CN111662899B

CN111662899B - Linker peptide mediated enzyme immobilized BaPAD catalyst and preparation method and application thereof

Info

Publication number: CN111662899B
Application number: CN202010515552.1A
Authority: CN
Inventors: 丁少军; 李璐璐
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2020-06-08
Filing date: 2020-06-08
Publication date: 2022-12-27
Anticipated expiration: 2040-06-08
Also published as: CN111662899A

Abstract

The invention discloses a connecting peptide mediated enzyme immobilized BaPAD catalyst and a preparation method and application thereof, belonging to the field of preparation and application of catalysts. The method comprises the steps of firstly inserting connecting peptide which is specifically adsorbed on zeolite into a plasmid pET28 a-bad carrier containing a gene coding phenolic acid decarboxylase BaPAD through Nco I/Xho I restriction enzyme cutting sites, and then immobilizing the connecting peptide on a pretreated Na-Y zeolite carrier through a fusion enzyme to obtain an enzyme immobilized BaPAD catalyst, wherein the phenolic acid decarboxylase BaPAD is from Bacillus atrophaeus. Compared with the method for catalyzing FA to produce 4-VG by whole cells, the biocatalyst prepared by the invention is used for catalyzing FA to produce 4-VG, and the concentration and the productivity of the 4-VG are improved to 1.97M and 22.8g/L/h under the same condition; the method has good reusability, and can meet the industrial production requirement of 4-VG.

Description

Connecting peptide mediated enzyme immobilized BaPAD catalyst and preparation method and application thereof

Technical Field

The invention belongs to the field of preparation and application of catalysts, and particularly relates to a connecting peptide mediated enzyme immobilized BaPAD catalyst and a preparation method and application thereof.

Background

Because the product 4-vinyl guaiacol has great toxicity to cell membranes, cells are cracked or killed in the process of producing 4-vinyl guaiacol (4-Vinylguaiacol, 4-VG) by using whole cells as a catalyst in a two-phase system, so that the conversion efficiency is gradually reduced or even lost in a long-term reaction, and the cells cannot be recycled. In addition, the cell membrane/wall of a whole cell may be a barrier to substrate or product diffusion, causing mass transfer limitations during the catalytic process. In contrast, the enzyme as a catalyst can directly enter the reaction environment, and the reaction conditions are simpler, but the industrial application of the free enzyme is often challenged by the poor stability of long-term operation, and the recovery and reuse of the enzyme face the technical obstruction. Furthermore, the high cost of enzyme purification is also another key factor affecting the economic sustainability of the process. Therefore, it is very critical to recover these catalysts in a downstream production process to improve operational stability, while improving recovery and stability can reduce costs and improve the efficiency of the overall bioreaction. In order to achieve these goals, immobilization technology is one of the most innovative and studied production methods to achieve industrial scale with higher productivity.

The use of immobilized enzymes in various fields has attracted a great deal of interest and research to varying degrees. There are many methods for immobilizing enzymes on solid carriers, and the most common method is adsorption method, but the traditional adsorption method has the disadvantages that the enzymes are not firmly adsorbed on the surface of the solid carrier, and the selectivity is poor, and adding a specific connecting peptide at the N-terminal or C-terminal of the BaPAD is a method for improving the disadvantages. The phenolic acid decarboxylase BaPAD from the Bacillus atrophaeus has great application value in the aspect of catalyzing ferulic acid to prepare 4-vinyl guaiacol, but the established method for producing 4-VG by taking the whole cell as a catalyst also has the problems that the cell is cracked and died in the reaction and cannot be recycled, and the boiling point of an organic phase and the boiling point of a product 4-VG are not greatly different, so that the downstream separation and purification are difficult and the like.

Disclosure of Invention

In view of the above problems in the prior art, the present invention provides a ligation-peptide-mediated enzyme-immobilized pad catalyst, and another technical problem to be solved by the present invention is to provide a method for preparing a ligation-peptide-mediated enzyme-immobilized pad catalyst; the last technical problem to be solved by the present invention is to provide the use of the above catalyst.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a method for preparing a connecting peptide mediated enzyme immobilized BaPAD catalyst comprises the following steps: firstly, connecting peptide which is specifically adsorbed on zeolite is inserted into a plasmid pET28 a-bad carrier containing a gene coding phenolic acid decarboxylase BaPAD through Nco I/Xho I restriction enzyme cutting sites, and then the enzyme immobilized BaPAD catalyst is obtained by immobilizing the enzyme immobilized BaPAD catalyst on a pretreated zeolite carrier through a fusion enzyme, wherein the phenolic acid decarboxylase BaPAD is from Bacillus atrophaeus.

