CN114480472A

CN114480472A - Process for producing fusion enzyme and fusion enzyme

Info

Publication number: CN114480472A
Application number: CN202210081967.1A
Authority: CN
Inventors: 高静; 李彦洁; 姜艳军; 周丽亚
Original assignee: Hebei University of Technology
Current assignee: Hebei University of Technology
Priority date: 2022-01-24
Filing date: 2022-01-24
Publication date: 2022-05-13

Abstract

The invention provides a preparation method of a fusion enzyme and the fusion enzyme, the preparation method of the fusion enzyme firstly constructs a fusion enzyme plasmid, then extracts the fusion enzyme plasmid, transfers the fusion enzyme plasmid into pichia pastoris, and carries out fermentation expression; and preparing a crude enzyme solution by using pichia pastoris, and separating and purifying the fusion enzyme. 6 XHis-SUMO labels are fused at the C ends of mutants of non-specific peroxidase (UPO), namely JaWaWa, SoLo and WamPa, the fusion enzyme is induced to express in pichia pastoris and is separated and purified at the later stage, the peroxidation activity of the enzyme can be removed by JaWa, WamPa has good stability in an organic solvent, and the SUMO labels can effectively improve the expression amount of the enzyme, so that the high-efficiency catalytic reaction is realized. In addition, JaWa, SoLo, WaThe mPa is fused with 6 XHis-SUMO label and then histidine and metal ion Ni can be utilized²⁺The chelation is used for separation and purification, and the enzyme catalytic activity after separation and purification is greatly improved.

Description

Process for producing fusion enzyme and fusion enzyme

Technical Field

The invention relates to the technical field of fusion enzyme construction and fermentation, in particular to a preparation method of fusion enzyme. Meanwhile, the invention also relates to a fusion enzyme.

Background

The selective mono-oxygenation reaction of inert carbon-hydrogen bonds is one of the main challenges of organic synthesis, and at present, the selective mono-oxygenation reaction of inert carbon-hydrogen bonds is mainly catalyzed by a chemical method and an enzymatic method, the reaction conditions of the chemical catalysis method are harsh, and the enzymatic catalysis method is relatively mild. The heme-dependent oxygenase is one of the commonly used selective oxygen functionalization catalysts, and among them, the non-specific peroxidase (UPO) has become a high-efficiency catalyst for catalyzing the monooxygenase reaction due to the simple electron transfer process and high economic benefit. UPO is widely available, among them, non-specific peroxidase (aaeoupo) from Agrocybe aegerita (Agrocybe aegerita) is a glycosylated (20% -40%) extracellular enzyme, has an isoelectric point between 5.2 and 6.2, and is widely concerned because it catalyzes a single-oxygenation reaction of various organic compounds with H2O2 as a co-substrate, and has high catalytic activity, a broad substrate spectrum, and heterologous expression.

In the application of AaeUPO, the expression level is low, and the substrate is difficult to dissolve in water; therefore, in order to improve the yield of the product and the catalytic efficiency of the enzyme, it is necessary to improve the expression level of AaeUPO and the tolerance and stability in an organic solvent. The existing technical means is to improve the expression quantity of the fusion enzyme by modifying enzyme molecules so as to obtain the fusion enzymes with different functions. However, in the prior art practice, there is a lack of a practical and effective solution for the preparation and purification of fusion enzymes; in addition, the catalytic activity of the enzyme obtained by the existing preparation method is not high, and the reaction catalytic efficiency in the preparation process is also low.

Disclosure of Invention

In view of the above, the present invention is directed to a method for preparing a fusion enzyme, so as to facilitate separation and purification of fusion enzymes with different functions.

In order to achieve the purpose, the technical scheme of the invention is realized as follows:

a method for preparing a fusion enzyme, the method comprising:

constructing fusion enzyme plasmids; extracting fusion enzyme plasmid, transferring the fusion enzyme plasmid into pichia pastoris, and performing fermentation expression; preparing crude enzyme liquid by using pichia pastoris, and separating and purifying the fusion enzyme.

Further, the construction of the fusion enzyme plasmid comprises the following steps:

a1. constructing fusion expression genes of 6 XHis-SUMO-JaWa, 6 XHis-SUMO-SoLo and 6 XHis-SUMO-WamPa;

a2. the fusion enzyme gene sequence is connected to E.coli DH5 alpha carrier by using restriction endonucleases BamH I and Not I to construct recombinant plasmid.

