阅读说明：本技术 一种连接肽介导的酶固定化BaPAD催化剂及其制备方法和应用 (Linker peptide mediated enzyme immobilized BaPAD catalyst and preparation method and application thereof ) 是由 丁少军 李璐璐 于 2020-06-08 设计创作，主要内容包括：本发明公开了一种连接肽介导的酶固定化BaPAD催化剂及其制备方法和应用,属于催化剂的制备与应用领域。该方法首先将对沸石特异性吸附的连接肽通过Nco I/Xho I限制性酶切位点插入到含有编码酚酸脱羧酶BaPAD基因的质粒pET28a-bapad载体上,然后通过融合酶在经过预处理的Na-Y沸石载体上固定化得到酶固定化BaPAD催化剂,其中酚酸脱羧酶BaPAD来自萎缩芽孢杆菌。本发明所制备的生物催化剂用于催化FA生产4-VG,和全细胞催化FA生产4-VG相比,在相同的条件下,将4-VG浓度和生产率提高到1.97M和22.8g/L/h；且具有很好的重复使用性,能够实现4-VG的工业化生产需求。(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 whole-cell catalysis of FA for producing 4-VG, 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 conditions; and the reusability is good, and the industrial production requirement of 4-VG can be met.)

1. A preparation method of a connecting peptide mediated enzyme immobilized BaPAD catalyst is characterized in that connecting peptide which is specifically adsorbed on zeolite is firstly inserted into a plasmid pET28a-BaPAD 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 fused enzyme on the pretreated zeolite carrier, wherein the phenolic acid decarboxylase BaPAD is from Bacillus atrophaeus.

2. The process of claim 1, wherein the linker peptide is formed by concatenating the n-repeats VKTQATSREEPPRLPSKHRPG and VKTQTAS, and 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.

3. The method of claim 2, wherein n is 4.

4. The method of claim 1, wherein the linker peptide is added to the Bacillus atrophaeus phenolic acid decarboxylase BaPAD N-terminus, and the linker peptide attached to the N-terminus is at the same end of BaPAD as the his tag.

5. The method of preparing a linker-mediated enzyme immobilized pad catalyst of claim 1, wherein the pretreatment of the zeolite support is: 10mg of the carrier was washed three times with 800. mu.L wash buffer, followed by 200mM, pH7.0, citric acid-Na₂HPO₄Washing 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, and has a pH of 7.5.

6. 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.

7. A linker-mediated enzyme-immobilized BaPAD catalyst prepared by the process of any one of claims 1-6.

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

9. 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.

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-Vinylguaiacol has great toxicity to cell membranes, cells are cracked or killed in the process of producing 4-Vinylguaiacol (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 a solid support, among which the adsorption method is most commonly used, but the conventional adsorption method has disadvantages that the adsorption of enzymes to the surface of a solid support is not so strong and the selectivity is poor, and adding a specific linker peptide to the N-terminus or C-terminus of BaPAD is one method for improving these 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 pET28a-BaPAD vector containing a gene for encoding 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 vector through a fusion enzyme, wherein the phenolic acid decarboxylase BaPAD is from Bacillus atrophaeus.

Further, a linker peptide is composed of n repeats VKTQATSREEPPRLPSKHRPG in tandem with VKTQTAS, and 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.

Further, n is 4.

Furthermore, a connecting peptide is added at the N-terminal of phenolic acid decarboxylase BaPAD of the Bacillus atrophaeus, and the connecting peptide connected at the N-terminal and the his label are positioned at the same end of the BaPAD.

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, pH7.0, citric acid-Na₂HPO₄Washing with buffer solution for three times, performing vortex oscillation in each process, and centrifuging at 8500rpm for 3min to remove supernatant; wash buffer comprised 10mM Tris-HCl, 100mM NaCl, 1% Triton X-100, pH 7.5.

Further, the enzyme immobilized BaPAD catalyst was stable in 0.5-5% Tween20 solution, and 2% SDS solution was able to desorb all the immobilized enzyme.

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.

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.

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 specifically bound with zeolite for catalyzing FA to produce 4-VG, the concentration and the productivity greatly exceed the results of whole cells and free enzyme as biocatalyst, 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 toluene-containing two-phase system, 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 met, and the application prospect is wide.

Drawings

FIG. 1 is a schematic diagram of 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-bapad;

FIG. 3 is a screening chart of positive clones 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 (15mg/g) of BaPAD and fusion enzyme to Na-Y zeolite in cell lysates;

FIG. 7 is a graph of the repetitive operational stability of Na-Y zeolite immobilized BaPAD and 4LP-BaPAD enzymatically decarboxylated ferulic acid in a two-phase system;

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.