Further, the linker peptide is composed of n repeating sequences VKTGATSEEPPRLPSKHRPG and VKTQTTAS concatenated together, and the amino acid residues represented by single letter symbols in the linker peptide are defined as follows: v is valine, K is lysine, T is threonine, Q is glutamine, A is alanine, S is serine, R is arginine, E is glutamic acid, P is proline, L is leucine, H is histidine, G is glycine; the zeolite carrier is Na-Y zeolite.

Further, n is 4.

Further, a linker peptide was added to the N-terminus of bacillus atrophaeus phenolic acid decarboxylase pad, and the linker peptide attached to the N-terminus was on the same side of pad as the his tag.

Further, the pretreatment of the zeolite carrier is as follows: 10mg of the carrier was washed three times with 800. Mu.L wash buffer, followed by 200mM, pH 7.0, citric acid-Na ₂ HPO ₄ Washing with buffer solution for three times, performing vortex oscillation in each process, and centrifuging at 8500rpm for 3min to remove supernatant; wash buffer comprises 10mM Tris-HCl,100mM NaCl,1% Triton X-100, pH 7.5.

Further, the enzyme immobilized BaPAD catalyst is stable in 0.5-5% Tween20 solution, and 2% SDS solution can desorb the immobilized enzyme completely.

The linker peptide mediated enzyme immobilized BaPAD catalyst prepared by the above method.

Application of a connection peptide mediated enzyme immobilized BaPAD catalyst in catalyzing ferulic acid to generate 4-VG.

Application of a connection peptide mediated enzyme immobilized BaPAD catalyst in catalyzing FA to generate 4-VG in a two-phase system containing equal volume of toluene in a bioreactor.

Has the advantages that: compared with the prior art, the invention has the advantages that: the invention takes BaPAD as an object, enzyme and Na-Y zeolite are immobilized as biocatalyst by adding connecting peptide which is specifically combined with zeolite, the concentration and the productivity greatly exceed the results of whole cells and free enzyme as biocatalyst for catalyzing FA to produce 4-VG, and compared with the whole cells for catalyzing FA to produce 4-VG, the concentration and the productivity of 4-VG are improved to 1.97M and 22.8g/L/h under the same conditions; the 4LP-BaPAD @ Na-Y zeolite prepared by the invention has good reusability in a two-phase system containing toluene, the activity is reduced to 73 percent after the FA reaction is catalyzed repeatedly for 10 cycles, the industrial production requirement of 4-VG can be realized, and the application prospect is wide.

Drawings

FIG. 1 is a diagram showing the construction of pET28 a-bad-2/3/4 lp;

FIG. 2 is a schematic diagram of the construction of pET28a-2/3/4 lp-bat;

FIG. 3 is a screening graph of positive clones of pET28 a-bad-1/2/3/4 lp (A) and pET28a-1/2/3/4 lp-bad (B);

FIG. 4 is an SDS-PAGE analysis of expression (A) and purification (B) of BaPAD and fusion enzyme;

FIG. 5 is a graph of the effect of pH and temperature on the activity of free and immobilized 4 LP-BaPAD;

FIG. 6 is a graph showing the selective binding (15 mg/g) of BaPAD and fusion enzyme to Na-Y zeolite in cell lysates;

FIG. 7 is a graph of the operating stability of Na-Y zeolite immobilized BaPAD and 4LP-BaPAD in a two-phase system for the enzymatic decarboxylation of ferulic acid;

FIG. 8 is a diagram of the preparation of 4-vinylguaiacol by catalyzing ferulic acid with immobilized enzyme in a two-phase system.

Detailed Description

The invention is further described with reference to specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. In the following examples, unless otherwise specified, all experimental procedures were carried out according to conventional methods.

Example 1 construction of a fusion enzyme of BaPAD fused to a linker peptide

(1) Construction of pET28a-bapad-lp and pET28a-lp-bapad

PCR was performed using pET28 a-bad as a template and BaPAD-F, baPAD-LP-R1, baPAD-LP-R2, LP-BaPAD-F1, LP-BaPAD-F2, and BaPAD-R (Table 1) as primers, and the reaction system was: 5 × 10 μ L of fastPfu buffer, 4 μ L of dNTP (2.5 mM), 1 μ L of pET28a-BaPAD, 1 μ L of BaPAD-F or LP-BaPAD-F, 1 μ L of BaPAD-LP-R1 or BaPAD-R, 1 μ L of fastPfu DNA polymerase (10U/. Mu.L), ddH ₂ O32. Mu.L. The reaction system is well mixed and put into a PCR instrument, and the reaction conditions are set as follows: pre-denaturation at 95 ℃ for 1.5min, denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, and extension at 72 ℃ for 2min for 30 cycles; finally 72 ℃ for 10min. The PCR product was detected by electrophoresis on a 1% agarose gel. The bapad-lp and lp-bapad gene fragments containing NcoI/XhoI enzyme cutting sites were recovered, purified and amplified using the Rapid PCR product Purification Kit (Easypure PCR Purification Kit) from the company, trans Gen, beijing.

