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

A technique for immobilizing enzymes and linking peptides, which is applied in biochemical equipment and methods, botanical equipment and methods, immobilized on or in inorganic carriers, and can solve the problems of separation and purification difficulties, poor selectivity, enzymes and solid carrier surfaces Inadequate adsorption and other problems, to achieve the effect of wide application prospects and good reusability

Active Publication Date: 2020-09-15
NANJING FORESTRY UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

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 a 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 the inability to reuse, 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, etc.

Method used

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  • Linker peptide mediated enzyme immobilized BaPAD catalyst, and preparation method and application thereof
  • Linker peptide mediated enzyme immobilized BaPAD catalyst, and preparation method and application thereof
  • Linker peptide mediated enzyme immobilized BaPAD catalyst, and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1B

[0025] The construction of the fusion enzyme that embodiment 1BaPAD is fused with connecting peptide

[0026] (1) Construction of pET28a-bapad-lp and pET28a-lp-bapad

[0027] 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. 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, ddH 2 O 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'...

Embodiment 2B

[0043] Expression and purification of embodiment 2 BaPAD and fusion enzyme recombinant protein

[0044] (1) Expression and purification of recombinant protein

[0045] 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.

[0046] 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.

[0047] Inoculate the 2% inoculum of the overnight culture into 50mL sterilized LBK liquid medium, and cultivate it to OD at 37°C with a shaker at 200rpm 600 is 0.6.

[0048] 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 bacte...

Embodiment 3

[0057] Example 3 Adsorption experiments of BaPAD and fusion enzymes on different carriers

[0058] (1) Affinity adsorption of BaPAD and fusion enzymes to different carriers

[0059] 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-NaCl 2 HPO 4 Buffer (200mM, pH 7.0) was washed three times, each process was vortexed, and centrifuged at 8,500rpm for 3min to remove the supernatant. Immobilization: 9 pure enzymes (50 μg, 100 μL citrate-Na 2 HPO 4 Buffer) was added to the washed carrier and incubated for 1 h with shaking on ice.

[0060] 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 obtaine...

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Abstract

The invention discloses a linker peptide mediated enzyme immobilized BaPAD catalyst, and a preparation method and application thereof, and belongs to the field of preparation and application of catalysts. The method comprises the following steps of: firstly, inserting linker peptide, which specifically adsorbs zeolite, into a plasmid pET28a-bapad vector containing an encoded phenolic acid decarboxylase BaPAD gene through an Nco I / Xho I restriction enzyme cutting site, and then, immobilizing to obtain the enzyme immobilized BaPAD catalyst on a pre-treated Na-Y zeolite carrier through fusion enzyme, wherein the phenolic acid decarboxylase BaPAD is from bacillus atrophaeus. The biocatalyst prepared by the invention is used for catalyzing FA to produce 4-VG; compared with a method for catalyzing FA to produce 4-VG in a whole-cell manner, the concentration and productivity of 4-VG are improved to 1.97 M and 22.8 g / L / h under the same condition; and the catalyst has good reusability, and canmeet industrial production requirements of 4-VG.

Description

technical field [0001] 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 [0002] 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 l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/14C12N9/88C12N15/70C12N15/60C12P7/22
CPCC12N11/14C12N9/88C12N15/70C12P7/22
Inventor 丁少军李璐璐
Owner NANJING FORESTRY UNIV
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