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

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

A kind of connecting peptide-mediated enzyme immobilized BaPAD catalyst and its preparation method and application

technical field

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

Background technique

Since the product 4-vinylguaiacol (4-VG) is very toxic to the cell membrane, the whole cell acts as a catalyst in the process of producing 4-vinylguaiacol (4-VG) in a two-phase system, and the cell lyses or dies , resulting in a gradual reduction or even loss of conversion efficiency in the long-term reaction, which cannot be reused. Alternatively, the cell membrane/wall of whole cells may act as a barrier to substrate or product diffusion, causing mass transfer limitation during catalysis. In comparison, enzymes as catalysts can directly enter the reaction environment, and the reaction conditions are relatively simple. However, the industrial application of free enzymes is often challenged by poor long-term operational stability, and the recovery and reuse of enzymes face technical obstacles. In addition, the high cost of enzyme purification is another key factor affecting the economic sustainability of the process. Therefore, it is very critical to recover these catalysts in the downstream production process and improve the operational stability, while improving the recovery and stability can reduce the cost and improve the efficiency of the overall biological reaction. To achieve these goals, immobilization technology has emerged as one of the most innovative and widely studied production methods to achieve industrial scale with higher productivity.

The application of immobilized enzymes in various fields has aroused great interest and different degrees of research. There are many methods for immobilizing enzymes on solid carriers, the most commonly used method is the adsorption method, but the traditional adsorption method has the disadvantages of weak adsorption on the surface of the enzyme and the solid carrier and poor selectivity. The addition of specific linker peptides at the C-terminus is one way to ameliorate these shortcomings. The phenolic acid decarboxylase BaPAD from Bacillus atrophaeus has great application value in catalyzing the production of 4-vinylguaiacol from ferulic acid, but the established method of producing 4-VG with whole cells as a catalyst still exists in the reaction of cells The problem of cracking death in the middle and not being able to be reused, the boiling point of the organic phase is not much different from the boiling point of the product 4-VG, which leads to difficulties in downstream separation and purification.

Contents of the invention

In view of the above-mentioned problems existing in the prior art, the technical problem to be solved by the present invention is to provide a kind of enzyme immobilization BaPAD catalyst mediated by the connecting peptide, and another technical problem to be solved by the present invention is to provide the enzyme immobilized BaPAD catalyst mediated by the connecting peptide. The preparation method of BaPAD catalyst; The last technical problem to be solved by the present invention is to provide the application of above-mentioned catalyst.

In order to solve the problems of the technologies described above, the technical scheme adopted in the present invention is as follows:

A preparation method of an enzyme-immobilized BaPAD catalyzer mediated by a connecting peptide, comprising the following steps: first, the connecting peptide adsorbed specifically to zeolite is inserted through the Nco I/Xho I restriction enzyme cleavage site into The plasmid pET28a-bapad carrier of the BaPAD gene is immobilized on the pretreated zeolite carrier by fusion enzyme to obtain the enzyme-immobilized BaPAD catalyst, and the phenolic acid decarboxylase BaPAD comes from Bacillus atrophaeus.

Further, the connecting peptide is composed of n repeated sequences VKTQATSREEPPRLPSKHRPG and VKTQTAS in series, and the amino acid residues represented by single-letter symbols in the connecting 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, the connecting peptide is added to the N-terminal of the phenolic acid decarboxylase BaPAD of Bacillus atrophaeus, and the connecting peptide connected to the N-terminal is at the same end of BaPAD as the his tag.

Further, the pretreatment of the zeolite carrier is as follows: 10 mg of the carrier is washed three times with 800 μL wash buffer, and then washed three times with 200 mM, pH 7.0 citric acid-Na ₂ HPO ₄ buffer, vortexed in each process, and centrifuged at 8500 rpm for 3 min to remove the net Supernatant; wash buffer including 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 completely desorb the immobilized enzyme.

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

Application of linker peptide-mediated enzyme-immobilized BaPAD catalyst in catalyzing the production of 4-VG from ferulic acid.

Application of linker peptide-mediated enzyme-immobilized BaPAD catalyst to catalyze 4-VG from FA in a biphasic system containing an equal volume of toluene in a bioreactor.