TABLE 1 primers used in the present application

Double digestion of the bapad-lp and lp-bapad gene fragments and expression plasmid pET28 a: the double enzyme cutting system of the bat-lp and lp-bat gene segments is as follows: ncoI 1.25. Mu.L, xhoI 1.25. Mu.L, 10 XBuffer D5. Mu.L, bapad-lp or lp-bapad 20. Mu.L, ddH ₂ O22.5. Mu.L. The expression plasmid pET28a has a double enzyme cutting system: ncoI 1.25. Mu.L, xhoI 1.25. Mu.L, 10 XBuffer D5. Mu.L, pET28a 10. Mu.L, ddH ₂ O32.5. Mu.L. Performing double enzyme digestion on the bapad-lp and lp-bapad gene fragments containing double enzyme digestion sites and the expression plasmid pET28a respectively, incubating for 30min at 37 ℃, and then respectively recovering and purifying enzyme digestion products by using a PCR product purification kit.

The double-enzyme-digested target genes bapad-lp, lp-bapad and pET28a are connected by T4 ligase, and the connection system is as follows: base-lp or lp-base 5.25. Mu.L, pET28a 1.75. Mu.L, 10 XT 4 Buffer 2. Mu.L, T4 DNA ligand 1. Mu.L. The reaction solution is prepared and mixed evenly, and then the mixture is subjected to metal bath for 30min at the temperature of 16 ℃. Recombinant plasmids pET28 a-bag-lp and pET28 a-lp-bag are obtained.

(2) Construction of pET28a-bapad-2/3/4lp and pET28a-2/3/4lp-bapad fusion enzymes

The same restriction enzyme sites are introduced at both ends of the bapad fragment: PCR with pET28 a-bad template and primers of BaPAD-F, baPAD-NcoI-R and 2/3/4LP-BaPAD-F, 2/4LP-BaPAD-R or 2/3/4LP-BaPAD-F, 3LP-BaPAD-R (Table 1), the reaction system: 5 Xfastpfu buffer 10. Mu.L, dNTP (2.5 mM) 4. Mu.L, pET28a-bapad 1. Mu.L, upstream and downstream primers 1. Mu.L each, fastPfu DNA polymerase (10U/. Mu.L) 1. Mu.L, ddH ₂ O32. Mu.L. The reaction conditions are as follows: pre-denaturation at 95 ℃ for 1.5min, denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, and extension at 72 ℃ for 2min for 30 cycles; finally 72 ℃ for 10min. The PCR product was detected by electrophoresis on a 1% agarose gel. And (4) recovering, purifying and amplifying the obtained gene fragment by using a PCR product purification kit. Are named bapad-XhoI and bapad-NcoI respectively.

The PCR products bapad-XhoI/bapad-NcoI and pET28a-2/3/4lp were obtained by single-enzyme digestion: pET28a-2/3/4lp and fragments bapad-XhoI and bapad-NcoI containing the same cleavage site at both ends were single-digested with restriction enzymes XhoI and NcoI, respectively. The single enzyme cutting systems of the bapad-XhoI gene fragment and the bapad-NcoI gene fragment are as follows: ncoI or XhoI 1.5. Mu.L, 10 XBuffer D5. Mu.L, bapad-XhoI or bapad-NcoI 20. Mu.L, ddH ₂ O23.5. Mu.L. The single enzyme cutting system of pET28a-2/3/4lp is as follows: ncoI or XhoI 1.5. Mu.L, 10 XBuffer D5. Mu.L, pET28a-2/3/4lp 10. Mu.L, ddH ₂ O 33.5μL。

The XhoI-digested base-XhoI and pET28a-2/3/4lp were ligated with T4 Ligase, and NcoI-digested base-NcoI and pET28a-2/3/4lp were ligated with T4 Ligase, and the base-XhoI or base-NcoI was ligated at 5.25. Mu.L, pET28a-2/3/4lp at 1.75. Mu.L, 10 XT 4 Buffer at 2. Mu.L, and T4 DNA Ligase at 1. Mu.L. The reaction solution is prepared according to the reaction system and is mixed evenly to react for 30min at 16 ℃, and the recombinant plasmids pET28 a-bad-2/3/4 lp (figure 1) and pET28a-2/3/4 lp-bad (figure 2) are obtained.

(3) Screening of Positive clones of recombinant plasmid

Coli top10 competent cells prepared in advance were thawed on ice.

Connecting the bapad-lp and lp-bapad PCR fragments with a vector pET28 a; the fragments of bad and pET28a-2/3/4lp, which were single-digested with NcoI and XhoI, respectively, were ligated and transformed into E.coli top10 competent, respectively.

The E.coli top10 competent cells introduced with the ligation product were subjected to ice bath for 30min, followed by heat shock at 42 ℃ for 2min, and 0.5mL of LB medium was added to the sterile platform, followed by shake cultivation at 37 ℃ and 200rpm for about 1 hour.

