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L-pantoic acid lactone dehydrogenase, mutant and application of L-pantoic acid lactone dehydrogenase

A technology of pantolactone and ester dehydrogenase, which is applied in the biocatalytic preparation of D-pantothenic acid precursor intermediate ketopantolactone, L-pantolactone dehydrogenase TpLPLDH, mutation In the field of body and its coding gene, it can solve the problems of low enzyme activity, serious inclusion body, incomplete transformation of L-pantoate lactone, etc., and achieve the effect of good solubility

Active Publication Date: 2022-04-15
ZHEJIANG UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the L-pantolactone dehydrogenase from Nocardia stellatus has been studied in detail (Kataoka M, et al. European Journal of Biochemistry 1992, 204, 799-806), the protein is present in the large intestine It is easy to form inclusion bodies when expressed in Bacillus, and the enzyme activity is low. The Km of the enzyme tested to catalyze the substrate L-pantoacid lactone is 26.8 mM, and the enzyme activity of the enzyme is 4.22 U / mg
Invention patent "from Nocardia farcinica L-pantolactone dehydrogenase and its application” (Application No.: CN201910366852.5), the patent only successfully expresses the protein, and the aldehyde and ketone reductase from Saccharomyces cerevisiae and the glucose dehydrogenase from Microbacterium Together, they catalyze the substrate L-pantoate lactone to generate the final substrate D-pantoate lactone. Without the addition of NADPH, L-pantoate lactone cannot be completely converted
There are few sources and information about L-pantolactone dehydrogenase, and the reported enzyme expression has serious inclusion bodies, which limits its application

Method used

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  • L-pantoic acid lactone dehydrogenase, mutant and application of L-pantoic acid lactone dehydrogenase
  • L-pantoic acid lactone dehydrogenase, mutant and application of L-pantoic acid lactone dehydrogenase
  • L-pantoic acid lactone dehydrogenase, mutant and application of L-pantoic acid lactone dehydrogenase

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1: Construction of L-pantolactone dehydrogenase engineering bacteria

[0031] The amino acid sequence of L-pantolactone dehydrogenase is shown as SEQ ID NO.1, and the codon-optimized gene SEQ ID NO.3 is constructed on the expression vector pET 28a, and a 6*his tag is added to the C-terminal for subsequent protein expression. The build process is as follows:

[0032] 1. Use F-1 and R-1 as primers (Table 1) to Tp LPLDH is a template to expand gene fragment 1;

[0033] 2. Use F-2 and R-2 as primers (Table 1), and use pET 28a as a template to expand vector fragment 2;

[0034] 3. Purify Fragment 1 and Fragment 2, and use NcoI and XhoI to digest the purified Fragment 1 and Fragment 2;

[0035] 4. Ligate the purified fragment 1 and fragment 2 with T4 ligase ligase, and transform the enzyme-linked product into E. coli BL21 competent cells;

[0036] 5. Pick a single colony and culture it overnight in a liquid medium containing 50 μg / μl kanamycin at 37 °C in a s...

Embodiment 2

[0039] Example 2: Construction of Mutant Library and Selection of Mutants

[0040] For the expression plasmid pET 28a- Tp LPLDH was used as a template, and the alanine at position 29 was saturatingly mutated. The build process is as follows:

[0041] 1. Use 25-29-F and 25-29-R in the following table 2 as primers, use pET 28a- Tp LPLDH is used as a template to expand the entire plasmid containing the mutated gene;

[0042] 2. Reaction system: PCR reaction system (25 µL): 1 µL forward primer (100 µM), 1 µL reverse primer (100 µM), 12.5 µL 2×Phanta buffer, 0.5 µL dNTP mixture (10 mM each) , 1 µL plasmid template, 0.5 µL DNA polymerase Phanta (Novazyme, China) and 8.5 µL ultrapure water;

[0043] 3. Reaction program: Pre-denaturation at 95°C for 5 min, followed by 30 cycles (denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 7 s), final extension at 72°C for 10 min, and incubation at 16°C;

[0044] 4. Add 1 µL DpnI (NEB, USA) and 2.5 µL Buffer ...