Beneficial effect: Compared with the prior art, the advantages of the present invention are: the present invention takes BaPAD as the object, and immobilizes the enzyme and Na-Y zeolite as a biocatalyst for catalyzing FA production by adding a linking peptide specifically bound to zeolite4 -VG, the concentration and productivity greatly exceed the results of whole cells and free enzymes as biocatalysts, compared with the whole cell catalyzed FA production of 4-VG, under the same conditions, the 4-VG concentration and productivity are increased to 1.97M and 22.8 g/L/h; and the 4LP-BaPAD@Na-Y zeolite prepared by the present invention has good reusability in the two-phase system containing toluene, and after 10 cycles of repeated catalytic FA reaction, the activity drops to the original 73% of the 4-VG can meet the industrial production requirements, and has a wide range of application prospects.

Description of drawings

Fig. 1 is the schematic diagram of constructing pET28a-bapad-2/3/4lp;

Fig. 2 is the schematic diagram of constructing pET28a-2/3/4lp-bapad;

Fig. 3 is the screening figure of pET28a-bapad-1/2/3/4lp (A) and pET28a-1/2/3/4lp-bapad (B) positive clones;

Fig. 4 is the SDS-PAGE analysis figure of expression (A) and purification (B) of BaPAD and fusion enzyme;

Fig. 5 is the figure of influence of pH and temperature on the activity of free and immobilized 4LP-BaPAD;

Fig. 6 is the selective binding (15mg/g) figure of BaPAD and fusion enzyme to Na-Y zeolite in the cell lysate;

Fig. 7 is the repeated operation stability diagram of Na-Y zeolite immobilized BaPAD and 4LP-BaPAD enzymatically decarboxyferulic acid in two-phase system;

Fig. 8 is a diagram showing the preparation of 4-vinylguaiacol from ferulic acid catalyzed by immobilized enzyme in a two-phase system.

detailed description

The present invention will be further described below in conjunction with specific embodiments. These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. In the following examples, unless otherwise specified, the experimental methods used are conventional methods.

The construction of the fusion enzyme that embodiment 1BaPAD is fused with connecting peptide

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

Using pET28a-bapad as a template, BaPAD-F, BaPAD-LP-R1, BaPAD-LP-R2 and LP-BaPAD-F1, LP-BaPAD-F2, BaPAD-R (Table 1) were used as primers for PCR respectively, and the reaction The system is: 5×fastPfu buffer 10μL, dNTP (2.5mM) 4μL, pET28a-bapad 1μL, BaPAD-F or LP-BaPAD-F1 1μL, BaPAD-LP-R1 or BaPAD-R1μL, fastPfu DNA polymerase (10U/μL) ₁ μL, ddH2O 32 μL. Mix the reaction system well and put it into the PCR instrument. Set the reaction conditions as follows: 1.5min pre-denaturation at 95°C, 30s denaturation at 94°C, 30s annealing at 57°C, 2min extension at 72°C, a total of 30 cycles; finally 10min at 72°C . PCR products were detected by 1% agarose gel electrophoresis. The bapad-lp and lp-bapad gene fragments containing NcoI/XhoI restriction sites were recovered, purified and amplified using Beijing Trans Gen Company's EasyPure PCR Purification Kit.

Table 1 The primers used in this application

Double enzyme digestion of bapad-lp and lp-bapad gene fragments and expression plasmid pET28a: The double enzyme digestion system of bapad-lp and lp-bapad gene fragments is: NcoI 1.25 μL, XhoI 1.25 μL, 10×Buffer D 5 μL, bapad- lp or lp-bapad 20 μL, ddH ₂ O 22.5 μL. The double digestion system of expression plasmid pET28a is: NcoI 1.25 μL, XhoI 1.25 μL, 10×Buffer D 5 μL, pET28a 10 μL, ddH ₂ O 32.5 μL. The bapad-lp and lp-bapad gene fragments containing double restriction sites and the expression plasmid pET28a were double-digested respectively, and the reaction conditions were incubated at 37°C for 30 minutes, and then the digested products were recovered and purified with a PCR product purification kit .