The cultured bacterial solution containing the ligation products was centrifuged at 4000rpm for 2min, about 400. Mu.L of the supernatant was removed, and the remaining cells were resuspended and spread on an LBK plate and cultured overnight in a 37 ℃ incubator.

Several single colonies were randomly picked from overnight LBK medium as templates and colony PCR was performed with the upstream and downstream universal primers T7/T7ter on pET28a vector. The specific operation is as follows: randomly picking several white single colonies from a culture plate, uniformly mixing the white single colonies in 20 mu L of sterile water, taking 10 mu L of the white single colonies as a template, and reacting the mixture in a reaction system: 2 x Hieff ^TM PCR Master Mix 13. Mu.L, universal forward primer T71. Mu.L, universal reverse primer T7ter 1. Mu.L, template (single colony aqueous solution) 10. Mu.L. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 2min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 2min for 30 cycles; finally 72 ℃ for 10min.

The PCR product was detected by 1.0% agarose gel electrophoresis, and as a result, as shown in FIG. 3, colonies corresponding to bands of 700bp or more were selected, and the colonies were subjected to tube shaking and sequencing.

Example 2 expression and purification of recombinant proteins of BaPAD and fusion enzymes

(1) Expression and purification of recombinant proteins

The recombinant plasmid with correct sequencing is selected to transform the competence of Escherichia coli BL21 (DE 3) in the same way as top 10. LBK plates containing 50. Mu.g/mL kanamycin were plated and incubated overnight in an incubator at 37 ℃.

White single colonies were picked from overnight-cultured LBK plates and incubated overnight at 37 ℃ on a shaker at 200rpm in 3mL of LBK broth containing 50. Mu.g/mL kanamycin.

The overnight-cultured broth was inoculated into 50mL of sterilized LBK liquid medium at an inoculum size of 2%, and shake-cultured at 37 ℃ and 200rpm to OD ₆₀₀ Is 0.6.

Adding IPTG with final concentration of 0.4mM, performing induced expression culture at 28 ℃ by a shaking table at 200rpm, centrifuging the bacterial liquid at 8500rpm for 10min after 12h, and collecting bacterial cells.

And (3) respectively resuspending the thalli by using 4mL lysine buffer, and carrying out ultrasonic crushing on an ice-water mixture, wherein the parameters of an ultrasonic crusher are 200w of power, 10min of time, 3s of ultrasonic time and 3s of interval time.

The formulation of lysine buffer in this example was 300mM NaCl,50mM Na ₂ HPO ₄ ·2H ₂ O, pH 8.0.

(2) SDS-PAGE analysis

After the cells induced to express were collected, the cells were sonicated and analyzed by SDS-PAGE, and as shown in FIG. 4 (A), the intensity of the bands in the 9 lanes was slightly different, indicating that the linker peptide had no significant effect on the expression of BaPAD and the fusion enzyme. The pure enzyme purified by Ni-NTA was also analyzed by SDS-PAGE as shown in FIG. 4 (B), and it was found that there were single bands at 22, 25, 28, 30, 32, 25, 27, 30 and 32kDa, respectively, corresponding to the theoretical molecular weights of BaPAD, baPAD-LP, baPAD-2LP, baPAD-3LP, baPAD-4LP, LP-BaPAD,2LP-BaPAD,3LP-BaPAD,4LP-BaPAD, respectively.

(3) Analysis of expression levels of BaPAD and fusion enzyme

As a result of analyzing protein concentrations of 9 kinds of pure enzymes purified by Ni-NTA by BCA method, the concentration of the pure enzyme BaPAD reached 178.1. Mu.g/mL, and concentrations of several kinds of pure enzymes containing a linker peptide were in the range of 143 to 206. Mu.g/mL, which were not much different from the results of SDS-PAGE, as shown in Table 2.

TABLE 2 expression levels of BaPAD and fusion enzymes

Enzyme	Expression level (μ g/mL)
		BaPAD	178.1
BaPAD-LP	206.2
		BaPAD-2LP	192.1
BaPAD-3LP	185.3
		BaPAD-4LP	143.5
LP-BaPAD	184.3
		2LP-BaPAD	199.0
3LP-BaPAD	174.3
		4LP-BaPAD	163.2

Example 3 adsorption experiment of BaPAD and fusion enzyme on different vectors

(1) Affinity adsorption of BaPAD and fusion enzyme to different carriers

Pretreatment of the solid carrier: 10mg of solid support was washed three times with 800. Mu.L wash buffer (10 mM Tris-HCl,100mM NaCl,1% Triton X-100, pH 7.5) and then with citric acid-Na ₂ HPO ₄ Buffer (200mM, pH 7.0) three times, each process vortex, 8,500rpm centrifugation for 3min to remove the supernatant. Immobilization: 9 pure enzymes (50. Mu.g, 100. Mu.L citric acid-Na) ₂ HPO ₄ Buffer) was added to the washed vehicle and incubated on ice for 1h with shaking.