Further, the fusion enzyme plasmid is extracted from escherichia coli, and the method comprises the following steps:

b1. extracting the fusion enzyme plasmid from the escherichia coli;

b2. cutting the circular plasmid by using Sal I endonuclease;

b3. judging the position of a strip of the linearized plasmid by using DNA electrophoresis;

b4. the linearized plasmid was purified and concentrated.

Further, the preparation of the crude enzyme solution by using pichia pastoris comprises the following steps:

f1. activating the strain on a flat plate, culturing overnight, inoculating into fresh BMGY culture medium, and culturing until OD is reached₆₀₀When the content reaches 1.8-2.5, adding BMMY for induction culture;

f2. and (3) centrifugally washing the BMGY culture medium, collecting supernatant, namely crude enzyme solution, and storing at the temperature of 3-5 ℃.

Furthermore, in the step f1, the time for inducing expression of the positive bacteria is 144-146 h.

Furthermore, in the process of separating and purifying the fusion enzyme, the fusion enzyme is separated and purified by utilizing the specific binding of a histidine tag and a nickel column.

Further, the step of transferring the fusion enzyme plasmid into pichia pastoris for fermentation expression comprises the following steps:

c. preparing pichia pastoris competent cells;

d. electrotransfering the fusion enzyme plasmid into a competent pichia pastoris cell;

e. extracting a plasmid in the pichia pastoris, and verifying the transduction condition of the plasmid;

further, in step c, the method comprises the following steps:

c1. inoculating glycerol strain with YPD culture medium, and activating Pichia pastoris cells;

c2. re-inoculating the activated Pichia cells into new YPD medium, waiting for OD₆₀₀When the value reaches 1.2-1.5, centrifuging;

c3. blowing and uniformly mixing precipitates obtained by standing after centrifugation with precooled sterile water, and centrifuging again;

c4. mixing pre-cooled sorbitol with the precipitate obtained in step c3 to form pichia pastoris competent cells;

and the step c3 is repeated for 2-4 times.

Furthermore, in step e, the plasmid extracted from pichia pastoris is replicated by using a PCR technique, and DNA electrophoresis is facilitated to verify the transduction condition of the plasmid.

Compared with the prior art, the preparation method of the fusion enzyme has the following technical advantages:

the expression quantity of the fusion enzyme can be improved by constructing the plasmid of the fusion enzyme, and the plasmid is fermented, expressed and copied by using pichia pastoris, so that the quantity of the fusion enzyme can be effectively improved; separating and purifying the prepared crude enzyme liquid by centrifugation and other modes; can be suitable for the purification and preparation of various fusion enzymes with different functions.

In addition, the expression level of the fusion enzyme can be improved by more than 1.28 times by utilizing the SUMO label, the peroxidation activity of the enzyme can be eliminated by the JaWa mutant, the catalytic efficiency of SoLo on 5 '-hydroxypropionic acid (5' -OHP) can be improved by 2 orders of magnitude compared with UPO, and the ee is>99 percent. WamPa itself has good stability in organic solvents and can catalyze reactions in organic reagents efficiently. JaWa, SoLo and WamPa are fused with 6 XHis-SUMO tag, and histidine and metal ion Ni can be utilized²⁺The chelation is used for separation and purification, so that the enzyme catalytic activity after separation and purification is greatly improved.

Another object of the present invention is to provide a fusion enzyme comprising a 6 XHis-SUMO tag bound to a mutant JaWa, SoLo, WamPa of a nonspecific peroxidase.

Compared with the prior art, the fusion enzyme is respectively composed of UPO mutants JaWa, SoLo and WamPa and a C-terminal 6 XHis-SUMO label, and is introduced into pichia pastoris for fermentation expression, so that the enzyme catalytic activity after separation and purification can be greatly improved.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an embodiment of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is a schematic diagram illustrating the preparation of a fusion enzyme according to an embodiment of the present invention;

FIG. 2 is a diagram showing a plasmid construction of the fusion enzyme according to example 1 of the present invention;

FIG. 3 is a DNA electrophoresis of the fusion enzyme plasmid of example 1 of the present invention;

FIG. 4 is a medium diagram of a plate for screening positive bacteria by MD plate described in example 2 of the present invention;