Embodiment 3

[0055] Embodiment 3: Mutant substrate catalytic ability test

[0056] Pick the engineering strain of embodiment 1 E. coli BL21(DE3) / pET-28b- Tp LPLDH and the mutant strain constructed in Example 1 E. coli BL21(DE3) / pET-28b- Tp LPLDH A29S A single colony was cultured in LB medium containing 50 μg / μL kanamycin at 37 °C in a shaker at 180 rpm overnight. Transfer the seed solution to 100 mL LB liquid medium containing 50 μg / μL kanamycin at 10% inoculum size, and culture it in a shaker at 37 °C and 180 rpm for 2-2.5 h to make the OD of the strain 600 Reach between 0.6-0.8. Add a final concentration of 0.1 mM isopropylthiogalactopyranoside (Isopropyl β -D-thiogalactoside, IPTG) at 28°C, 180 rpm for 12 h. then at 4 o C. Centrifuge at 12000rpm for 10 min to obtain protein containing L-pantolactone dehydrogenase activity Tp LPLDH and Tp LPLDH A29S wet bacteria;

[0057] Each strain was treated with pH 7.0, 50 mM phosphate buffer (0.2 M Na 2 HPO 4 , 0.2 M NaH 2 PO ...

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Abstract

The invention relates to L-pantoic acid lactone dehydrogenase TpLPLDH with good water solubility, a mutant and a coding gene thereof, and application of the L-pantoic acid lactone dehydrogenase TpLPLDH in preparation of a D-pantoic acid precursor intermediate ketone pantoic acid lactone through biological catalysis. The amino acid sequence of the L-pantoic acid lactone dehydrogenase TpLPLDH is as shown in SEQ ID NO. 1 (sequence identifier number 1). The novel protein with the activity of the L-pantoic acid lactone dehydrogenase has the advantages that the protein has the effect of the L-pantoic acid lactone dehydrogenase, L-pantoic acid lactone can be catalyzed to be dehydrogenated to generate keto-pantoic acid lactone, the solubility of the protein is good, and the protein can be used for preparing keto-pantoic acid lactone. The compound is almost completely dissolved in an aqueous solvent (such as a phosphate buffer). According to the mutant with the L-pantoic acid lactone dehydrogenase activity, after the mutant reacts in a system of a substrate L pantoic acid lactone with the final concentration of 1 mM for 30 min at the temperature of 30 DEG C and the speed of 1200 rpm, compared with a wild type, the mutant has the advantages that the substrate conversion rate is increased by 1.84 times, and the enzyme activity is increased by 1.12 times compared with the wild type.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to an L-pantolactone dehydrogenase TpLPLDH, a mutant and its coding gene, and its use in the biocatalytic preparation of D-pantothenic acid precursor intermediate ketopantolactone Applications. Background technique [0002] Pantothenic acid is a kind of water-soluble vitamin B group, and it is also one of the nutrients necessary for the normal growth of organisms. Pantothenic acid acts as a functional group in biological tissues and becomes a component of coenzyme A and acyl carrier protein (ACP). Coenzyme A is an important coenzyme involved in many reversible acetylation reactions in carbohydrate, fat and amino acid metabolism, and is important for endogenous metabolic energy exchange in various tissues. The main functions of pantothenic acid include promoting the utilization of nutrients, promoting the synthesis and decomposition of fatty acids, and participating in the c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P17/04C12R1/19
CPCC12N9/0006C12N15/70C12Y101/99027C12Y101/01168C12P17/04
Inventor 柳志强杨青朱芳莹张晓建郑裕国马石金杜军吴慧
Owner ZHEJIANG UNIV OF TECH
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