Calculation of enzyme load: the incubated sample was centrifuged at 8,500rpm for 3min, the supernatant was separated, and the protein concentrations of the supernatant before and after immobilization were measured by BCA method, followed by subtraction to obtain the immobilized enzyme concentration. Thus comparing the adsorption levels of 9 enzymes on different carriers.

Na-Y zeolite, ZSM-5 zeolite, beta zeolite, dense silica and mesoporous silica are selected as solid carriers, the concentration of pure enzyme is set to be 5mg/g of carrier, and the adsorption performance of 9 enzymes is analyzed. As can be seen from Table 3, both BaPAD and the fusion enzyme were completely adsorbed (100%) by Na-Y, ZSM-5 and zeolite beta. In addition, these enzymes exhibit 75-86% binding affinity for mesoporous silica, which is 2-3 times the affinity for dense silica. For both silica materials, the affinity of the enzyme containing the linker peptide was slightly stronger than the pad without the linker peptide, and the linker peptide demonstrated superiority in low enzyme concentration adsorption of the silica materials.

TABLE 3 Loading rates (%) of BaPAD and fusion enzymes on different vectors at low enzyme concentrations

(2) Maximum adsorption of BaPAD and fusion enzyme on Na-Y zeolite

10mg of Na-Y zeolite was treated with 800. Mu.L wash buffer (10 mM Tris-HCl,100mM NaCl,1% Triton X-100, pH 7.5) and citric acid-Na, respectively ₂ HPO ₄ Buffer (200mM, pH 7.0) three times, each process vortex, 8,500rpm centrifugation for 3min to remove the supernatant. 200. Mu.L of pure enzyme diluted to 1000. Mu.g/ml was added to each of the washed Na-Y zeolites so that the enzyme concentration in the system was 20mg/g, and the mixture was incubated on ice for 1 hour with shaking.

As can be seen from Table 4, the amount and loading rate of the enzyme immobilized on Na-Y zeolite increased with the increase of the linker peptide, regardless of whether the linker peptide was added to the N-terminus or the C-terminus of BaPAD, while the loading rate of the enzyme was increased by linking the linker peptide to the N-terminus compared to the linker peptide linked to the C-terminus, and the highest loading rate of 4LP-BaPAD reached 68.81%, and the corresponding concentration of the loaded protein was 13.8mg/g vector. In contrast, the loading of the original BaPAD without the linker peptide was only 31.93%. These results indicate that at high enzyme concentrations, the addition of linker peptide can increase the loading of the enzyme by Na-Y zeolite, and that the loading increases with the extension of the linker peptide.

TABLE 4 maximum loading of BaPAD and fusion enzymes on Na-Y Zeolite at high enzyme concentrations

(3) Biochemical property study of immobilized enzyme

S1, enzyme activity detection of immobilized enzyme:

enzyme activity determination step (taking FA as an example): reaction 1mL, containing 0.8mL of citric acid-Na ₂ HPO ₄ Buffer (pH 6.0), 100. Mu.L of 50mM sodium ferulate solution and 100. Mu.L of free and immobilized phenolic acid decarboxylase at a concentration of 20. Mu.g/mL. And reacting in a water bath kettle at 50 ℃ for 5min, and adding 2mL of methanol to stop the reaction. The content of generated 4-VG was determined by HPLC after filtration through a 0.22 μm filter. Samples treated under the same conditions with 100. Mu.L buffer instead of enzyme were used as controls.

And (3) detection of enzyme activity: the amount of the reaction product produced was measured by HPLC, and the amount of 4-VG produced was calculated from a standard curve using the measured peak area in the case of FA, to calculate the activity of immobilized phenolic acid decarboxylase (IU/mL).

The enzyme activities of 9 immobilized enzymes using Na-Y zeolite as carrier and p-coumaric acid (pCA), ferulic Acid (FA) and Caffeic Acid (CA) as substrates are shown in Table 5, it can be seen that the substrate specificity of the immobilized enzyme is not changed compared with that of the free enzyme, but the activity after immobilization is different from that of the free enzyme due to the position difference of the connecting peptide, the activity of BaPAD and BaPAD-1/2/3/4LP is still maintained 55-85%, while the activity after immobilization of 1/2/3/4LP-BaPAD is hardly reduced, even the activity of LP-BaPAD is slightly higher than that of the free enzyme, and in total, the activity of the fusion enzyme with the connecting peptide connected to the N terminal is changed to 95-105% of the free enzyme after immobilization. The reason may be that the connecting peptide and his tag attached to the C-terminal are distributed at both ends of the BaPAD, and the his tag may also have a certain adsorption effect on the zeolite material, so that the enzyme is anchored on the carrier and cannot flexibly contact with the substrate, resulting in slow decarboxylation reaction or poor substrate accessibility, so that the activity is much lower than that of the free enzyme; and the connecting peptide connected to the N end and the his label are positioned at the same end of the BaPAD, so that only one end of the fusion enzyme is fixed, the fusion enzyme is relatively more flexible, the substrate accessibility is better, and the activity remained after the immobilization is higher.