FIG. 5 is a DNA electrophoretogram of the plasmid extracted from Pichia pastoris, according to example 2 of the present invention;

FIG. 6 is a graph showing the optimization trend of the induced expression time according to example 3 of the present invention;

FIG. 7 is a graph comparing the stability of UPO and 6 XHis-WamPa in different organic solvents according to example 3 of the present invention;

FIG. 8 is a graph comparing UPO and 6 XHis-WamPa catalyzed conversion of naphthalene to 1-naphthol according to example 3 of the present invention;

FIG. 9 is a graph comparing the stability of UPO and 6 XHis-WamPa in different organic solvents according to example 3 of the present invention;

Detailed Description

It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.

In the time units used in this example, h represents hour, d represents day, and min represents minute.

In addition, unless otherwise specified, all terms and processes related to the present embodiment should be understood according to the conventional knowledge and conventional methods in the art.

The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.

This example relates to a method for preparing a fusion enzyme, an exemplary schematic diagram of which is shown in FIG. 1; the preparation method mainly comprises the following three steps:

firstly, constructing fusion enzyme plasmids;

secondly, extracting fusion enzyme plasmids, transferring the fusion enzyme plasmids into pichia pastoris, and performing fermentation expression;

and thirdly, preparing a crude enzyme solution by using pichia pastoris, and separating and purifying the fusion enzyme.

The fusion enzyme mainly comprises UPO mutants JaWa, SoLo and WamPa and a C-terminal 6 XHis-SUMO label, and the separation and purification process of the fusion enzyme is simple and efficient by utilizing the specific affinity action of histidine and metal ions.

Based on the general set-up principles of the above-described methods for preparing and purifying a fusion enzyme in this example, the preparation can be carried out by referring to the following specific steps.

Firstly, constructing fusion enzyme plasmids; the method mainly comprises the following steps:

Secondly, extracting fusion enzyme plasmids, transferring the fusion enzyme plasmids into pichia pastoris, and performing fermentation expression; the method mainly comprises the following steps:

the fusion enzyme plasmid is extracted by adopting escherichia coli, namely the fusion enzyme plasmid is extracted from the escherichia coli, and the method comprises the following specific steps:

b1. extracting the fusion enzyme plasmid from the escherichia coli;

b2. cutting the circular plasmid by using Sal I endonuclease;

b4. the linearized plasmid was purified and concentrated.

(II) transferring the fusion enzyme plasmid into pichia pastoris for fermentation expression, mainly comprising the following steps

c. Preparing pichia pastoris competent cells;

e. and extracting the plasmid in the pichia pastoris, and verifying the transduction condition of the plasmid.

Among them, in step c, the following steps are preferably employed:

c1. inoculating 1% of inoculum size from glycerol strain with YPD culture medium, and activating Pichia pastoris cells;

c2. 1% inoculum size of activated Pichia cells was re-inoculated into fresh YPD, and OD was waited₆₀₀When the value reaches 1.5, centrifuging the solution at 4 ℃ for 5 min;

c3. after centrifugation, the supernatant was decanted, leaving a precipitate. The precipitate was flushed well with pre-cooled sterile water. Centrifuging again, and repeating the operation for 2-3 times;

c4. and finally, uniformly mixing the precooled 1M sorbitol and the prepared pichia pastoris competent cells for use at present.

It is noted that the inoculation is preferably carried out at an inoculation amount of 1% in the process of inoculating into YPD medium, and the ratio of the inoculation amount is also appropriately adjusted according to the case. OD detection when activated Pichia cells were re-inoculated into New YPD₆₀₀The value of (b) can be set between 1.2 and 1.5, for example, 1.2, 1.3 or 1.5 is selected as the reference value. The temperature and time of centrifugation are preferably 4 ℃ and 5 min; of course, the adjustment may be made as appropriate.

In step d, the fusion plasmid needs to be electrically transferred into the competent pichia pastoris cell, and the following steps can be specifically adopted:

d1. the purified fusion plasmid, competent yeast cells, sorbitol were prepared in advance. In each tube of 80 u L yeast cell competence, adding 10L purified recombinant plasmid, blowing and mixing, suction into the treated electric rotating cup, in ice incubation for 5min, then electric rotation. After electrotransfer, the mixture is taken out, added with 1mL of sorbitol and put into a warm bath at 30 ℃ for 2 h.

d2. And coating part of the bacterial liquid after the electric conversion on a flat plate containing an MD culture medium for screening positive bacteria, carrying out positive culture, carrying out inverted culture in an incubator at 30 ℃ for 3-4 days after the bacterial liquid is completely absorbed by the culture medium, screening positive engineering strains, and storing at-80 ℃.