Ligate the double-digested target gene bapad-lp and lp-bapad and pET28a with T4 ligase, the ligation system is: bapad-lp or lp-bapad 5.25 μL, pET28a 1.75 μL, 10×T4 Buffer 2 μL, T4 DNA Ligase 1 μL . Prepare the reaction solution and mix well, and place in a metal bath at 16°C for 30 minutes. The recombinant plasmids pET28a-bapad-lp and pET28a-lp-bapad were obtained.

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

Introduce the same restriction site at both ends of the bapad fragment: with pET28a-bapad template, with 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) are primers for PCR, reaction system: 5×fastPfu buffer 10μL, dNTP (2.5mM) 4μL, pET28a-bapad 1μL, upstream and downstream primers 1μL , fastPfu DNA polymerase (10U/μL) 1 μL, ddH ₂ O 32 μL. The reaction conditions were: pre-denaturation at 95°C for 1.5min, denaturation at 94°C for 30s, annealing at 57°C for 30s, extension at 72°C for 2min, a total of 30 cycles; the final cycle at 72°C for 10min. PCR products were detected by 1% agarose gel electrophoresis. The PCR product purification kit was used to recover and purify the amplified gene fragments. They were named bapad-XhoI and bapad-NcoI, respectively.

Single enzyme digestion of the above PCR products bapad-XhoI/bapad-NcoI and pET28a-2/3/4lp: Respectively use restriction endonucleases XhoI and NcoI to single-digest pET28a-2/3/4lp and both ends contain the same enzyme cutting site Dot fragments bapad-XhoI and bapad-NcoI. The single enzyme digestion system of bapad-XhoI and bapad-NcoI gene fragments is: NcoI or XhoI 1.5 μL, 10×Buffer D 5 μL, bapad-XhoI or bapad-NcoI 20 μL, ddH ₂ O 23.5 μL. The single enzyme digestion system of pET28a-2/3/4lp is: NcoI or XhoI 1.5 μL, 10×Buffer D 5 μL, pET28a-2/3/4lp 10 μL, ddH ₂ O 33.5 μL.

Ligate bapad-XhoI and pET28a-2/3/4lp that have been digested with XhoI with T4 ligase, and connect bapad-NcoI and pET28a-2/3/4lp that have been digested with NcoI with T4 ligase, 5.25 μL of bapad-XhoI or bapad-NcoI, 1.75 μL of pET28a-2/3/4lp, 2 μL of 10×T4 Buffer, 1 μL of T4 DNA Ligase. Mix the reaction solution according to the reaction system and react at 16°C for 30 minutes to obtain recombinant plasmids pET28a-bapad-2/3/4lp (Figure 1) and pET28a-2/3/4lp-bapad (Figure 2).

(3) Screening of recombinant plasmid positive clones

Thaw the prepared E. coli top10 competent cells on ice.

Ligate the PCR fragments of bapad-lp and lp-bapad to the vector pET28a; connect the fragments of bapad and pET28a-2/3/4lp that were digested with NcoI and XhoI, respectively, and transform them into Escherichia coli top10 competent.

The Escherichia coli top10 competent cells that introduced the ligation product were placed in an ice bath for 30 minutes, then heat-shocked at 42°C for 2 minutes, added 0.5 mL of LB medium on a sterile operating table, and incubated at 37°C, 200 rpm, on a shaker for about 1 hour.

Centrifuge the cultured bacterial solution containing the ligation product at 4000 rpm for 2 minutes, remove about 400 μL of the supernatant, and resuspend the remaining bacteria on LBK plates, and place them in a 37°C incubator for overnight culture.

Randomly pick several single colonies from the LBK medium cultured overnight as templates, and carry out colony PCR with the upstream and downstream universal primers T7/T7ter on the pET28a vector. The specific operation is as follows: Randomly pick several white single colonies from the culture plate in 20 μL sterile water, mix well and take out 10 μL as a template, reaction system: 2×Hieff ^TM PCR Master Mix 13 μL, general upstream primer T71 μL, general downstream primer T7ter 1 μL, template (single colony aqueous solution) 10 μL. The reaction conditions were: pre-denaturation at 94°C for 2min, denaturation at 94°C for 30s, annealing at 55°C for 30s, extension at 72°C for 2min, a total of 30 cycles; final 10min at 72°C.

The PCR product was detected by 1.0% agarose gel electrophoresis, the result is shown in Figure 3, the colony corresponding to the band above 700bp was selected, shaken and sequenced.