TABLE 5 Activity of free and immobilized BaPAD and fusion enzymes on different substrates

S2, influence of pH on the Activity of free and immobilized 4 LP-BaPAD: free and immobilized 4LP-BaPAD enzymatic reactions with FA as substrate at 50 ℃ and different pH using citrate-Na at pH4.0-8.0 ₂ HPO ₄ Buffers, plotted with relative activity up to 100%.

Effect of temperature on activity of free and immobilized 4 LP-BaPAD: the enzymatic reactions were carried out in pH-optimal buffers at different temperatures (30-60 ℃) and plotted with a relative activity of up to 100%.

Effect of pH and temperature on activity of free and immobilized 4 LP-pads: detecting the activity of free and immobilized 4LP-BaPAD at pH4.0-8.0 and 30-60 deg.C with ferulic acid as substrate, and determining the highest enzyme activity as 100% (as shown in FIG. 5), wherein FIG. 5 (A) shows that the optimum pH of free 4LP-BaPAD is 5.0 and the optimum pH of immobilized 4LP-BaPAD is 5.5; as can be seen from FIG. 5 (B), the optimum temperatures of free and immobilized 4LP-BaPAD were 50 and 55 ℃ respectively, and the enzyme activity was high in the range of 45-55 ℃. The temperature and pH optima of 4LP-BaPAD were comparable to those of BaPAD without the linker peptide.

EXAMPLE 4 purification and immobilization of target proteins from crude enzyme solutions Using Zeolite materials in a one-step Process

(1) One-step purification method

Recombinant cells induced by IPTG were harvested after ultrasonicationThe supernatants of (1), crude enzyme, were diluted to 3000. Mu.g/mL, 0.1g of Na-Y zeolite washed with wash buffer (see the details of the procedure in the pretreatment of the solid support) was added to 0.5mL of the diluted cell lysate, incubated on ice for 1h,8, centrifugation at 500rpm for 3min, the supernatants were separated, and lysed buffer (300mM NaCl,50mM Na) ₂ HPO ₄ ·2H ₂ O, pH 8.0) was washed three times, the supernatant was collected, and the pellet was then resuspended in 0.5mL of lysine buffer. The same volume of cell lysate before immobilization was taken, and the supernatant after adsorption and the zeolite-immobilized resuspension were analyzed by SDS-PAGE.

As can be seen from FIG. 6, the BaPAD containing no linker peptide was present mainly in the postadsorption supernatant (unbound) after incubation with Na-Y zeolite, and only a very small amount of residual BaPAD was present in the protein-bound (immobilized) fraction, probably due to non-specific adsorption of Na-Y zeolite. Most of the fusion proteins to which the linker peptide was added were present in the bound portion after incubation with Na-Y zeolite, and the band of this portion increased with extension of the linker peptide, and for BaPAD-4LP and 4LP-BaPAD, only a small amount was present in the unbound portion. In addition, it can be seen from the SDS-PAGE patterns that all 8 fusion proteins show distinct single bands and are consistent with the theoretical molecular weight. These results indicate that the fusion enzyme with the linker peptide can purify the immobilized target protein from the cell lysate by the Na-Y zeolite one-step method.

(2) Adsorption selectivity of BaPAD and fusion enzyme on Na-Y zeolite

The method is a one-step purification method except for the final SDS-PAGE step. And measuring enzyme activity of the sample in each step by taking ferulic acid as a substrate, and obtaining the selectivity (%) of the BaPAD and the fusion enzyme on the Na-Y zeolite by calculating the fixed enzyme activity ratio of the supernatant fluid and the Na-Y zeolite.

The adsorption selectivity is defined by calculating the ratio of the activity of the immobilized enzyme to the original total enzyme activity. From Table 6, it can be seen that the initial total enzyme activity of BaPAD is the highest, reaching 318IU, but the selectivity of Na-Y zeolite adsorption is only 14%, while the Na-Y zeolite adsorption selectivity of fusion enzyme containing the connecting peptide is higher than that of the native BaPAD, and the selectivity is gradually improved along with the increase of the length of the connecting peptide, and the selectivity of the connecting peptide connected to the N-end is higher than that of the connecting peptide connected to the C-end. 4LP-BaPAD has the lowest initial total enzyme activity, but the highest selectivity for Na-Y zeolite, reaching 86%.