Similarly, in each of the above and following steps, the listed addition amount, temperature, time and the like are preferred specific parameter values, and these parameter values can be flexibly adjusted within a proper range, and should not be taken as a limitation to the protection scope of the preparation method of the present invention.

In addition, the processing of the electric rotor in the step d1 specifically includes the following steps:

d11. cleaning the electric rotary cup with alcohol for more than 3 times, and cleaning with ultrapure water for more than 3 times after drying;

d12. irradiating the electric rotary cup with ultraviolet lamp for 20 min.

Step e, designing and extracting the plasmid in the pichia pastoris, and verifying the plasmid transduction condition, wherein the following steps can be specifically adopted:

e1. extracting plasmids in pichia pastoris;

e2. the plasmid is massively replicated by adopting a PCR technology;

e3. the success of the electrotransformation of the plasmid was verified by DNA electrophoresis.

Herein, the step of extracting the plasmid in pichia pastoris of e1 includes:

e11. the yeast culture was centrifuged at 12000rpm for 2min and the supernatant discarded.

e12. And (4) carrying out high-speed vortex oscillation to break up the resuspended yeast cell mass. Add 300. mu.L of yeast lysate, vortex, shake and mix well.

e13. The lysate is placed in a water bath at 70 ℃ for 15-30min, and can be vortexed and uniformly mixed for several times to help the lysate. Incubate on ice for 5min to return to room temperature.

e14. After adding 100. mu.L of protein precipitation solution to the lysate returned to room temperature, the mixture was continuously shaken and mixed for 20 seconds at high speed on a vortex shaker, and then ice-cooled for 5 min.

Centrifuging at e15.13000rpm for 5-10min, sucking the supernatant into a new centrifugal tube, adding equal volume of room temperature isopropanol, reversing 30 times, and mixing evenly or until flocculent DNA precipitates. Centrifuge at 12000rpm for 1min, leave a precipitate, and discard the supernatant.

e16. Add 1mL 70% ethanol, reverse several times to rinse the DNA precipitation, 12000rpm centrifugation for 1min, pour the supernatant, drain the residual ethanol, air dry for a few minutes.

e17. Adding 40 μ L of DNA dissolving solution to rehydrate and dissolve DNA precipitate, flicking the tube wall, mixing, and incubating at 65 deg.C for 30-60min, wherein the flicking tube wall helps rehydrate DNA.

e18. Adding 10-15 μ L of RNase A (10mg/mL) or 1-2 μ L of RNase A (100mg/mL), mixing by inversion, and incubating at 37 deg.C for 30-60min to remove residual RNA.

The DNA may be stored at 2-8 ℃ and-20 ℃ if it is to be stored for a long period of time.

Further, the step e2 of verifying the success of the plasmid electrotransformation by the PCR technology comprises:

e21. designing an upper primer and a lower primer by using SnapGene software;

the pcr system is: 11.5 mu L of ultrapure water, 4 mu L of Buffer, 2 mu L of dNTP, 0.5 mu L of each of upper primer and lower primer, 0.5 mu L of Taq enzyme and 1 mu L of DNA plasmid template;

pcr reaction conditions: pre-denaturation at 94 ℃ for 5min, one cycle; denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 2min, and 30 cycles; extending at 72 ℃ for 10 min; keeping the temperature at 4 ℃.

Thirdly, preparing crude enzyme liquid of 6 XHis-JaWa, 6 XHis-SoLo and 6 XHis-WamPa fusion enzyme by using pichia pastoris, and separating and purifying the fusion enzyme; the method mainly comprises the following steps:

preparing a crude enzyme solution:

Wherein the activated strain is preferably inoculated into BMGY medium at an inoculum size of 1%,OD₆₀₀the reference value of (A) can be selected from 1.8, 2.0, 2.4, etc., preferably 2.0; the storage temperature is preferably 4 ℃.