Expression and purification of embodiment 2 BaPAD and fusion enzyme recombinant protein

(1) Expression and purification of recombinant protein

Select the recombinant plasmid with correct sequencing to transform Escherichia coli BL21 (DE3) competent, the method is the same as transforming top10 competent. Spread LBK plates containing 50 μg/mL kanamycin and culture overnight in a 37°C incubator.

Pick a single white colony from the overnight cultured LBK plate and place it in 3 mL of LBK liquid medium containing 50 μg/mL kanamycin, and culture it overnight at 37°C on a shaker at 200 rpm.

The overnight cultured bacterial solution was added to 50 mL of sterilized LBK liquid medium at an inoculum size of 2%, and cultured at 37° C. on a shaker at 200 rpm until the OD ₆₀₀ was 0.6.

Add IPTG with a final concentration of 0.4mM, induce expression culture at 28°C, 200rpm shaker, and after 12h, centrifuge the bacterial solution at 8500rpm for 10min to collect the bacterial cells.

The bacteria were resuspended in 4mL Lysis buffer, and ultrasonically crushed on the ice-water mixture. The parameters of the ultrasonic crusher were set at 200w power, 10min time, 3s ultrasonic time, and 3s interval.

The formula of Lysis buffer in this example is 300mM NaCl, 50mM Na ₂ HPO ₄ ·2H ₂ O, and the pH is 8.0.

(2) SDS-PAGE analysis

The cells induced to express were collected and ultrasonically disrupted, and the cell lysate was analyzed by SDS-PAGE, as shown in Figure 4 (A), the band intensities of the nine lanes were not significantly different, indicating that the linking peptides were effective for the expression of BaPAD and fusion enzymes No noticeable effect. The pure enzyme purified by Ni-NTA is also analyzed by SDS-PAGE, as shown in Figure 4 (B), it can be seen that there are single The bands correspond 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

The protein concentration of 9 kinds of pure enzymes purified by Ni-NTA was analyzed by BCA method. In the range of -206μg/mL, there is little difference, corresponding to the results of SDS-PAGE.

Table 2 Expression levels of BaPAD and fusion enzyme

enzyme Expression level (μg/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 experiments of BaPAD and fusion enzymes on different carriers

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

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

Calculation of enzyme load: After incubation, the sample was centrifuged at 8,500 rpm for 3 minutes, and the supernatant was separated. The protein concentration of the supernatant before immobilization and after adsorption was detected by BCA method, and the concentration of immobilized enzyme was obtained by subtraction. In order to compare the adsorption levels of 9 enzymes on different carriers.

Na-Y zeolite, ZSM-5 zeolite, β zeolite, dense silica and mesoporous silica were selected as solid supports, and the concentration of pure enzyme was set to 5 mg/g carrier to analyze the adsorption properties of 9 enzymes. It can be seen from Table 3 that both BaPAD and fusion enzyme can be completely adsorbed (100%) by Na-Y, ZSM-5 and β zeolite. In addition, these enzymes exhibited a binding affinity of 75-86% for mesoporous silica, 2-3 times higher than that for dense silica. For the two silica materials, the affinity of the enzyme containing the linker peptide was slightly stronger than that of BaPAD without the linker peptide, and the linker peptide showed superiority in the adsorption of silica materials at low enzyme concentrations.

The loading rate (%) of BaPAD and fusion enzyme on different carriers under the low enzyme concentration of table 3

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

10 mg Na-Y zeolite was washed three times with 800 μL wash buffer (10 mM Tris-HCl, 100 mM NaCl, 1% Triton X-100, pH 7.5) and citric acid-Na ₂ HPO ₄ buffer (200 mM, pH 7.0), each During this process, vortex, centrifuge at 8,500rpm for 3min to remove the supernatant. Add 200 μL of pure enzyme diluted to 1000 μg/ml to the washed Na-Y zeolite, so that the concentration of the enzyme in the system is 20 mg/g, and shake and incubate on ice for 1 h.