TABLE 6 selectivity of BaPAD and fusion enzymes for Na-Y zeolite adsorption

	Original Total enzyme Activity (IU)	Activity (IU) of immobilized enzyme	Selectivity (%)
				BaPAD	318.19±8.42	45.76±3.14	14.38
BaPAD-LP	312.15±5.68	85.42±0.33	27.36
				BaPAD-2LP	248.35±3.51	128.27±3.48	51.64
BaPAD-3LP	193.54±3.17	137.59±3.06	71.09
				BaPAD-4LP	191.25±0.53	135.52±2.32	70.86
LP-BaPAD	201.83±5.43	128.36±3.68	63.59
				2LP-BaPAD	186.42±4.98	155.63±2.37	83.48
3LP-BaPAD	185.84±3.76	150.57±2.22	81.02
				4LP-BaPAD	173.72±9.12	149.87±2.18	86.27

Example 5 stability of Na-Y Zeolite adsorption by LP-BaPAD and repeated Loading Studies

(1) Stability of adsorption

mu.L of 4LP-BaPAD was immobilized on the washed Na-Y zeolite at a concentration of 10mg/g of the carrier, and the amount of immobilized enzyme was obtained by measuring the supernatant protein concentration before and after adsorption. And then eluting the immobilized enzyme by using buffer solutions under different conditions, and examining the stability of the immobilized 4LP-BaPAD under different conditions by detecting the protein concentration and activity of the eluent.

As a result, as shown in Table 7, no immobilized protein was found to elute in Tween20 (pH 7) at a concentration ranging from 0.5% to 5%, and the immobilized enzyme was stable in the pH range of 3.0 to 9.0, but the immobilized enzyme could be partially eluted from the carrier by a 5M NaCl solution. The 4LP-BaPAD @ Na-Y zeolite was stable when treated with Tween20 solution which could disrupt the hydrophobic effect. SDS had a better desorption effect on the immobilized enzyme, and 100% was desorbed after 2% SDS treatment.

TABLE 7 stability of the 4LP-BaPAD @ Na-Y zeolite under different conditions of treatment

Elution conditions (pH/NaCl concentration, M)	Eluting the protein%
		pH3.0-9.0	0
3.0/5	0
		4.0/5	0
5.0/5	16.76
		6.0/5	49
7.0/5	38.6
		8.0/5	32.8
9.0/5	36.4
		7/0.5％Tween 20	0
7/1％Tween 20	0
		7/2％Tween 20	0
7/3％Tween 20	0
		7/4％Tween 20	0
7/5％Tween 20	0
		7/1％SDS	96
7/2％SDS	100

(2) Repetitive load

The resulting mixture was shaken and mixed with 100. Mu.L of 2% SDS solution and 4LP-BaPAD immobilized on Na-Y zeolite, centrifuged at 20s,8,500rpm for 3min with a vortex apparatus, and the desorption of the immobilized protein was completely repeated three times (verified by detection at 280nm with a spectrophotometer), after which the mixture was eluted with citric acid-Na ₂ HPO ₄ (pH 7.0) washing SDS remained on the zeolite, and then carrying out next round of adsorption,three cycles were repeated and the change in the amount of repeatedly loaded 4LP-BaPAD of Na-Y zeolite after desorption was compared by measuring the protein concentration before and after adsorption for each cycle.

2% SDS solution was used for desorption of 4LP-BaPAD from Na-Y zeolite. A typical cycle comprised adsorption and desorption, and the amount of zeolite reloaded protein was found to be nearly constant after three cycles, indicating the superior regenerability of 4LP-BaPAD on Na-Y zeolite.

(3) Reusability of immobilized BaPAD and 4LP-BaPAD

In this example, the operational stability of BaPAD and 4LP-BaPAD-Na-Y zeolite complexes as biocatalysts in a two-phase reaction system containing toluene was evaluated by a continuous reaction for 10 cycles. The yield of 4-vinylguaiacol during the continuous reaction is shown in FIG. 7. It can be seen that the conversion efficiency of immobilized BaPAD and 4LP-BaPAD gradually decreases as the reaction proceeds, and in the first cycle, 200mM substrate ferulic acid produces 175 and 180mM 4-vinylguaiacol under catalysis of immobilized BaPAD and 4LP-BaPAD, respectively, and in the second cycle, the data decreases to 145 and 175mM, while in the tenth cycle, the amount of 4-vinylguaiacol produced decreases to the initial 36% and 73% compared to the first reaction. Therefore, the operation stability of the immobilized phenolic acid decarboxylase containing the connecting peptide is far higher than that of the enzyme without the connecting peptide, and the immobilized 4LP-BaPAD can be used as a catalyst with extremely excellent catalytic capability and stability.