Step f1 relates to the induced expression of positive bacteria, and specifically comprises the following steps:

f11. YPD plate activation was performed on the engineered strain stored at-80 ℃ and cultured overnight in a biochemical incubator at 30 ℃, and then a single colony was picked up in YPD liquid medium containing ampicillin and cultured overnight in a shaker.

f12. The activated positive bacteria liquid is divided into a plurality of containers filled with fresh BMGY liquid medium in sequence according to the inoculation amount of 1 percent for amplification culture, and the bacteria liquid OD to be amplified cultured₆₀₀When the cell number reaches 2, collecting the cells, suspending the cells into an inducible expression medium BMMY, adding 1% of inducer methanol, adding the inducer methanol once every 24h, and carrying out induced culture at 30 ℃ for 5-6 d.

(II) separation and purification of the fusion enzyme:

g1. sample loading buffering is equipped: 20mM imidazole, 20mM sodium dihydrogen phosphate, 0.5M sodium chloride and elution buffer: 500mM imidazole, 20mM sodium dihydrogen phosphate and 0.5M sodium chloride, and performing suction filtration and ultrasonic degassing;

g2. filtering the crude enzyme solution with 0.22 μm water system filter membrane;

g3. injecting the filtered crude enzyme solution into an AKTA instrument, loading, eluting with 10%, 60% and 100% elution buffers respectively after loading, and collecting corresponding eluates; and respectively carrying out enzyme activity determination on each eluent, and collecting the eluent with the highest enzyme activity in each eluent. In this period, the optimum eluent concentration for separation and purification of the fusion enzyme was 60%.

By referring to the preparation process and arrangement, the purified fusion enzyme can be obtained, the 6 XHis tag in the fusion enzyme can be specifically combined with metal ions for separation and purification, the SUMO tag can improve the expression level of the fusion enzyme, and JaWa removes the peroxidation activity of the enzyme, thereby realizing the regioselective conversion of naphthalene into 1-naphthol; the catalytic efficiency of SoLo on 5 '-hydroxypropionic acid (5' -OHP) is improved by 2 orders of magnitude compared with UPO; WamPa has good stability in organic solvents.

The fusion enzyme is formed by combining a 6 XHis-SUMO label with a mutant JaWa, SoLo and WamPa of non-specific peroxidase; the fusion enzyme is composed of UPO mutants JaWa, SoLo and WamPa and a C-terminal 6 XHis-SUMO label, and can be introduced into Pichia pastoris for fermentation expression. The SUMO label improves the expression quantity of the fusion enzyme; JaWa abolishes the peroxidative activity of the enzyme; the catalytic efficiency of SoLo on 5 '-hydroxypropionic acid (5' -OHP) is improved by 2 orders of magnitude compared with UPO; WamPa has higher stability and activity in organic solvent, and the fusion enzyme has good separation and purification effects and strong specificity.

In addition, the expression level of the fusion enzyme can be improved by more than 1.28 times by utilizing the SUMO label, the JaWa mutant can remove the peroxidation activity of the enzyme, the regioselectivity conversion of naphthalene into 1-naphthol is realized, the catalytic efficiency of SoLo on 5 '-hydroxypropionic acid (5' -OHP) is improved by 2 orders of magnitude compared with UPO, and ee is higher than that of the USP>99 percent. Without ascorbic acid, SoLo is increased by 15 times over the TTN of wild type AaeUPO, whereas WamPa itself has good stability in organic solvents and is able to catalyze reactions efficiently in organic reagents. JaWa, SoLo and WamPa are fused with 6 XHis-SUMO tag, and histidine and metal ion Ni can be utilized²⁺The chelation is used for separation and purification, and the enzyme catalytic activity after separation and purification is greatly improved.

Molecular modification is still an effective way to improve a specific function of an enzyme. Based on the method, the specific functions of the enzyme can be optimized layer by layer through rational design or irrational design and a high-throughput screening technology, and finally the enzyme protein in a more ideal state is obtained.

Based on the above overall description of the preparation method, the following examples will specifically describe key steps in the preparation of the immobilized fusion enzyme of the present invention.

Example 1

This example relates to the preparation of 6 XHis-SUMO-JaWa, 6 XHis-SUMO-SoLo, 6 XHis-SUMO-WamPa fusion enzyme plasmids. The preparation principle of plasmid extraction from Escherichia coli, and subsequent linearization and purification is shown in FIG. 1.