As can be seen from Table 4, for the fusion enzyme with the linker peptide, no matter whether the linker peptide is added to the N-terminal or the C-terminal of BaPAD, with the growth of the linker peptide, the amount of enzyme immobilized on Na-Y zeolite and the loading rate will decrease. It increases accordingly, and compared with the connecting peptide connected at the C-terminal, connecting the connecting peptide at the N-terminal can improve the loading rate of the fusion enzyme enzyme, and the highest loading rate of 4LP-BaPAD can reach 68.81%. The loaded protein concentration was 13.8 mg/g carrier. On the contrary, the loading rate of the original BaPAD without linker peptide was only 31.93%. These results indicated that at high enzyme concentrations, the addition of linker peptides could increase the enzyme loading on Na-Y zeolite, and the loading increased with the extension of linker peptides.

The maximum loading of BaPAD and fusion enzyme on Na-Y zeolite under the high enzyme concentration of table 4

(3) Biochemical properties of immobilized enzymes

S1. Enzyme activity detection of immobilized enzyme:

Enzyme activity assay steps (taking FA as an example): 1 mL of reaction system, containing 0.8 mL of citric acid-Na ₂ HPO ₄ buffer solution (pH 6.0), 100 μL of 50 mM sodium ferulate solution and 100 μL of 20 μg/mL free and Immobilized phenolic acid decarboxylase. React in a water bath at 50°C for 5 minutes, and add 2 mL of methanol to terminate the reaction. After filtration with a 0.22 μm filter membrane, the content of the generated 4-VG was detected by HPLC. A sample treated under the same conditions without adding enzyme and replaced with 100 μL of buffer was used as a control.

Detection of enzyme activity: Utilize HPLC to detect the generation amount of reaction product, take FA as example, utilize the measured peak area to calculate the amount of the 4-VG that generates according to standard curve, thereby calculate the activity (IU) of immobilized phenolic acid decarboxylase /mL).

Nine kinds of immobilized enzymes with Na-Y zeolite as the carrier take p-coumaric acid (pCA), ferulic acid (FA) and caffeic acid (CA) as the enzyme activities of the substrates in Table 5. It can be seen that the immobilized The substrate specificity of the enzyme is generally unchanged compared with the free enzyme, but the activity after immobilization is different from that of the free enzyme due to the difference in the position of the connecting peptide, the activity of BaPAD and BaPAD-1/2/3/4LP Also retained 55-85%, and 1/2/3/4LP-BaPAD after immobilization activity almost did not decrease, even the activity of LP-BaPAD is slightly higher than the free enzyme, in general, the linker peptide linked to the N-terminal After immobilization of the fusion enzyme, the activity becomes 95-105% of that of the free enzyme. The reason may be that the connecting peptide and his tag attached to the C-terminal are distributed at both ends of 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 be flexibly attached to the substrate. The contact with the substrate will slow down the decarboxylation reaction or the accessibility of the substrate, so the activity is much lower than that of the free enzyme; and the connecting peptide connected to the N-terminus and the his tag are at the same end of BaPAD, so that only one end of the fusion enzyme is blocked Immobilization is relatively more flexible, and the substrate has better accessibility, so the activity retained after immobilization is higher.

Table 5 The activities of free and immobilized BaPAD and fusion enzymes to different substrates

S2. The effect of pH on the activity of free and immobilized 4LP-BaPAD: free and immobilized 4LP-BaPAD were enzymatically reacted with FA as a substrate at 50°C and different pH, and the buffer used was pH 4.0-8.0 The citrate-Na ₂ HPO ₄ buffer was plotted with the highest relative activity of 100%.

The effect of temperature on the activity of free and immobilized 4LP-BaPAD: Enzyme-catalyzed reactions were carried out at different temperatures (30-60° C.) in the optimal pH buffer solution, and the relative activity was plotted as the highest 100%.

Effects of pH and temperature on the activity of free and immobilized 4LP-BaPAD: Ferulic acid was used as a substrate to detect the activities of free and immobilized 4LP-BaPAD at pH 4.0-8.0 and 30-60°C, and the enzyme activity was the highest It is 100% (as shown in Figure 5), as can be seen from Figure 5 (A), 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 Figure 5 (B) The optimal temperatures of free and immobilized 4LP-BaPAD were 50 and 55°C, respectively, and the enzyme activity was higher in the range of 45-55°C. Compared with BaPAD without linker peptide, the optimum temperature and optimum pH of 4LP-BaPAD were similar.

