. Use of the directionally immobilized double enzyme of claim 1 in the synthesis of glutathione.

9. The method of claim 8, wherein L-cysteine, L-glutamic acid, glycine, mgSO ₄, ATP and sodium hexametaphosphate are added into 100mM Tris-HCl buffer solution with pH of 8.0 to form a reaction system, 2mol/L sodium hydroxide is used for adjusting the pH of the reaction system to 8.0, and the reaction is carried out for 10 hours under the condition of 25 ℃ and nitrogen introduction under stirring to obtain glutathione.

10. The use according to claim 9, wherein the final concentration of L-cysteine added, L-glutamic acid added, 100mM glycine added, mgSO ₄ added, ATP added, sodium hexametaphosphate added, and directional immobilized enzyme added are respectively 40-80mM, 60-100mM, 80-120mM, 40-80mM, 1-3mM, 20-50mM, and 10-30g/L.