(1) Mutants of non-specific peroxidase (AaeUPO) from Agrocybe aegerita, JaWaWa, SoLo, WamPa, were constructed and fused to the C-terminus with the 6 XHis-SUMO gene. Constructing a fusion enzyme gene sequence, connecting the fusion enzyme gene sequence to a plasmid pPIC9K by utilizing restriction enzymes BamHI and Not I, and constructing recombinant plasmids pPIC9k-6 XHis-SUMO-JaWa, pPIC9k-6 XHis-SUMO-SoLo and pPIC9k-6 XHis-SUMO-WamPa.

The schematic diagram of the constructed plasmid is shown in FIG. 2. The protein sequences of the fusion enzymes are listed in Table 1 below

TABLE 1 protein sequences of the fusion enzymes

(2) And transferring the constructed plasmid into a competent E.coli DH5 alpha strain, taking out partial bacterial liquid, coating the bacterial liquid on an LB culture medium plate containing ampicillin, culturing for half an hour in the front side, and performing inverted culture in an incubator at 37 ℃ for 12-16 hours after the bacterial liquid is completely absorbed by the culture medium. Positive strains were selected and stored at-80 ℃.

(3) E.coli DH5 alpha/pPIC 9K-6 XHis-SUMO-JaWa, E.coli DH5 alpha/pPIC 9K-6 XHis-SUMO-SoLo, E.coli DH5 alpha/pPIC 9K-6 XHis-SUMO-WamPa were taken out from a-80 ℃ refrigerator, activated with YPD medium containing ampicillin (10g/L peptone, 5g/L yeast extract, 10g/L NaCl and 2% agar powder), and cultured overnight in a shaker (37 ℃, 180 r/min).

(4) 1-4mL of overnight medium was centrifuged, the cells were collected, and the large intestine plasmid was extracted and linearized as indicated in the kit. The linearized plasmid is subjected to DNA electrophoresis, and the position of a band is used for judging whether the size of the DNA is correct or not. The DNA electrophoresis of the fusion plasmid is shown in FIG. 3.

(5) Purifying the extracted linearized plasmid to increase its concentration. The collected linearized plasmids were stored at-80 ℃ until use.

Example 2

This example relates to electrotransformation of the fusion plasmid into competent Pichia cells and obtaining positive bacteria. The colony pattern of positive bacteria on MD plate is shown in FIG. 4. The plasmid extraction map and the PCR-verified electrophoresis map of the positive bacteria are shown as a and b in FIG. 5, respectively.

(1) First, Pichia pastoris was activated with YPD medium and inoculated from 1% inoculum size into the medium from glycerol.

(2) Inoculating to two new YPDs with 1% inoculum size, culturing Pichia pastoris until OD is reached₆₀₀When the temperature reaches 1.2, the bacterial liquid is centrifuged. The centrifugation conditions were 5000rpm, 5min, 4 ℃. After centrifugation, the supernatant was decanted, leaving a precipitate. And (3) blowing and beating the precipitate uniformly by using 10-15mL of precooled sterile water, continuing to centrifuge, and repeating the operation for 2-3 times. Finally, it was resuspended with precooled sorbitol. The prepared pichia pastoris is competent to be used in time.

(3) The purified fusion plasmid, competent yeast cells, sorbitol were prepared in advance. In each tube of 80 u L yeast cell competence, adding 10L purified recombinant plasmid, blowing and mixing, suction into the treated electric rotating cup, in ice incubation for 5min, then electric rotation. After electrotransfer, the mixture is taken out, 1mL of sorbitol is added, and the mixture is put into a warm bath at 30 ℃ for 2 hours.

(4) And coating part of the bacterial liquid after the electric conversion on a flat plate containing an MD culture medium for screening positive bacteria, carrying out positive culture, carrying out inverted culture in an incubator at 30 ℃ for 3-4 days after the bacterial liquid is completely absorbed by the culture medium, screening positive engineering strains, and storing at-80 ℃. Since MD medium is unable to provide histidine to the microorganism, pichia pastoris that has been successfully electroporated can provide histidine itself for growth on MD plates. The morphology of Pichia pastoris colonies on MD plates is shown in FIG. 3, with milky white colonies, smooth edges, and slightly raised centers.