Example 4 One-step purification and immobilization of target protein from crude enzyme solution using zeolite material

(1) One-step purification method

The supernatant collected after sonication of recombinant cells induced by IPTG, that is, the crude enzyme, was diluted to 3000 μg/mL, and 0.1 g of Na-Y zeolite washed with wash buffer (see the pretreatment of solid carrier for details) was added. Into 0.5mL diluted cell lysate, incubated on ice for 1h, centrifuged at 8,500rpm for 3min, separated the supernatant, washed three times with Lysis buffer (300mM NaCl, 50mM Na ₂ HPO ₄ 2H ₂ O, pH 8.0), collected the supernatant The supernatant, and then the pellet was resuspended with 0.5 mL of Lysisbuffer. Take the same volume of cell lysate before immobilization, supernatant after adsorption and zeolite-immobilized resuspension were analyzed by SDS-PAGE method.

It can be seen from Figure 6 that after incubation of BaPAD without linking peptide with Na-Y zeolite, it mainly exists in the supernatant (unbound) after adsorption, and only a very small amount of residual BaPAD exists in the part of binding protein (immobilized) , may be due to the nonspecific adsorption of Na-Y zeolite. Most of the fusion proteins with linker peptides were present in the binding part after incubation with Na-Y zeolite, and the bands in this part were enhanced with the extension of the linker peptide, for BaPAD-4LP and 4LP-BaPAD, only a small amount present in the unbound fraction. In addition, it can be seen from the SDS-PAGE figure that all 8 fusion proteins show obvious single bands, which are consistent with the theoretical molecular weight. These results indicated that the fusion enzyme added with connecting peptide could purify and immobilize the target protein from cell lysate by one-step method of Na-Y zeolite.

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

The method is the same as the one-step purification method, except for the final SDS-PAGE step. The enzyme activity of the samples in each step was measured using ferulic acid as a substrate, and the selectivity (%) of BaPAD and fusion enzymes for Na-Y zeolite adsorption was obtained by calculating the ratio of supernatant and Na-Y zeolite immobilized enzyme activity.

Adsorption selectivity was defined by calculating the ratio of the activity of the immobilized enzyme to the original total enzyme activity. It can be seen from Table 6 that the initial total enzyme activity of BaPAD is the highest, reaching 318IU, but its selectivity to Na-Y zeolite adsorption is only 14%, while the fusion enzyme containing connecting peptide has high selective adsorption to Na-Y zeolite Compared with native BaPAD, and with the increase of the length of the connecting peptide, the selectivity gradually increased, and the selectivity of the connecting peptide at the N-terminus was higher than that at the C-terminus. Although 4LP-BaPAD had the lowest original total enzyme activity, it had the highest adsorption selectivity to Na-Y zeolite, reaching 86%.

Table 6 The selectivity of BaPAD and fusion enzymes to Na-Y zeolite adsorption

Raw total enzyme activity (IU) Activity of immobilized enzyme (IU) 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 4LP-BaPAD adsorption Na-Y zeolite stability and repeated loading research

(1) Adsorption stability

100 μL of 4LP-BaPAD was immobilized on washed Na-Y zeolite at a concentration of 10 mg/g carrier, and the amount of immobilized enzyme was obtained by measuring the supernatant protein concentration before and after adsorption. Afterwards, the immobilized enzyme was eluted with different buffer solutions, and the stability of the immobilized 4LP-BaPAD under different conditions was investigated by detecting the protein concentration and activity of the eluate.

The results are shown in Table 7. In the concentration range of 0.5% to 5% Tween 20 (pH 7), no immobilized protein was found to be eluted, and the immobilized enzyme was also eluted in the range of pH 3.0 to 9.0. Stable, however, 5M NaCl solution can partially elute the immobilized enzyme from the support. 4LP-BaPAD@Na-Y zeolite is stable when treated with Tween 20 solution which can destroy the hydrophobic interaction. SDS has a good desorption effect on immobilized enzymes, and can be 100% desorbed after being treated with 2% SDS.