Example 6 conversion of FA with 4LP-BaPAD @ Na-Y zeolite in a bioreactor to produce 4-VG

(1) Preparation of fusion enzyme 4LP-BaPAD

200mL of LBK culture medium prepared in 8 flasks were inoculated with 2% by volume of activated 4LP-BaPAD seed cell suspension and cultured to OD at 37 ℃ ₆₀₀ = about 0.6, add IPTG at a final concentration of 0.4mM, and induce culture at 28 ℃ for 12h. After centrifugation at 8,000rpm for 10min at 4 ℃ and removal of the supernatant, the cells were resuspended in 4mL lysine buffer and sonicated on ice. After centrifugation at 10,000rpm for 10min at 4 ℃, about 200mL of the supernatant was collected as crude enzyme, and the total amount of crude protein was about 700mg by BCA assay.

Among them, LBK culture solution is LB liquid medium, at 121 degrees C sterilization for 20 minutes, cooling to about 60 degrees C, adding the final concentration of 50 u g/mL kanamycin.

(2) Preparation of catalyst 4LP-BaPAD @ Na-Yzeolite

70g of Na-Y zeolite was weighed, washed three times with lysine buffer (pH 8.0), 200mL of the crude enzyme solution was added, magnetically stirred on ice for 1h, the protein concentration of the supernatant was measured after centrifugation, and the total unadsorbed protein amount was calculated to be about 220mg, using 70g 4LP-BaPAD @ Na-Y zeolite with 480mg of total protein immobilized.

(3) Preparation of 4-VG with Ferulic Acid (FA) as substrate

Reaction system: the 5L fermentor contained 1L of toluene and 1L of 70g 4LP-BaPAD @ Na-Y zeolite catalyst containing an initial concentration of 200mM FA and 480mg total protein immobilized.

Reaction conditions are as follows: adjusting the reaction temperature to 30 ℃ through a water circulation system of the fermentation tank, respectively arranging a stirring rotor for the water phase and the organic phase at the rotation speed of 150rpm, and supplementing ferulic acid powder through the change of the pH value of an instrument control panel in the reaction process to control the pH value within the range of 6.5 +/-0.1. Samples were taken hourly and samples centrifuged at 10,000rpm for 5min to separate the organic and aqueous phases thoroughly.

The detection method comprises the following steps: the organic and aqueous phases were diluted with methanol and subjected to HPLC detection, and the concentrations of generated 4-VG and residual FA were calculated from the standard curve.

In a two-phase biotransformation system using toluene as an organic phase, ferulic acid is used as a substrate to prepare the high-concentration 4-vinyl guaiacol. HPLC analysis indicated a total of 1.97M product, 98.95% conversion, and 22.8g/L/h productivity after 13 hours of continuous reaction, as shown in FIG. 8.

Claims

1. A process for preparing the linking peptide mediated enzyme immobilized BaPAD catalyst includes such steps as passing the linking peptide adsorbed to zeolite by itNcoI/XhoI restriction sites were inserted into plasmid pET28a-BaPAD vector containing the gene coding for phenolic acid decarboxylase BaPAD, the linker peptide consisting of n repeats VKTAATSEEPPPRHPSKPG and VKTQTAS are linked in tandem, the amino acid residues represented by the single letter symbols in the linker peptide are defined as follows: v is valine, K is lysine, T is threonine, Q is glutamine, A is alanine, S is serine, R is arginine, E is glutamic acid, P is proline, L is leucine, H is histidine, G is glycine; the zeolite carrier is Na-Y zeolite; n is 4; the connecting peptide is added at the N-end of phenolic acid decarboxylase BaPAD of the Bacillus atrophaeus, the connecting peptide connected at the N-end and the his label are positioned at the same end of the BaPAD, and then an enzyme immobilized BaPAD catalyst is obtained by immobilizing fusion enzyme on a pretreated zeolite carrier, wherein the phenolic acid decarboxylase BaPAD is from the Bacillus atrophaeus; the pretreatment of the zeolite carrier comprises the following steps: 10mg of the carrier was washed three times with 800. Mu.L wash buffer, followed by 200mM citric acid-Na, pH 7.0 ₂ HPO ₄ Washing with buffer solution for three times, performing vortex oscillation in each process, and centrifuging at 8500rpm for 3min to remove supernatant; the wash buffer comprises 10mM Tris-HCl,100mM NaCl,1% Triton X-100, pH 7.5.

2. The method of preparing a linked peptide mediated enzyme immobilized pad catalyst of claim 1, wherein the catalyst is stable in 0.5-5% tween20 solution and 2% SDS solution is capable of desorbing all of the immobilized enzyme.

3. A linker-mediated enzyme-immobilized pad catalyst prepared by the process of any one of claims 1-2.

4. Application of a connection peptide mediated enzyme immobilized BaPAD catalyst in catalyzing ferulic acid to generate 4-VG.

5. Use of a linker peptide mediated enzyme immobilized BaPAD catalyst in a two-phase system comprising an equal volume of toluene in a bioreactor to catalyze the production of FA to 4-VG.