(5) The plasmid in Pichia pastoris was extracted using yeast culture, which was first centrifuged at 12000rpm for 2min and the supernatant discarded. And (4) carrying out high-speed vortex oscillation to break up the resuspended yeast cell mass. Add 300. mu.L of yeast lysate, vortex, shake and mix well. The lysate is placed in a water bath at 70 ℃ for 15-30min, and can be vortexed and uniformly mixed for several times to help the lysate. Incubate on ice for 5min to return to room temperature. After adding 100. mu.L of protein precipitation solution to the lysate returned to room temperature, the mixture was continuously shaken and mixed for 20 seconds at high speed on a vortex shaker, and then ice-cooled for 5 min. 13000rpm for 5-10min, the supernatant was aspirated into a new centrifuge tube, and an equal volume of room temperature isopropanol was added, and the mixture was inverted 30 times to mix or until a flocculent DNA precipitate appeared. Centrifuge at 12000rpm for 1min, leave a precipitate, and discard the supernatant. Add 1mL 70% ethanol, reverse several times to rinse the DNA precipitation, 12000rpm centrifugation for 1min, pour the supernatant, drain the residual ethanol, air dry for a few minutes. Adding 40 μ L of DNA dissolving solution to rehydrate and dissolve DNA precipitate, flicking the tube wall, mixing, and incubating at 65 deg.C for 30-60min, wherein the flicking tube wall helps rehydrate DNA. Adding 10-15 μ L of RNase A (10mg/mL) or 1-2 μ L of RNase A (100mg/mL), mixing by inversion, and incubating at 37 deg.C for 30-60min to remove residual RNA. The DNA may be stored at 2-8 ℃ and below-20 ℃ if it is to be stored for a long period of time.

(6) Then, the success of plasmid electrotransformation was verified by PCR technology. First, the upper and lower primers were designed using SnapGene software. The PCR system is as follows: 11.5 μ L of ultrapure water, 4 μ L of Buffer, 2 μ L of dNTP, 0.5 μ L of each of upper and lower primers, 0.5 μ L of Taq enzyme, and 1 μ L of DNA plasmid template. And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 5min, one cycle; denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 2min, and 30 cycles; extending at 72 ℃ for 10 min; the temperature is kept at 4 ℃. And (4) carrying out DNA electrophoresis on the PCR product, and judging whether the PCR product is a positive bacterium or not according to the position of the strip.

Example 3

This example relates to the preparation of fusion enzymes and their determination of stability in organic solvents.

(1) The activated positive bacteria liquid is divided into a plurality of containers filled with fresh BMGY liquid medium in sequence according to the inoculation amount of 1 percent for amplification culture, and the bacteria liquid OD to be amplified cultured₆₀₀When the bacterial cell number reaches 2, collecting the bacterial cell, re-suspending the bacterial cell into an inducible expression culture medium BMMY, adding 1% of inducer methanol, and adding the inducer methanol once every 24 hours for 30 hoursThe culture was induced and the induction time was optimized (48-172h), and as can be seen from FIG. 6, the specific enzyme activity was highest at 144h and then did not increase any more.

(2) The reaction was carried out at room temperature, and each reaction mixture contained 6U of purified enzyme, 1mM ethylbenzene, 20% acetonitrile and 4mM H₂O₂In 100mM potassium phosphate buffer at pH 7.0 (1mL final volume). After 24h, the reaction was stopped with 20 μ L of 37% hydrochloric acid and the mixture was analyzed by GC. The results of the experiment are shown in fig. 7, where JaWa produced only 4% of acetophenone, whereas UPO produced as much as 36% of acetophenone, thus demonstrating that JaWa effectively removed the peroxidative activity of the enzyme.

(3) The reaction was carried out at room temperature, and each reaction mixture contained 6U of purified enzyme, 1mM naphthalene, 20% acetonitrile and 1mM H₂O₂In 100mM potassium phosphate buffer at pH 7.0 (1mL final volume). After 3h, the reaction was stopped with 20 μ L of 37% hydrochloric acid and the mixture was analyzed by GC. As shown in FIG. 8, the 1-phenol produced by SoLo is 87% and the 1-phenol produced by UPO is only 34%, thus demonstrating that SoLo effectively improves the efficiency of enzyme catalyzing naphthalene.