Table 7 Stability of 4LP-BaPAD@Na-Y zeolite under different conditions

Elution condition (pH/NaCl concentration, M) Eluted 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) Repeated load

Use 100 μL of 2% SDS solution to shake and mix with 4LP-BaPAD immobilized on Na-Y zeolite, vortex for 20 s with a vortex instrument, centrifuge at 8,500 rpm for 3 min, and repeat three times to completely desorb the immobilized protein (by spectrophotometry The meter was detected at 280nm to verify), and then the residual SDS on the zeolite was washed with citric acid-Na ₂ HPO ₄ (pH 7.0), and then the next round of adsorption was performed, and three cycles were repeated. By detecting the protein concentration before and after each cycle of adsorption , to compare the change in the amount of Na-Y zeolite repeatedly loaded with 4LP-BaPAD after desorption.

2% SDS solution was used for desorption of 4LP-BaPAD from Na-Y zeolite. A typical cycle consisted of adsorption and desorption, and the amount of protein reloaded on zeolite was found to be almost unchanged after three cycles, indicating the superior reproducible performance 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 composites as biocatalysts in a two-phase reaction system containing toluene was evaluated by continuous reaction for 10 cycles. The yield of 4-vinylguaiacol during the continuous reaction is shown in Figure 7. It can be seen that the conversion efficiency of immobilized BaPAD and 4LP-BaPAD decreases gradually as the reaction continues. In the first cycle, 200mM substrate ferulic acid is catalyzed by immobilized BaPAD and 4LP-BaPAD to produce 175 and 180mM 4-vinylguaiacol, the second cycle of this set of data decreased to 145 and 175mM, while in the tenth cycle, the production of 4-vinylguaiacol was reduced compared to the first reaction For the initial 36% and 73%. Therefore, the operational stability of the immobilized phenolic acid decarboxylase containing the connecting peptide is much higher than that of the enzyme without the connecting peptide, and the immobilized 4LP-BaPAD can be used as a catalyst with excellent catalytic ability and stability.

Example 6 Using 4LP-BaPAD@Na-Y zeolite to convert FA into 4-VG in a bioreactor

(1) Preparation of fusion enzyme 4LP-BaPAD

Prepare 200mL×8 bottles of sterilized LBK culture solution, add activated 4LP-BaPAD seed cell solution according to the volume ratio of 2%, cultivate at 37°C until OD ₆₀₀ =0.6, add IPTG with a final concentration of 0.4mM, and incubate at 28 ℃ induction culture for 12h. Centrifuge at 8,000 rpm for 10 min at 4°C, remove the supernatant, resuspend the cells with 4 mL Lysis buffer, and sonicate on ice. Centrifuge at 10,000 rpm at 4°C for 10 min, and collect about 200 mL of the supernatant, which is the crude enzyme. The total amount of crude protein detected by the BCA method is about 700 mg.

Among them, the LBK culture medium is the LB liquid medium which is sterilized at 121° C. for 20 minutes, then cooled to about 60° C. and added with kanamycin at a final concentration of 50 μg/mL.

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

Weigh 70g Na-Y zeolite, wash it three times with Lysis buffer (pH 8.0), add 200mL crude enzyme solution, stir magnetically on ice for 1h, detect the protein concentration of the supernatant after centrifugation, and calculate the total unadsorbed protein amount of about 220 mg, the catalyst used was 70 g of 4LP-BaPAD@Na-Y zeolite immobilized with 480 mg of total protein.

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

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

Reaction conditions: adjust the reaction temperature to 30°C through the water circulation system that comes with the fermenter, one stirring rotor for the water phase and one organic phase, and the rotation speed is 150rpm, add ferulic acid powder during the reaction process through the change of the pH value of the instrument control panel , so that its pH is controlled within the range of 6.5±0.1. Samples were taken every hour, and the samples were centrifuged at 10,000 rpm for 5 minutes to fully separate the organic phase and the aqueous phase.

Detection method: Dilute the organic phase and the aqueous phase with methanol respectively, and perform HPLC detection, and calculate the concentrations of generated 4-VG and residual FA according to the standard curve.

In a two-phase biotransformation system with toluene as the organic phase, ferulic acid was used as the substrate to prepare high-concentration 4-vinylguaiacol. HPLC analysis showed that after 13 hours of continuous reaction, as shown in Figure 8, a total of 1.97M products were generated, the conversion rate was 98.95%, and the productivity was 22.8g/L/h.

Claims (5)

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.

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