(4) The stability of UPO and WamPa in different organic solvents (acetonitrile, acetone, DMSO, DMF, methanol) was determined. The total activity of the enzyme was 1mL, which contained 0.3mM ABTS, 2mM hydrogen peroxide, 30% organic solvent, pH 4.5 citrate-sodium citrate buffer, and 100. mu.L of enzyme was added and incubated at room temperature for 2 h. The experimental results are shown in fig. 9, and it is evident that WamPa is superior to UPO in stability in each organic solvent.

Example 4

This example relates to the isolation and purification of the fusion enzyme.

(1) Sample loading buffering is equipped: 20mM imidazole, 20mM sodium dihydrogen phosphate, 0.5M sodium chloride and elution buffer: 500mM imidazole, 20mM sodium dihydrogen phosphate, 0.5M sodium chloride and suction filtration and ultrasonic degassing.

(2) The crude enzyme solution was filtered through a 0.22 μm aqueous membrane.

(3) The AKTA instruments were first equilibrated with loading buffer by its individual pumps and tubing to fill the instrument with loading buffer, followed by attachment of the nickel column and continued equilibration at 1mL/min for 10-15 min.

(4) And (3) injecting the filtered crude enzyme liquid into an AKTA instrument after the balance is good, loading, eluting by 10%, 60% and 100% of elution buffer after the loading is finished, and collecting the eluent. And measuring the enzyme activity of the eluent, and collecting the eluent with high enzyme activity.

In actual operation, the enzyme solution is mainly collected when 60% of eluent is eluted, and the collected enzyme activity is very high at the moment, which indicates that the purity is very pure. And the liquid eluted by the eluent at other concentrations has almost no enzyme activity. The enzyme activity, protein content and specific enzyme activity after separation and purification are compared with those of the crude enzyme solution as shown in the following table 2.

TABLE 2 comparison of the effect of SUMO labeling and separation and purification on enzyme activity, specific enzyme activity and protein content

In conclusion, the expression quantity of the fusion enzyme can be improved by constructing the plasmid of the fusion enzyme, and the plasmid is fermented, expressed and copied by using pichia pastoris, so that the quantity of the fusion enzyme can be effectively increased; the crude enzyme solution prepared is separated and purified by centrifugation and the like, and is suitable for the purification and preparation of various fusion enzymes with different functions. Moreover, the enzyme is introduced into pichia pastoris for fermentation expression, so that the enzyme catalytic activity after separation and purification can be greatly improved.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A method for producing a fusion enzyme, comprising:

constructing fusion enzyme plasmids;

extracting fusion enzyme plasmid, transferring the fusion enzyme plasmid into pichia pastoris, and performing fermentation expression;

preparing crude enzyme liquid by using pichia pastoris, and separating and purifying the fusion enzyme.

2. The method for preparing the fusion enzyme according to claim 1, wherein the constructing the fusion enzyme plasmid comprises the steps of:

3. The method for preparing the fusion enzyme according to claim 1, wherein the fusion enzyme plasmid is extracted from Escherichia coli, and comprises the steps of:

b1. extracting the fusion enzyme plasmid from the escherichia coli;

b2. cutting the circular plasmid by using Sal I endonuclease;

b4. the linearized plasmid was purified and concentrated.

4. The method for preparing the fusion enzyme according to claim 1, wherein the preparation of the crude enzyme solution using pichia pastoris comprises the following steps:

5. The method for producing the fusion enzyme according to claim 4, wherein:

in step f1, the time for inducing expression of the positive bacteria is 144-146 h.

6. The method for producing the fusion enzyme according to claim 1, wherein:

in the process of separating and purifying the fusion enzyme, the specific binding of the histidine tag and the nickel column is utilized for separation and purification.

7. The method for preparing the fusion enzyme according to any one of claims 1 to 6, wherein the transforming the fusion enzyme plasmid into Pichia pastoris for fermentative expression comprises the following steps:

c. preparing pichia pastoris competent cells;

8. The method for preparing the fusion enzyme according to claim 7, comprising the steps of, in the step c:

and the step c3 is repeated for 2-4 times.

9. The method for producing the fusion enzyme according to claim 7, wherein:

in step e, the plasmid extracted from the pichia pastoris is replicated by adopting a PCR technology, and the DNA electrophoresis is facilitated to verify the transduction condition of the plasmid.

10. A fusion enzyme, characterized by: the fusion enzyme is formed by combining 6 XHis-SUMO label with mutant JaWa, SoLo and WamPa of non-specific peroxidase.